F113. 1 4.. '21.. f .. ks wwrvfiwwvmwflflx ’ l or...) , Au" . w .5: . I! ma s... .4: gr. 1. filo I to 3‘... . 3 (a... .1. 1.... wfi. , - ‘nali . ‘ . . “who! «LBJ»?! i. 39.3.: ‘ . a... “ Pl .- . avaii...» . ..., unwr.-....n .14; «7...? .. 8...... a... I An, 2.. . 3; t... .5 .l. Wsfi:l:..”|3!.h91 ... . . !. I, :11 I! : . en .. U. s I 4i! Affoht .54. .r ¢ .. r . 4-. A! Wu). 53!: hex! on; a . 3.77... ”New... I; oitnffluflu» . . .ll: 49...! ., 1 to 1.3”.) : i. 32,514.51 9 a Furs This is to certify that the dissertation entitled DIABETIC OSTEOPOROSIS AND BONE ADIPOSITY presented by SERGIU BOTOLIN has been accepted towards fulfillment of the requirements for the Ph.D. degree in PHYSIOLOGY iKaOZflSP/z/{C 65/6‘ Major Protéssor’s Signature 12/05/2006 Date MSU is an Affirmative Action/Equal Opportunity Institution m LIBRARY Michigan State University h. PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. [mmmm J” 0 Di Wu“ DATE DUE DATE DUE AN 1 1 2011] C.— 121700 2/05 p:/ClRC/DateDue.indd-p.1 DIABETIC OSTEOPOROSIS AND BONE ADIPOSITY By Sergiu Botolin A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 2006 ABSTRACT DIABETIC OSTEOPOROSIS AND BONE ADIPOSITY By Sergiu Botolin Diabetes mellitus (IDDM type I) is a chronic disease, which is characterized by a lack of insulin production. One of the long-term complications of IDDM is diabetic osteopenia and osteoporosis, which are associated with increased fracture rate and delayed fracture healing. Pathophysiological mechanisms of diabetic bone loss are still unclear leading to a lack in adequate therapeutic strategy. In the present work, we investigated mechanisms of diabetic bone loss in streptozotocin-induced wild type, BaIb/c male mice. This mouse model of diabetes type I is closed to real diabetic situation, it is relatively inexpensive, easy to induce and manipulate, and has a potential for future genetic manipulation. Our histological and imaging data demonstrated that streptozotocin induced diabetes is associated with bone loss in diabetic mice. This is associated with decrease in bone expression of osteocalcin and Runx-2, decreased serum levels of osteocalcin, and normal to decreased levels of osteoclast resorptive activity markers such as DPD, PYD and TRAP-5b. We were the first to report that diabetic bone loss is associated with increased bone marrow adiposity and increased expression of adipogenic markers PPARyZ, Resistin and aP2. This increase in adiposity occurred in parallel with an increase in serum triglyceride and free fatty acid levels and a general lipolysis of fat depots. Inhibition of PPARy pathways with a synthetic PPARy antagonist BADGE, led to a complete normalization of the serum levels of triglycerides and free fatty acids. PPARy and aP2 expression in tibia bone from diabetic animals treated with BADGE was decreased to normal levels and so were bone marrow adipocyte numbers. Diabetes associated bone loss and osteogenic markers expression in bones and serum of diabetic animals did not show an improved pattern, suggesting that hyperlipidemia and bone marrow adiposity are not necessarily directly linked to bone density status and are not the cause of type I diabetic bone loss. To further characterize this experimental diabetic model, we investigated the immediate response of adipose and bone markers to streptozotocin induced diabetes. We found that increase of aP2 and decrease of osteocalcin mRNA expression in streptozotocin injected animals, occurred as early as day 5 after the first injection. PPARy2 mRNA was significantly increased at day 7 after the first injection. Interestingly, Runx-2 expression was decreased 24 hours after the injection, suggesting an immediate component of diabetic bone loss following streptozotocin administration. Taken together, this work brings novelty into the field of diabetic bone loss, revealing new insides in pathogenesis of diabetic osteoporosis, opening new possibilities in treatment and management programs of diabetes. ...dedicated to my wife Daniela and my son Paul, and my parents, for support and encouragement that allowed me to reach as far as I have... ACKNOLEDGEMENTS I would like thank all professors which participated in my scientific education. In particular, I would like to thank all my present and past lab mates. Thank you Regina Irwin and Christopher Ontiveros for being my friends and supporters. I would like to thank Dr. Laura McCabe, which was my teacher, my mentor and my friend, and which had the deepest impact on my scientific educafion. TABLE OF CONTENTS LIST OF TABLES .................................................................................. ix LIST OF FIGURES .................................................................................. x ABBREVIATIONS ................................................................................. xiii CHAPTER I. LITERATURE REVIEW ......................................................... 1 1.Bone ................................................................................................ 1 1.1 Overview ................................................................................ 1 1.1.1 Functions .................................................................... 1 1.1.2 Structure ..................................................................... 2 1.2 Bone cells .............................................................................. 3 1.2.1 Overview .................................................................... 3 1.2.2 Osteoblast differentiation ..................... , .......................... 4 1.3 Bone Marrow ......................................................................... 6 1.3.1 Overview ......................................................... - ........... 6 1.3.2 BMSC ....................................................................... 7 1.3.3 Adipocytes differentiation ............................................... 8 2. Osteoporosis .................................................................................... 9 2.1 Overview ............................................................................... 9 2.1.1 Definition ................................................................... 9 2.1.2 Epidemiology ............................................................ 10 2.1.3 Classification ............................................................. 11 2.2 Osteoporosis and bone marrow adiposity .................................... 12 3. Diabetes ......................................................................................... 13 3.1 Overview .............................................................................. 13 3.1.1 Definition .................................................................. 13 3.1.2 Epidemiology ............................................................. 14 3.1.3 Classification ............................................................. 14 3.2 Diabetic complications ............................................................ 15 3.3 Diabetic dyslipedimia .............................................................. 16 4. Diabetic Bone Loss ........................................................................... 17 4.1 Overview ............................................................................. 17 4.1.1 Clinical studies ........................................................... 18 4.1.2 Animal model work ...................................................... 19 4.1.3 Proposed mechanisms ................................................ 20 vi 4.1.3.1 Impaired osteoblasts function ............................ 20 4.1.3.2 Hypoinsulinemia ............................................ 21 4.1.3.3 IGF ............................................................... 21 4.1.3.4 Vascular factors .............................................. 23 4.1.3.5 Nutrition ....................................................... 23 4.1.3.6 Glycation ...................................................... 24 5. Diabetes type I experimental models and osteoporosis ............................. 25 REFERENCES ................................................................................... 31 CHAPTER II. MATERIALS AND METHODS ............................................ 40 CHAPTER III. INCREASED BONE ADIPOSITY AND PPARyZ EXPRESSION IN TYPE I DIABETIC MICE ........................... 54 Abstract ........................................................................... 55 Introduction ....................................................................... 57 Results ............................................................................ 60 Discussion ........................................................................ 64 References ....................................................................... 71 Figure Legends .................................................................. 80 Figures ............................................................................ 83 CHAPTER IV. INHIBITION OF PPARy PREVENTS TYPE I DIABETIC BONE MARROW ADIPOSITY BUT NOT BONE LOSS ................................................... 93 Abstract .......................................................................... 94 Introduction ..................................................................... 96 Results ........................................................................... 100 Discussion ...................................................................... 103 References ..................................................................... 1 08 Figure Legends ................................................................ 112 Figures ........................................................................... 115 CHAPTER V DIABETIC ADIPOSITY AND DMSO TREATMENT lN STREPTOZOTOCIN DIABETIC MICE .............................. 123 Introduction ..................................................................... 124 Results .......................................................................... 126 Discussion ..................................................................... 128 References ..................................................................... 130 Figure Legends ............................................................... 132 Figures ........................................................................... 133 vii —-——— CHAPTER VI. BONE RESPONDS IMMEDIATELY TO STREPTOZOTOCIN-INDUCED DIABETES ........................ 135 Abstract .......................................................................... 136 Introduction ..................................................................... 137 Results ........................................................................... 140 Discussion ...................................................................... 143 References ..................................................................... 148 Figure Legends ................................................................ 152 Figures ........................................................................... 155 CHAPTER V. CHRONIC HYPERGLYCEMIA MODULATES OSTEOBLAST GENE EXPRESSION THROUGH OSMOTIC AND NON-OSMOTIC PATHWAYS ..................... 161 Abstract .......................................................................... 162 Introduction.......................... ........................................... 164 Results ........................................................................... 168 Discussion ...................................................................... 173 References ..................................................................... 179 Figure Legends ................................................................ 186 Figures ........................................................................... 189 CHAPTER Vl. DISCUSSION ............................................................. 193 REFERENCES ............................................................ 207 viii LIST OF TABLES Table 1. Tibia uCT analysis ............................................................................... 85 Table 2. Tibia histology analysis ........................................................................ 88 Table 3. Tibia pCT analysis ............................................................................. 120 ix LIST OF FIGURES Figure 1. Diabetes increases serum glucose, serum osmolality and serum triglycerides and is associated with decrease body weight .......................................................................... 75 Figure 2. Diabetes reduces trabecular bone volume .......................................... 76 Figure 3. Systemic and tibia specific measures of osteoclast activity in diabetic bone ........................................................................ 78 Figure 4. Osteocalcin mRNA and serum levels are decreased in diabetes, in contrast to runx-2 and alkaline phosphatase (Alk Phos) mRNA levels ...................................................................... 81 Figure 5. Adipocyte markers, PPARy2, aP2 and Resistin are increased in diabetic bone ............................................................. 82 Figure 6. Adiposity is increased in diabetic tibia, in contrast to peripheral adipose tissue lipolysis. ..................................................... 83 Figure 7. Peripheral adipose tissue is decreased in diabetic mice ..................... 84 Figure 8. BADGE treatment prevents hyperlipidemia in diabetic mice, but does not correct hyperglycemia or prevent weight loss ............... 113 Figure 9. BADGE treatment blocks induction of adipogenic gene expression in diabetic bones ............................................................. 114 Figure 10. PPARy2 antagonist treatment prevents type I diabetes-induced bone adiposity ...................................................... 115 Figure 11. BADGE treatment is unable to rescue osteogenic gene expression in diabetic bone .................................................... 116 Figure 12. BADGE treatment does not prevent type I diabetes-induced bone loss ........................................................... 117 Figure 13. Chronic BADGE injection is the most effective treatment regime in preventing type I diabetes-induced hypertriglyceridemia and aP2 expression ...................................... 119 Figure 14. BADGE treatment beginning at the time of streptozotocin injection is unable to prevent osteocalcin mRNA suppression in diabetic mice ............................................... 120 Figure 15. Diabetic bone response to BADGE and DMSO treatment. Figure 16. Bone effects of BADGE and DMSO treatment in control mice. Figure 17. STZ injections induce hyperglycemia and body weight loss 3 days after the first injection .......................................... 143 Figure 18. Early loss of femoral fat depots is associated with normal serum free fatty acids levels ................................................. 144 Figure 19. Muscle tissue weight loss in result to STZ injections .................. 145 Figure 20. Early increase in adipocyte markers PPARv2 and aP2 in bones from STZ injected mice. .............................. 146 Figure 21. Serum osteoblast maturation and bone resorption markers are decreased in STZ injected animals 5 days post injection ................................................... ‘ ....... 147 xi Figure 22. Figure 23. Figure 24. Figure 25. Figure 26. Figure 27. Figure 28. Figure 29. Figure 30. Immediate decrease in Runx-2 mRNA is followedby a decrease in osteocalcin mRNA in STZ injected animals ............. 148 Osteoblast morphology and number does not change under chronic hyperglycemia, but alkaline phosphatase activity is increased ............................................................ 182 Expression of early and mid stage markers of osteoblast differentiation is modulated by chronic hyperglycemia ................ 183 Expression of early and mid stage markers of osteoblast differentiation is modulated by 48-hour hyperglycemic conditions..184 Expression of late stage markers of osteoblast differentiation is modulated by chronic hyperglycemia and, in the case of MMP-13, hyperosmolarity ........................... 185 Expression of late stage markers of osteoblast differentiation is modulated by 48-hour hyperglycemic conditions .................... 186 Expression of adipocyte-phenotype marker, PPARy2, is increased under chronic hyperglycemic conditions ..................... 187 Expression of VEGF and GAPDH is modulated by chronic hyperglycemia ..................................................... 188 Expression of late VEGF and GAPDH in differentiated osteoblasts is reduced under 48-hour hyperglycemic conditions....189 xii ABBREVIATIONS a-MEM P9 AML AP AP-1 aP2 BMP beB C/EBP CDNA Col l DPD OC PPARy PYD RT-PCR Runx SOX9 TRAP-5b a minimal essential media microgram microliter acute myeloid leukemia factor alkaline phosphatase activator protein 1 adipocyte fatty acid binding protein bone morphogenic protein core binding factor beta CAAT/enhancer binding protein complementary deoxynucleic acid collagen I deoxypyridinoline osteocalcin peroxisome proliferators activator receptor gamma pyridinoline crosslinks reverse transcription polymerase chain reaction runt related transcription factor sex determining region Y box 9 tartrate resistant acid phosphatase 5 b xiii CHAPTER I LITERATURE REVIEW 1.Bone 1.1 Overview 1.1.1 Functions Bone is a highly specialized and dynamic tissue, which throughout evolution made possible the appearance of vertebrates. It has a variety of important functions in mammalian body including mechanical, protective, chemical, and hematological functions. Bone’s mechanical properties confer rigidity to the skeleton, provide support for muscle attachment, and serve as a lever for muscles. In addition, by providing hard to penetrate “housing”, bones such as skull, thoracic chest, pelvis and long bones protect brain, thoracic organs, pelvic organs and bone marrow from external mechanical forces. Bone’s chemical function is the basis for calcium homeostasis and metabolism. The hematological function of bone is derived from the ability of bone to aid in development and storage of blood cells. 1.1.2 Structure In order to perform the above mentioned functions, bone has two structural components: organic and inorganic. The extracellular matrix represents the organic component and it is composed of 95 % collagen type I fibers and 5 % proteoglycans and numerous noncollagenous proteins. The inorganic component imbeds collagen fibers and is composed of calcium and phosphate salts that deposit in the form of hydroxyapatite. Based on the structure, functional demand and body localization there are 2 types of bone: cortical (compact) and trabecular (cancellous). Cancellous or trabecular bone is a highly porous and highly cellular form of bone. In contrast, cortical bone has densely packed collagen fibrils. These structural differences are the basis of mechanical and protective functions of the cortical bone and metabolic function of cancellous bone respectively. In a typical long bone, the cortex or exterior of the shaft (diaphysis) and flared ends (metaphysis) are composed of compact bone while the interior, particularly near the articulating ends, is filled with cancellous bone. 1.2 Bone cells 1.2.1 Overview There are 4 main cell types in adult bone: osteoblasts, osteocytes, bone lining cells and osteoclasts. Osteoblasts, osteocytes and lining cells are involved in formation and maintaince of the bone and are of mesenchymal cell origin. Osteocalsts are cells responsible for bone resorption and are of hematopoetic origin. Osteoblasts are the cells responsible for the formation and organization of the extracellular matrix of bone and its subsequent mineralization. They are derived from mesenchymal precursor cells in bone marrow. During mineralization, portions of osteoblasts become trapped in lacunae within the matrix of bone as osteocytes that are connected by a system of canaliculi. These cells are responsible for intercellular communication, and regulate the response of bone to the mechanical environment. In response to different stimuli osteocytes secrete growth factors that activate the lining cells or stimulate the osteoblasts. Lining cells are former osteoblasts which line the entire surface of the bone and can participate in initiation of bone remodeling. They have a flat shape and are responsible for immediate release of calcium from the bone if the blood calcium is too low. They also protect the bone from chemicals in the blood which dissolve crystals (such as pyrophosphate). Osteoclasts are the cells responsible for the resorption of bone matrix, and are presented as large, motile, multinucleated cell, located on bone surfaces, tightly associated with the calcified matrix. They are derived from haematopoetic stem cells in marrow and are formed by the fusion of mononuclear cells. Osteoclastic bone resorption initially involves mineral dissolution, followed by degradation of the organic phase. These degradation processes depend on lysosomal enzyme secretion and an acid microenvironment. Tartrate resistant isoenzyme of acid phosphatase (TRAP5b) and cysteine-proteinases such as cathepsins, are actively involved in this lytic process and therefore are used as a markers of the osteoclast activity. 1.2.2 Osteoblast differentiation Osteoblasts are derived from bone marrow stem cell (BMSC) which can give rise to adipocytes, chondrocytes, muscle cells and other cell types (1). These cells are distinguished by their ability to self-renew, divide and differentiate into executive mature cells. The first osteoblast related executive cell is the colony-forming unit-fibroblast (CFU-F), which carries the ability to form colonies of fibroblastic cells in culture(2),(3). Many of these colonies have the ability to form a mineralized matrix in vitro and bone tissue in vivo. The second executive cell is CFU-osteoblast (CFU-OB) which gives rise to the matrix mineralizing colonies. CFU-OBs have a high proliferative and finite self-renewal capacity classifying them as osteoblast progenitors amplifying cells rather than stem cells. CFU-OBs number is altered by many physiological and pathological conditions. It is been demonstrated that CFU-OB number decreases with aging in humans and rodents, and it is also decreased in reduced bone remodeling and formation following an excess of glucocorticoids (4),(5). The increase in CFU-OB number is associated with increase in bone remodeling in sex steroid depletion (6). Osteogenic pathway requires a broad spectrum of regulatory signals which commit, maintain commitment and provide maturation of osteoprogenitor cells. It is been shown that the program to follow osteogenic commitment and avoid differentiation into other cell types is controlled at transcriptional level (7). Major transcriptional controllers are Runx/bea/AML — runt homology domain transcriptional factors. Runx-1 is associated with hematopoiesis, Runx-3 with neurogenesis, and Runx-2 is the principal player in directing osteoblast proliferation and differentiation (7). Runx-2 expression can be tracked to the early event of commitment of mesenchymal cell to the bone lineage. Experiments by Otto et al (1997) and Yamaguchi et al (2000) demonstrated that absence of Runx-2 in mouse embryos lead to a total absence of osteoblast and a subsequent lack of bone tissue (8),(9). Therefore Runx-2 is considered the “master switch” in osteogenesis as PPARy and CEBPa in adipogenesis and SOX9 in chondrogenesis (10),(11),(12). Controlling function of Runx-2 in osteogenic commitment is accentuated by its ability to activate osteogenic genes in pluritotent stem cells and by the ability to redirect muscular and adipogenic commitment towards osteoblastic (13),(14),(15). A committed osteoblast has to differentiate into a mature osteoblast. Differentiation status of an osteoblast is tightly associated with osteocalcin expression and production. Osteocalcin is the second most abundant bone matrix protein after collagen and it is expressed at high levels only in differentiated osteoblasts and odontoblasts (16),(17). This protein has a high affinity for hydroxyapatites in bone and because of this it is considered that osteocalcin has a critical role in regulation of the mineral phase of bone formation (18). Osteocalcin gene regulation is viewed as a transcriptional regulation where strong suppression signals predominate in early osteoblasts, while enhancing factors in mature osteoblasts (18). Among these transcription factors are AP-1 family members (19), MSX-2 (20), DLX-5 (21 ), CCAAT/enhancer binding proteins (22), and RUNX-2 (23). 1.3 Bone Marrow 1.3.2 Overview Both cortical and trabecular bone provide perfect anatomical niches for bone marrow tissue. The two bone marrow tissues in the adult are the red and yellow bone marrow. During infancy and early childhood, all bone marrow is red (24). In the adult, red bone marrow is found in portions of the vertebrae, sternum, ribs, skull, scapulae, pelvis, and proximal limb bones, collectively termed as flat and irregular bones (25). Red bone marrow is the natural environment for hematopoietic stem cells (HSCs), from which blood cells are derived. Mesenchymal stem cells (MSCs) give rise to the cells of marrow stroma which support and maintain hematopoietic tissue. Stroma consists off the reticular cells, osteocytes, adipocytes, vascular endothelium and extracellular matrix (26). Other marrow areas contain a fatty tissue known as yellow marrow. Yellow marrow is found in the hollow center of the diaphysis (medullary cavity). Normally, yellow marrow does not have any blood-producing function. However, under certain conditions, as a hemorrhage, yellow marrow can be converted to red marrow and assume the responsibility of producing blood cells. With aging there is an increase in content of yellow marrow and subsequent increase in bone marrow adiposity (24). 1.3.2 Bone Marrow Stem Cells BMSCs. Mesenchymal Stem Cells (MSCs) have orthodox and unorthodox types of plasticity. Orthodox plasticity involves differentiation of MSCs into cartilage, bone, myelosupportive stroma and fat tissue. Reticulocytes, osteoblasts, chondrocytes and adipocytes are considered final executor cells of these tissues and are thought to be completely differentiated (26). The unorthodox view on marrow stromal cell plasticity comes from the recent studies that demonstrate a myogenic (27), neural (28) and hepatogenic (29) pathways of differentiation. 1.3.3 Adipocytes differentiation As mentioned above bone marrow stroma consists of reticular cells, osteocytes, adipocytes, vascular endothelium and extracellular matrix. The most abundant stromal cell found in adult human is the adipocyte and it plays an important role in health and disease (24),(30). With aging the number and the size of marrow adipocytes increases until eventually occupies 50% of the human marrow (31). Adipose tissue was viewed for a long time as an inert tissue which contains a constant number of adipocytes and acts as a passive energy depot by realizing or storing lipids. Present knowledge in the field demonstrates that adipose tissue is a very dynamic endocrine organ. Moreover, several studies demonstrate that physiological and pathological adipose tissue expansion is accompanied by increase in adipocyte number. Increase in adipocyte number is achieved by increase in differentiation of adipocytes from the marrow stromal stem cell. In vitro studies demonstrated that a committed preadipocyte has to go into growth arrest first. After this phase, preadipocytes receive a combination of adipogenic signals leading to a progressive manifestation of the characteristics of mature adipocytes (32). Many of the changes that occur during preadipocyte differentiation take place at transcriptional level. The “master switches” in adipogenesis are CEBPs and PPARy2 (33),(34). Following ligand binding, PPARyZ heterodimerizes with the retinoid X receptor and serves as a transcriptional regulator of adipocyte-specific genes such as insulin-responsive glucose transporter (GLUT4), stearoyl CoA desaturase 1 (SCD1), and the fatty acid binding protein (aP2) (35). Fatty acid-binding proteins (FABPs) are a family of proteins expressed in a tissue-specific manner. They bind fatty acids and are involved in shuttling fatty acids to cellular compartments, modulating intracellular lipid metabolism, and regulating gene expression (36). The adipocyte FABP (aP2) has been shown to affect insulin sensitivity, lipid metabolism and lipolysis, and has recently been shown to play an important role in atherosclerosis (37). A unique feature of PPARs is that ligand-binding pockets are usually large, which allows the receptor to accommodate a variety of different ligands (38). Among those, are natural PPARy ligands such as free fatty acid (39), prostaglandin J2 (PGJ2) (40), modified tyrosine and leucine derivatives (41 ), polyunsaturated fatty acids and their oxidation products (40). Synthetic PPARy ligands such as thiazolodinediones are used as antidiabetic drugs for the treatment of type 2 Diabetes and have been recently demonstrated to have a negative effect on bone quality. 2. Osteoporosis 2.1 Overview 2.1.1 Definition Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. The World Health Organization (WHO) defines osteoporosis as bone mineral density (BMD) values which fall well below the average for the 25 year old female (stated statistically as 2.5 standard deviations below the average). If the BMD value is between 1 and 2.5 standard deviations below the average, the bone is said to be osteopenic. A classical way to measure BMD is by X-ray densitometry of bone. 2.1.2 Epidemiology In the US. today, 10 million individuals are estimated to already have the disease. Almost 34 million more are estimated to have low bone mass, placing them at increased risk for osteoporosis. Of the 10 million Americans estimated to have osteoporosis, 8 million are women and 2 million are men. One in two women and one in four men over age 50 will have an osteoporosis-related fracture in her/his remaining lifetime. 34-50 % of postmenopausal white women have ostepenia. Both low bone mass conditions increase fracture risks, with osteoporosis having the greater impact (World Health Organization). During the first year following a hip fracture the mortality rate is 36% for men and 21% for women. The cost of fractures due to the disease is $13.8 billion per year, and that is expected to double over the next 25 years (National Osteoporosis Foundafion) 10 2.1.3 Classification Osteoporosis can be classified in various ways based on diagnostic categories, etiology, and stage. These classifications include the WHO classification, classification as either primary osteoporosis or secondary osteoporosis, and the Mayo Clinic classification. WHO distinguishes 4 groups: 1) Normal: BMD or bone mineral content (BMC) not more than 1 SD below the young adult mean. 2) Osteopenia: BMD or BMC between 1 and 2.5 SD below young adult mean. 3) Osteoporosis: BMD or BMC 2.5 SD or more below the young adult mean and 4) Severe osteoporosis (or established osteoporosis): BMD or BMC 2.5 SD or more below the young adult mean in the presence of one or more fragility fractures. Primary osteoporosis is the most common form of osteoporosis and is diagnosed when other disorders known to cause osteoporosis are not present. Secondary osteoporosis is diagnosed when the condition is related to another illness or to the use of medications or drugs. Researchers at Mayo Clinic suggested that primary osteoporosis be classified as Type I or Type II osteoporosis [Riggs and Melton, 1986] (42). Type I - postmenopausal osteoporosis (high turnover osteoporosis) affects women after menopause and is associated with wrist fractures and vertebral crush fractures. Type II - senile (low turnover) osteoporosis affects men and women older than the age of 70 and is associated with hip fractures and vertebral wedge fractures. 11 2.2 Osteoporosis and bone marrow adiposity Bone is a highly dynamic tissue which is maintained so by simultaneous processes: bone resorption and bone formation. Equilibrium between these two processes maintains a stable bone mass whereas a misbalance between these two will lead to either bone loss or excessive bone formation. Based on the mechanistic classification of osteoporosis, type I or high turnover osteoporosis is associated with increased bone resorption which can not be compensated by normal bone formation. Type II or low turnover osteoporosis is caused by a decrease of bone formation on a foundation of normal bone resorption. Type I has a direct link with increased osteoclasts number and function where type two is linked to decreased osteoblast number and activity. Several studies demonstrate that increase in marrow adipocytes number is observed in almost all conditions associated with bone loss. Wronski et al found an increase in bone marrow adipocyte number in overectomized rats (43). Minaire et al demonstrated an increase in bone marrow adiposity associated with immobilization (44). Wang et al describe an increase in adipocyte number in femoral head in glucocorticoid induced avascular necrosis of the femoral head in rabbits (45). From all situations associated with bone loss the most consistent and well accepted is age-related or senile osteporosis. Human aging is associated with a progressive decrease in bone mass with an increase in fracture rates. Multiple studies demonstrate that bone volume decreases with age in both males and females. 12 Recent studies demonstrate an age related increase in adipose tissue volume fraction over a decrease in trabecular bone fraction. ln neonatal mamals, adipocytes are absent in bone marrow. However, the number of adipocyte in bone marrow increases with age, leading to fatty marrow (24). Interestingly, studies by Moerman et al demonstrate that during aging, status of BMSCs changes with respect to increased commitment towards adipocyte differentiation and decreased osteoblast differentiation. Morover, aged BMSCs have a decreased expression of osteoblast specific factors such as Runx-2 and DLX5 and an increased expression of adipocyte specific marker such as PPARyZ and aP2(46) 3. Diabetes 3.1 Overview 3.1.1 Definition Diabetes mellitus is a group of diseases characterized by high levels of blood glucose resulting from defects in insulin production, insulin action, or both. In order to confirm diagnosis of diabetes, two tests are used: Fasting Plasma Glucose Test (FPG) and an Oral Glucose Tolerance Test (OGT'I'). The American Diabetes Association recommends the FPG because it is easier, faster, and less expensive to perform. With the FPG test, a fasting blood glucose level between 13 100 and 125 mg/dl indicates a pre-diabetic state. A person with a fasting blood glucose level of 126 mg/dl or higher has diabetes. 3.1.2 Clasification There are 3 types of diabetes mellitus: Type I — Insulin Dependent Diabetes Mellitus (IDDM), type II - non-insulin-dependent diabetes mellitus, and gestational diabetes. Type 1 or juvenile-onset diabetes is a chronic disease, which is characterized by lack of insulin production and increased serum glucose concentration. IDDM usually strikes children and young adults, although disease onset can occur at any age. Type 1 diabetes may account for 5% to 10% of all diagnosed cases of diabetes. Type 2 diabetes or adult-onset diabetes, accounts for about 90% to 95% of all diagnosed cases of diabetes. It is usually associated with insulin resistance with a later loss of pancreatic ability to produce insulin. NIDDM is associated with older age, obesity, family history of diabetes, history of gestational diabetes, impaired glucose metabolism, physical inactivity, and race/ethnicity. Gestational diabetes is a form of glucose intolerance that is diagnosed in some women during pregnancy. 3.1.3 Epidemiology A total of 20.8 million people or 7.0% of the population in United States have diabetes. American Diabetes Association estimates 41 million of pre- 14 diabetics in USA. 1.5 million new cases of diabetes were diagnosed in people aged 20 years or older in 2005. For the year 1999 there were 5.3 million people with IDDM worldwide. About one in every 400 to 600 children and adolescents below 20 years of age have type 1 diabetes. About 5 to 10 percent of patients with diabetes in USA have type 1 diabetes (IDDM). 3.2 Diabetic complications IDDM is a life long disease with multiple long-term complications which affect all the organs and systems in the body. The most requiring medical attention are complications of heart and circulation, kidney (nephropathy), neuropathy, peripheral artery disease, intermittent claudication, injuries in the feet, retinopathy, mental function and dementia, infections, depression and osteoporosis. An important step in preventing and delaying these complications is an adequate diabetes management. The accepted tool for the diabetic management assessment is the use of glycosylated hemoglobin (HbA1c) values. For people without diabetes, normal HbA1c values range from 4% to 6%. Satisfactory controlled diabetes is considered when HbA1c values are below 7%. Since it is presumed that the glycosylation of albumin, low-density lipoprotein (LDL), proteins of the erythrocyte membrane, lens, and myelin sheath causes abnormal structures and functions of involved cells and tissues, monitoring HbA1c levels may help to predict and manage diabetic complications. 15 3.3 Diabetic dyslipedimia IDDM is associated with a striking increase in the risk of atherosclerotic disease that is of uncertain origin. Multiple research groups continuously reported associations of IDDM with different lipid abnormalities. Winocour et al also reported that hyperlipideamia is common in insulin-dependent diabetes mellitus, and may be particularly apparent in older patients and/or those with early renal dysfunction or poor glyceamic control (47). Significant positive associations were observed between hypertension and plasma triglycerides, total cholesterol and LDL-C in men and women. Triglyceride levels were predicted (in order of importance) by insulin dose, age at diagnosis, HbA1 and body mass index (48). More recently Decsi et al analyzed fatty acid concentrations in serum and erythrocyte membranes from diabetic and control children, and found significantly increased concentrations of linoleic acid and a-Iinolenic acid in serum of diabetic children. This was associated with a significant decrease in serum levels of arachidonic acid and docosahexaenoic acid (49). Ebeling et al described an association of IDDM with increased intramuscular triglyceride content (50). Experimental work in different animal models also supports these findings. Ebara et al have reported abnormalities of fasting lipid levels, most commonly hypertriglyceridemia, in poorly controlled or uncontrolled diabetes in streptozotocin-diabetic hamsters. These findings were absent in animals with good glycemic control (51 ). It was reported that early stages of insulin deficiency 16 are accompanied by increase in both adipose tissue lipolysis and free fatty acid mobilization (52),(53). It was also reported that both aloxan induced and streptozotocin induced diabetic rats have a increased intramuscular triglyceride content and elevated serum free fatty acid concentration (54), (55). Kurtz et al observed a moderate increase of plasma free fatty acid levels in prediabetic NOD mice, which are a genetic model of autoimmune IDDM. Interestingly, this increase in FFA evolved on a foundation of normal insulin and glucose plasma levels (56). Recently, Pighin et al observed an increase in serum FFA 6 days after the first injection of sreptozotocin, while insulin and glucose levels remained normal. At this time they also observed a marked increase in muscle triglyceride content and enhanced lipolysis of fat pads. At day 12, when hyperglycemia was observed, there was a greater increase in serum FFA concentration accompanied by increased serum triglyceride levels (57). 4. Diabetic Bone Loss 4.1 Overview While bone loss related to type 2 diabetes remains controversial, it is well accepted that one of the long-term complications of IDDM is diabetic osteopenia and osteOporosis. Diabetic bone loss has been shown to be associated with increased fracture rate and delayed fracture healing. l7 4.1.1 Clinical studies For a very long time researchers have reported that bone loss is a frequent complication of type I diabetes. Bone loss was reported to accompany diabetes in both children and adults. At the beginning, these works didn’t have a high degree of trust from the scientific community because of the old and inferior methods of treatment, poor management of glycemic control and old imaging techniques. Therefore, new studies in this field are of great importance. These new studies confirm that IDDM is associated with decreased bone mineral density and decreased bone turnover. Kemnik et al showed that of 35 middle- aged patients with uncomplicated IDDM, more than a half of males and females had osteopenia of femoral neck and/or lumbar spine. This decrease in BMD was associated with decreased serum levels of alkaline phosphatase and osteocalcin (58). Studies by Tuominen et al found that patients that develop IDDM after 30 years of age also have a decreased BMD when compared with age-matched controls (59). Women with type 1 diabetes were 12.25 times more likely to report a fracture as compared to nondiabetic women (the Iowa Women's Health Study). It is well supported now that lower bone mass develops over the first few years of IDDM. Decreased bone mass is also reported to be associated with IDDM in adolescents and children. In pediatric studies there are still unclear issues about the relationship between glycemic control and BMD. Some report a tight 18 relationship between levels of HbA1c and BMD, whereas other studies didn’t find any correlation between those two. Now it is well recognized that patients with type 1 diabetes have lower BMD and higher risk of fractures. 4.1.2 Animal model work Most of the experimental data available on diabetic bone loss comes from studies in diabetic rats. All these studies suggest that diabetes type 1 is associated with a decrease in bone mass. Long-term studies by Einhorn et al. described defects in bone mineralization, decreased calcium-to-phosphate composition of the ash, decreased ash content in tibial metaphysic and reduced strength-related properties (60). Reports by Dixit et al and Hou et al, show that bone strength is diminished in IDDM rats at the femur and the femoral neck (61),(62). Verhaeghe et al in multiple reports demonstrated that irrespective of the model used, insulin deficient rats exhibit reduced or absent bone formation and this decline is appreciated in relation to all bone surfaces examined (trabecular, periosteal, and endocortical). It is has been shown that the overall bone loss in IDDM is due to a deficit in mineralized surface area, a decrement in the rate of mineral apposition, deceased osteoid surface, depressed osteoblast activity, and decreased numbers of osteoclasts, which in turn lead to a depression of bone remodeling. These facts are reinforced by the data that osteoblast marker osteocalcin is decreased in serum from diabetic rats, and so is urinary deoxypyridinoline, an index of bone resorption (63),(64),(65). 19 4.1.3 Proposed mechanisms 4.1.3.1 Impaired osteoblasts function The idea of an impaired osteoblasts function and bone turnover has been supported by studies in both humans and animals. In several studies dynamic bone histology in diabetic rats showed decreased osteoblasts number, osteoid formation and bone mineral apposition rate (66),(67). Recent studies by Thrailkill used a tibial distraction osteogenesis (DO) model in NOD (non obese diabetic) mice to assess de novo bone formation and osteoblasts function. They found that total new bone in the DO gap was reduced histologically and radiographically in diabetic mice compared with nondiabetic mice. This was accompanied by decreased serum osteocalcin levels (68). Studies by Mishima et al found that volume of bone formed and the number of osteoclasts was significantly lower in streptozotocin diabetic rats than in the controls. They conclude that streptozotocin-induced diabetes mellitus reduces the rate of bone turnover. Studies in humans showed a similar pattern (69). Bouillon et al concludes that in diabetic patients osteoblast function is significantly decreased and it associated with a maturation defect. This conclusion is based on the fact that early osteoblast marker, PlCP, remained normal in all types of diabetes, whereas a marker of mature osteoblast such as osteocalcin, is decreased in diabetes irrespective of age (70). 20 4.1.3.2 Hypoinsulinemia Insulin is a skeletal growth factor, and there are plenty of studies demonstrating its anabolic action on bone. Studies by Mishima et al found that volume of bone formed and the number of osteoclasts were significantly lower in streptozotocin diabetic rats than in the controls, but all these modifications were reversed by insulin treatment (69). Studies by Lu et al used a bone marrow ablation model and found that bone formation along the expression of osteocalcin, collagen type | and Runx-2 were reduced in streptozotocin induced diabetic mice after marrow ablation. Insulin treatment substantially reversed the effect of diabetes on the expression of these factors (71). Studies in diabetic NOD mice by Thrailkill et al showed that reduced serum osteocalcin was normalized by insulin treatment. Total new bone in the DO gap along with trabecular bone volume and thickness, cortical thickness, cortical strength have been prevented from change by insulin treatment (68). 4.1.3.3 IGF It has been shown that insulin-like growth factor (IGF-1, IGF-2) can independently influence bone metabolism. Studies by Kemink et al demonstrate a significantly lower plasma IGF-1 levels in diabetes type I patient with femoral neck osteopenia (58). IGFs are part of an IGF-l / IGF-binding protein system. 21 Specifically, IGF-1 is inhibitory to lGFBP-1 and lGFBP-4 and stimulatory to lGFBP-3 and lGFBP-5. Studies by Jehle et al investigated serum levels of insulin-like growth factor system components and their relationship to bone metabolism in Type 1 and Type 2 diabetes mellitus patients. They found that type 1 diabetics showed significantly lower IGF-I and lGFBP-3 but higher lGFBP-1 levels compared with Type 2 patients or healthy controls. IGFBP-5 levels were markedly lower in both diabetic groups than in controls, whereas IGFBP-4 levels were similar in diabetics and controls (72). Liver lGF-1—deficient (LID) mice exhibited relatively normal growth and development, despite having 75% reductions in serum IGF-1 levels. Studies in double IGF-1 gene disrupted mice showed further reductions in serum IGF-1 levels and asignificant reduction in linear growth. The proximal growth plates of the tibiae of IGF deficient mice were smaller in total height as well as in the height of the proliferative and hypertrophic zones of chondrocytes. There was also a 10% decrease in bone mineral density and a greater than 35% decrease in periosteal circumference and cortical thickness in these mice. IGF-1 treatment for 4 weeks restored the total height of the proximal growth plate of the tibia. Thus, authors conclude that a threshold concentration of circulating IGF-1 is necessary for normal bone (73). Scheiwiller et al showed that normal growth of diabetic rats is restored by infusion of recombinant human (rh)lGF-l without normalization of the blood sugar level (74). 22 4.1.3.4 Vascular factors Microangiopathy is a major complication of type I diabetes. The role of microangiopathy in evolution of osteoporosis is still controversial. Studies in Cohen diabetic rat, a non obese model of noninsulin-dependent diabetes mellitus, demonstrate that osteoporosis precedes the onset of microangiopathy, questioning its importance in pathogenisis of osteoporosis in this animal model (75). On the other hand, clinical studies demonstrate that among relatively healthy older women decreased vascular flow in the lower extremities may be associated with an increased rate of bone loss at the hip and calcaneus (76). Shih et al found a significant correlation between marrow perfusion of vertebral bone marrow and BMD in postmenopausal female subjects (77). Studies by Tanko et al showed a positive relationship between severe osteoporosis in the hip and advanced atherosclerosis (78). Since advanced atherosclerosis is associated with severe alteration of blood flow, it is plausible to speculate that a vascular component plays an important role in the pathogenesis of osteoporosis. 4.1.3.5 Nutrition Correct and adequate nutrition has been well accepted to have a primordial role in a normal growth and development. Studies in semi-starved rats 23 demonstrate a bone loss and a reduction in skeletal growth after 7 days of food depletion (79). Studies in diabetic rats by Nyomba et al showed a decreased duodenal Ca uptake with decreased 1,25-(OH)2D3 levels, decreased duodenal 9K Ca-binding protein, and decreased number of 1,25-dihydroxyvitamin D3 [1,25-(OH)203j-binding sites in duodenum, although the binding affinity was above normal. Plasma vitamin D-binding protein levels were decreased by 62% in diabetic rats, due to a marked decrease in production rate, while the plasma half-time remained normal (80). Schneider et al showed that serum concentration of 1,25-dihydroxyvitamin D was depressed in untreated diabetic rats and was restored to control levels by insulin treatment (81). Furthermore, studies by Chertow et al shoed that Vitamin D deficiency is associated with impaired insulin secretion (82). Verrotti et al showed that diabetics with persistent microalbuminuria (MA) had significantly lower 25-OHD and 1,25 (OH)2D3 as well as serum osteocalcin when compared with controls and subjects with normoalbuminuria (83). It was also demonstrated that urinary calcium and magnesium excretion is lower in children born to mothers with insulin dependent diabetes mellitus than those born to non-diabetic mothers (84). 4.1.3.6 Glycation Diabetes mellitus type I is associated with formation and accumulation of advanced glycation end products (AGES). There is a growing body of evidence to show that AGES-AGE receptor (RAGE) interactions are involved in the 24 development of atherosclerosis and diabetic microangiopathy (85). Kim et al have shown that pharmacological and/or genetic deletion/mutation of the receptor attenuates the development of hyperglycemia in NOD mice. In these mice, interruption of ligand-RAGE interaction prevents or delays the chronic complications of the disease in both macro- and microvessel structures (86). Hein et al found that osteoporotic patients have significantly higher serum AGEs concentrations than healthy subjects (87). Santana et al showed involvement of AGEs to diminished bone healing in type I diabetes, possibly mediated by RAGE (88). Furthermore, Yamagishi et al have recently found that AGES-RAGE interactions induced human mesenchymal stem cell apoptosis and subsequently prevented cognate differentiation into adipose tissue, cartilage, and bone (89). Moreover, Odetti et al found increased concentrations of AGEs (pentosidine) in cortical bone during aging (90). 5. Diabetes type I experimental models and osteoporosis Type I diabetes mellitus in humans is characterized by a specific destruction of pancreatic B-cells through an immune-mediated damage (91). Despite the long and silent evolution, at the time of clinical presentation there is little surviving B-cell mass and absolute insulinopenia is established. The first experimental reproduction of insulinopenic state in an animal was accidentally achieved in 1880 by Joseph von Merring and Oskar Minkowski. Von Merring was 25 working on the absorption of fat from the intestine when Minkowski suggested he remove the pancreas of a dog. After the surgery the animal developed polyuria and polydipsia and was found to have diabetes mellitus (92). This event was followed by a long period of trials to destroy pancreatic tissue and induce hypoinsulinemia by different methods and in different animals. To accomplish this task destruction of pancreatic B-cells was performed by surgical excision of pancreatic tissue, by chemical destruction and by genetic manipulations of the immune system. Surgical removal of pancreas is rarely used because of the ethical issues, high complication rate such as infections and because of the availability of other more efficient methods. Among the chemicals that upon injection induce diabetes mellitus are alloxan, streptozotocin, vacor, dithizone and 8-hydroxyquinolone. The most widely used is streptozotocin (STZ) which is a nitrosurea derivative isolated from Streptomyces achromogenes that has a broad-spectrum antibiotic and anti-neoplastic activity (93). Streptozotocin-based drug combinations remain the first line standard in combined treatment of insulinomas (94). STZ is powerful alkylating agent which is been shown to induce multiple DNA strand breaks (95). It was also shown to interfere with glucose transport (96) and glucokinase function (97). A single large dose or multiple small doses of STZ are used to induce diabetes in rodents. Although thought that multiple doses treatment induces diabetes trough immunological pathways rather than direct toxic effects, STZ will produce diabetes even in the absence of functional T and B cells (98). Also, STZ induced diabetes can not be transferred to synergenic recipients by the transfer of splenocytes like in 26 spontaneous animal diabetic models (99). Today, four murine models of spontaneous autoimmune diabetes are used by the scientific community for the study of IDDM. These are NOD (non-obese diaebetic) mouse, BioBreeding- diabetes-prone (BB-DP) rat, Komeda diabetes-prone (KDP) rat, and LEW.1AR1 rat. The most intensively studied are NOD mouse and BB rat. In both animal types it is well established that T lymphocytes are both initiators and effectors of B-cell destruction (92). Most of the experimental osteoporosis related studies are performed in STZ induced or BB rats, because of the convenience with bone sample size, blood volume and animal size. Dynamic bone histology studies in diabetic rats show decreased osteoblast number, osteoid formation, and bone mineral apposition rate (66),(67). ln diabetic rats it was also shown that in parallel with decreased bone formation there is a decrease in osteoclast number. These findings were reversed by insulin treatment. Rat studies demonstrated that insulin treatment normalizes BMD and markers of bone turnover (69),(100). Studies by Bouillon et al showed reduced serum IGF-1 levels in diabetic rats while Scheiwiller et al showed that skeletal effects of hypoinsulinemia are corrected with IGF-1 infusion in diabetic rats (101 ),(74). Bone related studies in diabetic type I mice are scarce. In 1984 Yoon et al were able to induce severe hyperglycemia and hypoinsulinemia in mice infected with the D variant of encephalomyocarditis (EMC) virus. They observed that alkaline phosphatase activity and the rate of mineralization in the proximal tibial epimetaphysis were markedly decreased in the diabetic mice 30 and 180 days 27 post infection (102). In 1998 Lalla et al, showed increased alveolar bone loss in diabetic vs. non-diabetic mice infected with P. gingivalis at 2 and 3 months after infection (103). In 1999 Maor et al reported reduced expression of the GLUT4 and IGF-l receptor in the bone growth center. They studied both in vivo and in vitro early endochondral bone formation in control and streptozotocin-induced young diabetic mice. Further, in the skeletal growth centers of streptozotocin- induced diabetic mice, GLUT4, IGF-1, and IGF-1 and insulin receptor levels, but not GLUT1 were markedly reduced. The decrease in GLUT4 and in IGF-I and insulin receptors was associated with severe histological changes in the mandibular condyles and humeral growth plate (104). Studies by Lu et al in 2003 used a bone marrow ablation model to investigate mechanisms of delayed fracture healing in streptozotocin induced diabetes in mice. They found an equal amount of immature mesenchymal tissue at day 4 in both the experimental and control groups. At day 6 they saw a burst of bone formation in the control group that was significantly reduced in the diabetic group. This deficit was evident at the molecular level as shown by diminished expression of osteocalcin and collagen type I. When transcription factors were examined, core-binding factor alpha1 (bea1)/runt domain factor-2 (Runx-2) and human homolog of the Drosophila distal-less gene (Dlx5) expression were substantially reduced in the diabetic, compared with control, groups on day 4 and 6. Interestingly, insulin treatment substantially reversed the effect of diabetes on the expression of bone matrix osteocalcin, collagen type I and transcription factors bea1/Runx2 and Dlx5 (71). Recently, Thrailkill et al (2005) reported that during tibial distraction 28 osteogenesis (DO) total new bone in the DO gap was reduced histologically and radiographically in diabetic mice compared with nondiabetic mice but preserved by insulin treatment. Serum osteocalcin concentrations were also reduced in diabetic mice and normalized with insulin treatment. Evaluation of the contralateral tibiae by microCT and mechanical testing, demonstrated reductions in trabecular bone volume and thickness, cortical thickness, cortical strength, and an increase in endosteal perimeter in diabetic animals. All these findings were prevented by insulin treatment (68). Again, their conclusion is that bone formation during DO is impaired in a model of type 1 diabetes and preserved by systemic insulin administration. Taken this together, we set our goal to characterize bone status in streptozotocin induced diabetic mouse model and further investigate possible pathophysiological mechanisms of diabetes related bone loss. Chapter I is dedicated to a literature review on bone, diabetes and diabetic bone loss. In Chapter II we discuss material and methods used to accomplish this work. Chapter III describes bone status changes in streptozotocin induced diabetes. We report significant bone loss in diabetic animals and reveal a new finding associated with this status such as increased bone marrow adiposity. Chapter IV and Chapter V are focused on manipulation of marrow adiposity by inhibition of adipogenesis with a PPARyZ antagonist BADGE. Bone quality and marrow adiposity status were investigated under BADGE treatment conditions in relation to untreated animals. We were able to successfully inhibit increased bone marrow adiposity with this treatment but we lack restoration of bone status. In 29 chapter VI we set our goal to characterize immediate changes associated with streptozotocin induced onset of diabetes. We found that bone related changes occur as early as 24 hours after beginning of the induction of diabetes, whereas adipogenesis markers became altered as early as day 5. Chapter VII investigates the effect of chronic hyperglycemia on cultured MC3T3 osteoblasts in vitro. 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Mutat Res 5122121-34 Wang Z, Gleichmann H 1998 GLUT2 in pancreatic islets: crucial target molecule in diabetes induced with multiple low doses of streptozotocin in mice. Diabetes 47250-6 Zahner D, Malaisse WJ 1990 Kinetic behaviour of liver glucokinase in diabetes. l. Alteration in streptozotocin-diabetic rats. Diabetes Res 14:101- 8 Reddy S, Wu D, Elliott RB 1995 Low dose streptozotocin causes diabetes in severe combined immunodeficient (SCID) mice without immune cell infiltration of the pancreatic islets. Autoimmunity 20:83-92 38 99. 100. 101. 102. 103. 104. Arata M, Bruno L, Pastorale C, Pagliero F, Basabe JC 2001 Effect of modified diabetic splenocytes on mice injected with splenocytes from multiple low-dose streptozotocin diabetic donors. Exp Biol Med (Maywood) 226:898-905 Glajchen N, Epstein S, Ismail F, Thomas S, Fallon M, Chakrabarti S 1988 Bone mineral metabolism in experimental diabetes mellitus: osteocalcin as a measure of bone remodeling. Endocrinology 1232290-5 Bouillon R 1992 Diabetic bone disease. Low turnover osteoporosis related to decreased IGF-I production. Verh K Acad Geneeskd Belg 54:365-91; discussion 391-2 Yoon JW, Notkins AL 1983 Virus-induced diabetes in mice. Metabolism 32:37-40 Lalla E, Lamster IB, Feit M, Huang L, Schmidt AM 1998 A murine model of accelerated periodontal disease in diabetes. J Periodontal Res 33:387-99 Maor G, Karnieli E 1999 The insulin-sensitive glucose transporter (GLUT4) is involved in early bone growth in control and diabetic mice, but is regulated through the insulin-like growth factor I receptor. Endocrinology 140:1841-51 39 CHAPTER II Materials and Methods Diabetic mouse model Diabetes was induced in adult (15 week old) male Balb/c mice (Harlem Laboratories, Houston, TX) by daily intraperitoneal injection with streptozotocin (40 ug/g body weight in 0.1 citrate buffer), a pancreatic beta cell cytotoxin, for 5 days (1, 2). Controls were injected with buffer alone. Seven days after the last injection, non-fasting blood glucose measurements were‘performed using blood obtained from the lateral saphenous vein and a glucometer (Accu-Check instant, Boehringer Mannheim Corporation, Indianapolis, IN). This day was considered as day zero. Mice with blood glucose levels greater than 300 mg/dl were considered diabetic. For experiments described in chapter II, 2 or 4 weeks mice (controls and diabetics) were fasted for 6 hours and then euthanized. Tibiae were immediately removed, freed from soft tissue, and either fixed in formalin (for histology and uCT analyses) or snap-frozen in liquid nitrogen and stored at -80°C (for RNA analyses). At 4 weeks the liver and subcutaneous femoral fat pads were dissected from the surrounding tissues. Tissues were weighed and snap frozen in liquid nitrogen or fixed in PBS buffered formalin. For experiments described in chapter lll, half of control (non-diabetic) and diabetic mice were treated daily with an intraperitoneal injection of BADGE at a concentration of 30 4O mg/kg in 15% DMSO (3). BADGE treatment regimes included: chronic (beginning at the time of diabetes confirmation, 12 days after the first injection of streptozotocin), early (treatment only at days 1-7 following the confirmation of diabetes) and late (treatment only at days 21-28 following the confirmation of diabetes). An additional study incorporated BADGE treatment beginning at the time of the first streptozotocin injection. Each group contained 8-11 animals to obtain statistical significance as determined by statistical power analyses. Mice (controls and diabetics, treated and untreated) were fasted for 6 hours prior to euthanasia. Tibiae were immediately removed, freed from soft tissue, and either fixed in formalin (for histology and pCT analyses) or snap-frozen in liquid nitrogen and stored at -80°C (for RNA analyses). For the experiments described in chapter IV, adult (15 week old) male BALB/c mice were intraperitonealy injected daily with streptozotocin (4O pg/g body weight in 0.1 citrate buffer) for 5 days (1, 2). Controls were injected with citrate buffer alone. Twenty-four hours after the first injection animals were considered as 1 day post injection. Mice were harvested 1, 3, 5, 7, 9, 11, and 17 days post after the first injectionMice were euthanized and tibiae were immediately removed, freed from soft tissue, and snap-frozen in liquid nitrogen and stored at -80°C (for RNA analyses). . All mice were kept on a light/dark (12h/12h) cycle at 23°C, and received food (standard lab chow) and water ad libitum. Animal studies were conducted in accordance with the Michigan State University All-University committee on Animal Use and Care. 41 Plasma measurements Blood was obtained from mice at the time of euthanasia at every time point. Blood serum was prepared from each sample by letting it to clot for 10 minutes at room temperature and then centrifugation for 10 min at 3000 rpm. Serum was stored frozen at -—20°C for immediate analyses and at -80°C for further analyses. Glucose concentration in serum samples was determined using a Glucose Assay Kit (Sigma, Saint Louis, MO). Serum osmolality, defined as the expression of the total number of solute particles dissolved in one kilogram of solvent (International Federation of Clinical Chemistry), was determined using a vapor pressure osmometer (Wescor, lnc., Logan, UT). Serum osteocalcin levels were measured using Mouse Osteocalcin EIA Kit (Biomedical Technologies Inc. Stoughton, MA, USA) according to manufacturer instructions. Quantitative determinations of serum glycerol, total triglycerides and true triglyceride (expressed as equivalent triolein concentration) levels were performed using a Serum Triglyceride Determination Kit (Sigma, Saint Louis, MO). Quantitative determination of non-esterified (free) fatty acids in mouse serum was performed according to manufacturer instructions using Wako NEFA C test kit (Wako Chemicals USA, Inc. Richmond, VA, USA). Osteoclast activity- systemic measurements For measurement of tartrate resistant acid phosphatase (TRAP), serum from diabetic and control mice was used. Active serum TRAP5b, produced by 42 mouse osteoclasts, was measured according to manufacturer protocol using a solid phase immunofixed enzyme activity assay, MouseTRAP (SBA Sciences, Turku, Finland). For urine deoxypyridinoline (DPD) measurements, mice were housed in metabolic cages to allow collection of 24-hour urine samples. Urine specimens were stored at —80°C. DPD and creatinine urine levels were determined using Metra DPD Enzyme Immunoassay kit and Metra Creatinine Assay kit, respectively (Quidel Corporation, CA). DPD levels were expressed relative to creatinine levels. Serum PYD, which are serum pyridinoline crosslinks, was measured using Metra® PYD kit (Quidel Corporation, San Diego, CA, USA) according to manufacturer instructions. RNA Analysis Tibiae were crushed under liquid nitrogen conditions using a Bessman Tissue Pulverizer and RNA extracted using the method of Chomczynski and Sacchi as previously described (4, 5). Synthesis of cDNA was performed by reverse transcription with 2 pg of total RNA using the Superscript II kit with oligo dT(12-13) primers as described by the manufacturer (lnvitrogen, Carlsbad, CA). cDNA (1 pl) was amplified by PCR in a final volume of 25 pl using the iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) with 10 pmol of each primer (Integrated DNA Technologies, Coralville, IA). Osteocalcin was amplified using 5’-ACG GTA TCA CTA TTT AGG ACC TGT G-3’ and 5’-ACT TTA TTT TGG AGC T60 43 TGT GAC-3’ (6). Runx2 was amplified using 5’- GAO AGA AGC TTG ATG ACT CTA AAC C-3’ and 5’- TCT GTA ATC TGA CTC TGT CCT TGT G-3’ (7). Alkaline phosphatase was amplified using 5’-CGT AAT CTA CCA TGG AGA CAT TTT C-3’ and 5'-GAC TGT GGT TAC TGC TGA TCA TTC-3’. Collagen l was amplified using 5’-AAG CCT CTT TCT CCT CTC TGA CC-3’ and 5’-AGA AAG GAT CTC CTG GTG CTG AT-3’. Matrix metalloprotease 13 (MMP-13, collagenase 3) was amplified using 5’-CCA CTG TCC TTG TAG TGG CTG TTA- 3’ and 5’-GGA GCC ACA GAT GAG CAC AGA TAA-3’. PPARv2 was amplified using 5’-TGA AAC TCT GGG AGA TTC TCC TG-3’ and CCA TGG TAA TTT CTT GTG AAG TGC-3’ (8). Adipocyte fatty acid-binding protein 2 (aP2) was amplified using 5’-GCG TGG AAT TCG ATG AAA TCA-3’ and 5’-CCC GCC ATC TAG GGT TAT GA-3’ (9). Resistin was amplified using 5'- GCTGCTGCCAAGGCTGAT-3’ and 5’-TCTCCTTCCACCATGTAGTI'TCC-3’ (10). TRAP5 was amplified using 5’-AATGCCTCGACCTGGGA-3' and 5’- CGTAGTCCTCCTTGGCTGCT-B’ (11). Cathepsin was amplified using 5’-GCA GAG GTG TGT ACT ATG A-3’ and 5’-GCA GGC GTT GTT CTT ATT-3’ (12). Glyceraldehyde phosphate dehydrogenase (GAPDH) was amplified using 5’- GTG TAC ATG GTT CCA GTA TGA CTC C-3’ and 5’-AGT GAG TTG TCA TAT TTC TCG TGG T-3’ and vascular endothelial growth factor (VEGF) was amplified using 5’-ATA TCA GGC TTI' CTG GAT TAA GGA C-3’ and 5’-CAG ACG AAA GAA AGA CAG AAC AAA G-3'. Cyclophilin, which was not modulated under present experimental conditions, was used as a control for RNA levels; it was amplified using 5’-ATT CAT GTG CCA GGG TGG TGA C-3’ and 5’-CCG T'I'I' 44 GTG TTT GGT CCA GCA-3’ (13, 14). Real time PCR was carried out for 40 cycles using the iCycler (Bio-Rad, Hercules, CA) and data were evaluated using the iCycler software. Each cycle consisted of 95°C for 15 seconds, 60°C for 30 seconds (except for runx2 and osteocalcin, which had an annealing temperature of 65°C) and 72°C for 30 seconds. RNA-free samples, a negative control, did not produce amplicons. Melting curve and gel analyses were used to verify single products of the appropriate base pair size. Tissue and mineralized bone histology and histomorphometry Proximal tibiae isolated from 4 week control and diabetic mice were fixed in absolute ethanol at room temperature, dehydrated, and embedded in methylmethacrylate as previously described (15). For dynamic measurements, mice were injected intraperitoneally with 0.2 mls of 10 mg/ml calcein (Sigma) in sterile saline at 6 days prior to and at 2 days prior to euthanasia. Sections of 3- and 7-micrometer thickness were cut with a Microm microtome, model HM360 (Microm, C. Zeiss, Thornwood, NY) and stained with the modified Masson- Goldner trichrome stain (16). Static and dynamic parameters of bone structure, formation, and bone resorption were measured at standardized sites under the growth plate using the semi-automatic method (Osteoplan ll, Kontron, Munich, Germany) at a magnification of 200X (15, 17). For adipose analyses, visible adipocytes, greater than 50pm, were counted. All parameters comply with the guidelines of the nomenclature committee of the American Society of Bone and Mineral Research (18). 45 For light microscopy proximal tibiae isolated from control and diabetic mice were fixed in 10% neutral buffered formalin. Fixed samples were processed on an automated Thermo Electron Excelsior tissue processor for dehydration, clearing and infiltration using a routine overnight processing schedule. Samples were then embedded in Surgipath embedding paraffin on a Sakura Tissue Tek ll embedding center. Paraffin blocks were sectioned at 5 microns on a Reichert Jung 2030 rotary microtome. Slides were stained with hematoxylin & eosin. Visible adipocytes, greater than 30 pm, were counted in the trabecular region ranging from the growth plate to 2 mm away distally. Subcutaneous femoral fat depots were isolated from surrounding tissue and fixed in 10% neutral buffered formalin. Fixed samples were processed on an automated Thermo Electron Excelsior tissue processor for dehydration, clearing and infiltration using a routine overnight processing schedule. Samples were then embedded in Surgipath embedding paraffin on a Sakura Tissue Tek lI embedding center. Paraffin blocks were sectioned at 5 microns on a Reichert Jung 2030 rotary microtome. Slides were stained with hematoxylin & eosin. Livers were dissected out, sectioned and placed in Tissue Tek O.C.T. freezing media on a sectioned cork; corks were snap frozen in liquid nitrogen. Frozen tissue-corks were stored at -80 degrees Celsius. Tissues were then sectioned on a -20 degree Celsius Sakura Tissue Tek Cryostat at 10 microns. Sections were placed on adhesive slides, air dried for 30 minutes, fixed in 37- 40% formaldehyde for 1 minute, rinsed in running tap water for 5 minutes, and stained with hematoxylin 8 eosin. 46 Micro-computed tomography (pC1) analysis Fixed bones and mouse legs were scanned using a GE Explore Locus pCT system at a voxel resolution of 20 um obtained from 720 views. Beam angle of increment was 0.5 and beam strength was set at 80 kvp and 450 pA. Each run included control and diabetic bones and a calibration phantom to standardize grayscale values and maintain consistency. Based on autothreshold and isosurface analyses of multiple bone samples, a fixed threshold was used to separate bone from bone marrow. All cortical bone analyses were made in a defined 3-mm3 cube in the mid-diaphysis 1 mm proximal of the tibial-fibular junction. The polar cross-sectional moment (moment of inertia, MOI) was also determined at the tibial-fibular junction using the parallel axis theorem. Trabecular bone analyses were done in a region of trabecular bone defined at 0.17 mm (approximately 1% of the total length) under the growth plate of the proximal tibia extending 2 mm toward the diaphysis, and excluding the outer cortical shell. Bone mineral content, mineral density and volume fraction values were computed by a GE Healthcare MicroView software application for visualization and analysis of volumetric image data. 47 Cell Culture System MC3T3-E1 cells (19), subcloned for maximal alkaline phosphatase staining and mineralization, were used for all studies. Osteoblasts were seeded at 5,000 cells/cm2 surface area and fed daily with alpha-MEM containing 7% fetal calf serum (a final glucose concentration of 5.5 mM). After 7 days, the media is supplemented with 25 ug/ml ascorbic acid and 2 mM beta-glycerol phosphate (Sigma, St. Louis, MO) to promote osteoblast differentiation and bone formation. Treatment with glucose or mannitol began 24 hours after seeding (on day 1) and continued with each feeding until day 7, 14, 21 or 28. Determination of alkaline phosphatase activity and mineralization Alkaline phosphatase staining was performed by incubating cell layers for 30 minutes at 37 °C with 0.5 mg/ml naphthol AS-MX phosphate disodium salt with 1 mg/ml Fast Red TR salt (Sigma Chemical Co., St Louis, MO) in a 10.2 M Tris buffer, pH 8.4. Alkaline phosphatase quantitation was performed by solubilizing the precipitated salt in 100% trichloracetic acid and reading at 540 nm as previously described (20). Mineralization was determined by using a 0.02M solution of alizarin red in water and incubating at room temperature for 20 minutes. Wells were rinsed several times to get our unincorporated dye. Quantitation was performed by extracting the dye from the wells with 10% cetylpyridinum choride solution and reading at 570 nm. Oil Red-O solution was used to stain for lipid and quantitated by adding 100% ethanol to each well and reading at 508 nm. 48 DNA Analysis DNA quantification was performed with CyQUANT® Cell Proliferation Assay Kit (Molecular Probes, Oregon, USA) according to instructions provided by manufacturer. Specifically, frozen cell pellets were thawed to room temperature and lysed in 1 ml of 1X CyQUANT GR working solution and span for 5 minutes at 3000 RPM. Samples were analyzed for fluorescence using a fluorescence microplate reader with filters appropriate for 480 nm excitation and 520 nm emission. Values were expressed relative to day 7 control levels. In vitro examination of streptozotocin on pluripotent C3H10T1/2 cells C3H10T1/2 cells (21) were obtained from American Type Culture Collection (Manassas, VA) and maintained in Eagle’s Basal Medium (Sigma, Saint Louis, MO )with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA ) at 37° in a humidified atmosphere of 5% C02/95% air. Cells were plated at a density of 100,000 cells per 6 well. To induce osteoblast differentiation, BMP2 (Sigma) was added at a concentration of 100 ng/ml twenty-four hours after plating (21). Cells were fed every other day with or without an additional 100 ng/ml BMP2. Cells were grown for 14 days. Streptozotocin concentrations were 0.01, 0.1, 0.5 and 1.0 mg/ml. Streptozotocin was added with every feeding. Cells were fed and treated 24 hours before harvesting for RNA analyses or lipid staining. 49 Statistical analysis All statistical analyses were performed using Microsoft Excel data analysis program for t-test analysis. Values are expressed as a mean i SE. 50 References 1. 10. Pechhold K, Patterson NB, Blum C, Fleischacker CL, Boehm BO, Harlan DM 2001 Low dose streptozotocin-induced diabetes in rat insulin promoter-mCDBO-transgenic mice is T cell autoantigen-specific and CD28 dependent. J Immunol 166:2531-2539 Szkudelski T 2001 The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas. Physiol Res 502537-546 Cuzzocrea S, Pisano B, Dugo L, lanaro A, Maffia P, Patel NS, Di Paola R, Ialenti A, Genovese T, Chatterjee PK, Di Rosa M, Caputi AP, Thiemermann C 2004 Rosiglitazone, a ligand of the peroxisome proliferator-activated receptor-gamma, reduces acute inflammation. Eur J Pharmacol 483279-93 McCabe LR, Kockx M, Lian J, Stein J, Stein G 1995 Selective expression of fos- and jun-related genes during osteoblast proliferation and differentiation. Exp Cell Res 218:255-262 Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159 Ontiveros C, McCabe LR 2003 Simulated microgravity suppresses osteoblast phenotype, Runx2 levels and AP-1 transactivation. J Cell Biochem 882427-437 Ontiveros C, lnrvin R, Wiseman RW, McCabe LR 2004 Hypoxia suppresses runx2 independent of modeled microgravity. J Cell Physiol 2002169-176 Kast-Woelbern HR, Dana SL, Cesario RM, Sun L, de Grandpre LY, Brooks ME, Osburn DL, Reifel-Miller A, Klausing K, Leibowitz MD 2004 Rosiglitazone induction of lnsig-1 in white adipose tissue reveals a novel interplay of peroxisome proliferator-activated receptor gamma and sterol regulatory element-binding protein in the regulation of adipogenesis. J Biol Chem 279:23908-23915 Maeda K, Uysal KT, Makowski L, Gorgun CZ, Atsumi G, Parker RA, Bruning J, Hertzel AV, Bernlohr DA, Hotamisligil GS 2003 Role of the fatty acid binding protein mal1 in obesity and insulin resistance. Diabetes 522300-307 Akune T, Ohba S, Kamekura S, Yamaguchi M, Chung Ul, Kubota N, Terauchi Y, Harada Y, Azuma Y, Nakamura K, Kadowaki T, Kawaguchi H 2004 PPARgamma insufficiency enhances osteogenesis through 51 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. osteoblast formation from bone marrow progenitors. J Clin Invest 113:846- 855 Wiren KM, Zhang XW, Toombs AR, Kasparcova V, Gentile MA, Harada S, Jepsen KJ 2004 Targeted overexpression of androgen receptor in osteoblasts: unexpected complex bone phenotype in growing animals. Endocrinology 14523507-3522 Yoshimatsu M, Shibata Y, Kitaura H, Chang X, Moriishi T, Hashimoto F, Yoshida N, Yamaguchi A 2006 Experimental model of tooth movement by orthodontic force in mice and its application to tumor necrosis factor receptor-deficient mice. J Bone Miner Metab 24:20-27 Lewis ML, Hughes-Fulford M 2000 Regulation of heat shock protein message in Jurkat cells cultured under serum-starved and gravity-altered conditions. J Cell Biochem 772127-134 Trogan E, Choudhury RP, Dansky HM, Rong JX, Breslow JL, Fisher EA 2002 Laser capture microdissection analysis of gene expression in macrophages from atherosclerotic lesions of apolipoprotein E-deficient mice. Proc Natl Acad Sci U S A 99:2234-2239 Botolin S, Faugere MC, Malluche H, Orth M, lVIeyer R, McCabe LR 2005 Increased bone adiposity and peroxisomal proliferator-activated receptor- gamma2 expression in type I diabetic mice. Endocrinology 146:3622-3631 Goldner J 1938 A modification of the Masson trichrome technique for routine laboratory purposes. Am J Pathol 142237-243 Manaka RC, Malluche HH 1981 A program package for quantitative analysis of histologic structure and remodeling dynamics of bone. Comput Programs Biomed 132191-202 Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche HH, Meunier PJ, Ott SM, Recker RR 1987 Bone histomorphometry: standardization of nomenclature, symbols and units. J Bone Miner Res 62595-610 Sudo H, Kodama HA, Amagai Y, Yamamoto S, Kasai S 1983 In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol 96:191-198. McCabe LR, Banerjee C, Kundu R, Harrison RJ, Dobner PR, Stein JL, Lian JB, Stein GS 1996 Developmental expression and activities of specific fos and jun proteins are functionally related to osteoblast maturation: role of Fra- 2 and Jun D during differentiation. Endocrinology 137:4398-4408 52 21. Date T, Doiguchi Y, Nobuta M, Shindo H 2004 Bone morphogenetic protein-2 induces differentiation of multipotent C3H10T1/2 cells into osteoblasts, chondrocytes, and adipocytes in vivo and in vitro. J Orthop Sci 92503-508 53 CHAPTER III Increased bone adiposity and PPARv2 expression in type I diabetic mice. Sergiu Botolin‘, Marie-Claude Faugere3, Hartmut Malluche3, Michael Orthz, Ron Meyer1 and Laura R McCabe1 Michigan State University"2 Departments of Physiology and Radiology1 Molecular Imaging Research Center1 Department of Animal Science2 University of Kentucky, Department of Pathology3 54 Abstract Decreased bone mass, osteoporosis and increased fracture rates are common skeletal complications in patients with insulin-dependent diabetes mellitus (IDDM, type I diabetes). IDDM develops from little or no insulin production and is marked by elevated blood glucose levels and weight loss. Here we use a streptozotocin-induced diabetic mouse model to examine the effect of type I diabetes on bone. Histology and micro-computed tomography demonstrate that adult diabetic mice, exhibiting increased plasma glucose and osmolality, have decreased trabecular bone mineral content compared to controls. Bone resorption could not completely account for this effect as resorption markers (tartrate resistant acid phosphatase (TRAP) 5b, urine deoxypyridinoline (DPD) excretion, TRAP5 mRNA) are unchanged or reduced at 2 and/or 4 weeks post-diabetes induction. However, osteocalcin mRNA (a marker of late stage osteoblast differentiation) and dynamic parameters of bone formation were decreased in diabetic tibias, while osteoblast number and runx2 and alkaline phosphatase mRNA levels did not differ. These findings suggest that final stages of osteoblast maturation and function are suppressed. We also propose a second mechanism contributing to diabetic bone loss: increased marrow adiposity. This is supported by increased expression of adipocyte markers (PPARv2, resistin and aP2) and the appearance of lipid dense adipocytes in diabetic tibias. In contrast to bone marrow, adipose stores at other 55 sites are depleted in diabetic mice as indicated by decreased body, liver and peripheral adipose tissue weights. These findings suggest that type I diabetes contributes to bone loss through changes in marrow composition resulting in decreased mature osteoblasts and increased adipose accumulation. 56 Introduction Bone is a highly specialized and dynamic tissue that undergoes continual remodeling throughout its lifetime. Remodeling processes in bone are a sum of two activities: bone resorption and bone formation. Osteoblasts are responsible for bone formation while osteoclasts resorb bone. An equilibrium between osteoblast and osteoclast activities maintains bone mass and quality. On the other hand, alteration of one part of the equilibrium can lead to bone loss (decreased formation and/or increased resorption) or to increased bone formation (decreased bone resorption and/or increased bone formation) (12). Changes in this balance leading to bone loss and osteoporosis can occur under conditions such as aging, skeletal unloading/disuse, and disease (1-5). Diabetes mellitus (IDDM type I) is a chronic disease, which is characterized by a lack of insulin production. In the absence of insulin, insulin sensitive cells exhibit markedly reduced glucose uptake resulting in increased serum glucose levels. One of the long-term complications of IDDM is diabetic osteopenia and osteoporosis (6-18), which are associated with increased fracture rate (19-22) and delayed fracture healing (23-25). Recent clinical studies found that 67% of men and 57% of women with IDDM suffer from osteopenia of the femoral neck and/or lumbar spine (9) and 14-20% of IDDM patients age 20-56 met the criteria for more extensive bone loss, osteoporosis (9, 18). In diabetic patients, osteoblast function is significantly decreased and is thought to be associated with a maturation defect. This conclusion is based on the fact that serum levels of an early osteoblast marker, peptide of pro-collagen (PICP), 57 remain normal in all types of diabetes, whereas serum levels of a late stage marker of osteoblast maturation, osteocalcin, are decreased in patients at a range of ages (26). These pathological findings are even more pronounced in diabetic rats (27-30), which also exhibit decreased bone volume and osteoid surfaces with no reduction in osteoclast number (27, 31) and significantly less osseointegration of bone implants than controls (32). These findings suggest that a decrease in osteoblast number and/or function/maturation could be a major contributor to bone loss in IDDM. Osteoblasts are derived from mesenchymal stem cells. These pluripotent stem cells can give rise to adipocytes as well as a variety of other cell types (33-36). During the process of osteogenesis and osteoblast differentiation, expression of runx2, a transcription factor required for these processes (37, 38), is increased. Late stage differentiation is marked by elevated osteocalcin expression (39-41). On the other hand, overexpression of peroxisome proliferator-activated receptor v2 (PPARv2), a member of the nuclear receptor transcription factor family (42), induces adipogenesis over osteoblastogenesis in pluripotent cells (43). Interestingly, it PPARv2 is expressed in osteoblasts it can suppress the mature osteoblast phenotype and induce genes associated with an adipocyte-like phenotype, such as fatty acid binding protein 4 (FABP4/aP2), fatty acid synthase (FAS) and lipoprotein lipase (LPL) (43). Selection of adipogenesis over osteoblastogenesis is thought to contribute to bone loss associated with a variety of conditions including age related and disuse-associated osteoporosis (44-48). 58 While studies have examined the influence of type I diabetes on human and rat bone, few have directly addressed the influence of diabetes on the skeletal system of mice, an animal model that is amendable to genetic manipulation. Here we use a streptozotocin-induced type I diabetic mouse model to examine the influence of this disease on bone. Consistent with human and rat studies, we found a decrease in diabetic bone volume (histologically and by micro-computed tomography (pCT) analyses). In addition, we found a decrease in osteocalcin mRNA levels, and we are the first to report an increase in expression of adipocyte markers PPARy2, resistin and aP2 and an increase in visible adipocytes in type I diabetic compared to control tibias. In contrast to bone marrow, peripheral adipose tissue was decreased in size, suggesting that the marrow environment and adipose stores are regulated differently from peripheral sites. These findings support our hypothesis that type I diabetes causes a shift in skeletal composition marked by increased adipose storage and decreased mature osteoblasts. 59 Results Diabetes type I was induced in adult Balb/c mice, using a multiple (5 day) streptozotocin (40 pg/g body weight) injection procedure (49). Streptozotocin selectively destroys pancreatic B cells (50). One week later, non-fasting blood glucose measurements were taken from the saphenous vein to confirm diabetes at levels greater than 300 mg glucose/dl. Control (vehicle-injected) mice tightly maintained blood glucose levels near 180 mg/dl (178 1 5.3) while streptozotocin- injected mice had blood glucose levels averaging 445 i 27 mg/dl. Serum glucose levels remained elevated through out the study (Figure 1). Similarly, blood osmolality determined by a vapor pressure osmometer, was also significantly elevated in diabetic compared to control mice, averaging 325 mmol/kg compared to 302 mmol/kg, respectively (Figure 1). This suggests that tissues and cells of the diabetic mice are exposed to hyperosmotic conditions. In addition to the above analyses, we also examined serum triglyceride levels. Diabetic mice exhibited a significant increase in triglyceride levels throughout the study, consistent with previous studies in diabetic mice and humans (67, 68). In contrast, body weights of diabetic mice were decreased throughout the time course of the study (Figure 1). To determine the effect of streptozotocin-induced diabetes on the mouse skeleton, pCT analyses were carried out on the tibia, which has regions of predominantly cortical bone (the mid-diaphysis) and regions of trabecular bone (the metaphysis). Because differences in bone length (as a result of altered development) could add an additional variable to our analyses, we first measured 60 and compared the length of control and diabetic tibias. Table I demonstrates that tibial length does not significantly differ between control and diabetic (4 weeks post-induction) animals, consistent with the animals being on the flat part of the growth curve at the time of diabetes induction. Examination of cortical bone parameters, bone mineral content, bone mineral density, and moment of inertia, demonstrated no significant difference between control and diabetic animals (Table I). In contrast, all trabecular bone parameters, bone mineral content, bone mineral density and bone volume, were significantly decreased in diabetic compared to control mice (Table l). The pCT images in Figure 2 visually depict the reduction in trabecular bone within the secondary spongiosa of diabetic tibias in lateral and transverse sections. Consistent with our pCT analyses, histological analyses of diabetic (4 week post-injection) proximal tibia demonstrate a decrease in bone volume marked by decreased trabecular bone volume and thickness (Table II). Osteoblast number and osteoid parameters (volume, surface and thickness) did not show a significant difference between control and diabetic animals (Table II). However, dynamic measurements indicate a trend toward decreased osteoblast function/bone formation, which was significant when label incorporation/surface area was determined (3.5 versus 1.4% in control and diabetic animals, respectively) (Table II). Next, we examined systemic and tibial parameters of osteoclast activity. Urine deoxypyridinoline (DPD), a breakdown product of collagen released systemically during osteoclast resorption and is readily measured in the urine, 61 indicated that osteoclast activity is not increased in diabetic animals 14 days post-induction (Figure 3A). Serum levels of active TRAP5b (produced by active osteoclasts) were not significantly increased in diabetic animals at any time studied and were actually decreased on days 14 and 21. Histological examination of the proximal tibia further supports that osteoclast activity is not increased; this is indicated by the lack of a difference in osteoclast number, erosion surface or erosion depth between control and diabetic animals (4 weeks post-induction) (Figure 33). Consistent with this finding, measurement of TRAP5 mRNA levels in control and diabetic tibia did not differ (Figure BB). To assess if diabetes suppresses osteoblast differentiation, we extracted total RNA from control and diabetic tibias and measured markers of progressive osteoblast differentiation: runx2, alkaline phosphatase and osteocalcin mRNA levels. While we did not detect a change in runx2 or alkaline phosphatase, a significant decrease in osteocalcin mRNA levels was evident in diabetic compared to control tibiae (Figure 4). This was consistent with measurements of serum osteocalcin which were also decreased in diabetic mice (Figure 4). This suggests that there is a decrease in mature osteocalcin expressing osteoblasts in the bones of diabetic animals, which supports our dynamic measurements indicating a trend toward decreased bone formation. Further examination of gene expression levels in mouse tibias indicated an increase in PPARyZ mRNA levels, a marker of adipocytes, in diabetic compared to control tibias (Figure 5). Analysis of two other adipocyte markers, resistin and aP2, also demonstrated increased expression in diabetic tibia (Figure 5). To confirm that 62 these effects do not result from direct effects of streptozotocin on mesenchymal and osteoblast cells, we chronically treated pluripotent C3H10T1/2 cells (induced toward the osteoblast lineage or uninduced) with 0.01 to 1 mg/ml streptozotocin. This treatment did not induce adipogenic genes or reduce osteocalcin expression (data not shown) indicating that streptozotocin does not directly influence gene expression in these cells. In agreement with RNA analyses, histological sections of diabetic tibia demonstrated an increase in marrow adiposity compared to controls (Figure 6). Quantitation of adipocytes demonstrated that diabetic tibias contain nearly 3-fold more adipocytes (greater than 50 pm in diameter) per area of bone marrow than control tibias (14.4 :t 4.6 versus 4.8 :t 1.7 adipocytes per mm area of bone marrow, respectively). Interestingly, adipose tissue obtained from control femoral fat pads exhibits lipid dense adipocytes in contrast to diabetic adipose tissue, which exhibits lipid sparse adipocytes (Figure 6). While liver architecture appeared somewhat different in diabetic compared to control animals (Figure 6), it did not exhibit adiposity as determined by histological examination, by oil-red-O staining (data not shown), and by decreased weight (1.31 :I: 0.02 versus 1.46 i 0.02 for controls, p 0.001). The reduction in peripheral adiposity was further demonstrated by pCT analysis of transverse sections of mouse legs and by isolating and weighing the fat pads, which were reduced by greater than 3 fold in size/weight (Figure 7). 63 Discussion Bone loss is a known negative consequence of diabetes type I. The mechanism accounting for this outcome is unknown, making it difficult to develop optimal therapies. Diabetes is associated with a variety of pathologies including little or no insulin, decreased IGF-1, hyperglycemia, and increased blood osmolality (as we show in Figure 1). An animal model is needed that is amenable to genetic manipulation to allow identification of mechanisms accounting for diabetes-associated bone loss through knockout and/or transgenic approaches. Therefore, we set out to characterize the bone phenotype in wild type, BaIb/c streptozotocin-induced diabetic mice. Our histological analyses demonstrated a significant decrease in mouse trabecular bone volume (by 32%) 4 weeks after the induction of diabetes. This amount is similar to bone loss reported in diabetic rats which ranges from 10- 50% (69, 70). Our pCT analyses confirm diabetes-associated bone loss in regions of trabecular bone (density decreased by 24% and volume by 69%), but no statistically significant changes were observed in regions of cortical bone. This is consistent with trabecular bone being more metabolically active compared to cortical bone, and perhaps with extended time cortical bone loss would become apparent. Alternatively, our results could indicate that trabecular bone (intimately associated with the marrow environment and local cytokine and growth factor stimuli) is more responsive to the diabetic environment than cortical bone (which is thought to be controlled more by systemic factors) (71 ). 64 Reports on diabetic osteoclast activity have been variable but most studies indicate no change or a decrease in activity based on histology and/or secretion of deoxypyridinoline, a breakdown product of bone collagen matricies (69). Herrero et al (28) demonstrate decreased pyridinium crosslinks after 12 weeks of diabetes in rats. Similarly, Verhaeghe et al. (29) demonstrate a decrease in urinary deoxypyridinoline secretion in rats diabetic for 8 weeks. Our studies also did not demonstrate an increase in diabetic urine deoxypyridinoline levels, however this measurement can be problematic in diabetics where urine volumes are significantly increased compared to controls, filtration rates may be altered as a result of nephropathy, and collagen breakdown may be occurring in other tissues as well. Therefore, as additional measures of osteoclast activity, we examined TRAP5 mRNA and serum levels. TRAP5 mRNA levels did not differ between control and diabetic tibias at days 14 (figure 3B) and 28 (not shown). Active TRAP5b levels in mouse serum, which is a specific systemic measure of osteoclast activity and correlates with other markers of bone turnover (72, 73), did not increase 4 weeks after induction of diabetes (when bone loss was evident), nor were they elevated at time points prior to this (5, 14 and 21 days). In fact, at day 14 and 21 we observed a significant decrease in active serum TRAP5b levels. A reduction in osteoclast activity could contribute to decreased osteoblast maturation through reduced osteoclast production of osteoblast enhancing factors (74, 75). Alternatively, the reduction in osteoclast activity could be secondary to reduced osteoblast maturation and its associated decrease in osteoclast signaling (75, 76). It should also be noted that while 65 measurements of osteoclast activity at days 5, 14, 21 and 28 were unchanged or reduced, our data does not exclude the possibility that osteoclast activity is increased at earlier time points. In sum, these results support the hypothesis that osteoclast activity is not increased and could possibly be decreased under diabetic conditions at the time points studied. Examination of osteoblast maturation revealed a decrease in osteocalcin expression, a marker of late stage osteoblast differentiation. Both tibial osteocalcin mRNA and systemic serum osteocalcin levels were reduced by over 40%. This reduction correlates with the decrease in mineral apposition rate and label incorporation in diabetic histological sections. Given the high metabolic rate of mice, whereby a 4 week period is roughly equivalent to, 1 year in human terms, it is possible that this suppression can alone account for the bone loss that we observed. Our findings of suppressed osteocalcin expression are supported by nearly all studies measuring serum osteocalcin in diabetic rats and humans, which report on average a 50% decrease in osteocalcin levels compared to controls (27, 28, 77). This decrease is independent of the level of exercise the animals receive (29). Diabetes-associated suppression of osteoblast maturation is also suggested in bone implant/healing studies done in rats, which indicate that diabetic animals exhibit decreased calvarial defect healing (30), and decreased bone formation around titanium or hydroxyapatite bone implants (78- 80). In marrow ablated mice, both runx2 and osteocalcin mRNA levels were found to be decreased in diabetic relative to control bones at 4 and 6 days after ablation, a time at which bone formation was dramatically induced in controls 66 (81). In our studies we did not detect a difference in runx2 mRNA levels between control and diabetic tibias. Our mRNA analyses were done at 2 weeks, to examine changes in gene expression that occur prior to major bone loss, so it may be that the animals were not diabetic long enough to detect a suppression in runx2 mRNA levels in whole bone. The suppression of osteoblast differentiation in diabetes is also consistent with our in vitro analyses examining the role of hyperglycemia in the regulation of osteoblast phenotype. Specifically, we demonstrated that acute hyperglycemic as well as hyperosmotic conditions can suppress late stage markers of osteoblast differentiation in vitro, such as osteocalcin (82). Here we show that diabetic mice do indeed exhibit elevated plasma osmolality, which could contribute to modulation in gene expression. The above data supports the hypothesis that diabetics lose bone through the suppression of late stage osteoblast differentiation, while progression through earlier stages appears unaffected (as indicated by unaltered runx2 and alkaline phosphatase mRNA levels). However, we also propose a second pathway that contributes to diabetic bone loss, which involves the regulation of marrow adiposity. Specifically, our studies demonstrate for the first time that the number of lipid dense adipocytes and expression of adipocyte markers (PPARy2, resistin and aP2) are increased in diabetic type I bone. From our experiments we cannot distinguish whether lipid sparse adipocytes that are always present in the marrow are accumulating lipid and becoming visible or whether mesenchymal pluripotent cells are becoming adipocytes. The later could occur at the expense of osteoblast lineage selection (eventually leading to decreased osteoblast number 67 with more extended experimental time points) or in addition to selecting the osteoblast pathway (thereby not reducing the number of immature osteoblast cell number). Selection of adipogenesis over osteoblastogenesis is a common theme that has been reported in other conditions of bone loss, including age- related osteoporosis and disuse (44-46, 48, 83). PPARy can play a role in this selection based on studies demonstrating that PPARy insufficiency results in the enhancement of osteogenesis and suppression of adipogenesis in mice (84) and on studies demonstrating that elevation of PPARyZ levels can promote adipogenesis in pluripotent mesenchymal cells in vitro (43, 85). Thus, PPARy2 levels have a dominant suppressive influence on osteogenesis. In our studies, at least two possibilities can explain the function of PPARyZ elevation: either it functions as an inducer of adipogenesis or it represents a marker of increased adipocyte number and/or adipogenesis. It is known that PPARy levels and adipogenesis can be regulated by a variety of factors including bone morphogenic protein (86-88), estrogen (89, 90), parathyroid hormone-related peptide (91) and TFF-B (92). In addition, low serum IGF-1 can contribute to reduced bone mineral density, osteoblast differentiation and increase the number of adipocytes in bone (93, 94). The role of the later factor, IGF-1, is of particular interest to our type I diabetic animal studies because diabetic animals and humans exhibit reduced IGF-1 levels (95, 96). It is interesting that reduced IGF-1 and insulin may promote marrow adipogenesis in vivo, in contrast to cell culture studies where lack of these factors reduces the probability of adipogenesis to occur (97). 68 While elevation of PPARy2 is clearly critical for allowing PPARy2 signaling to occur, it is also important to have PPARyZ ligands available to bind to this receptor and activate its function (98). Our studies demonstrate that triglycerides are elevated in diabetic mice, and it is known that fatty acids can activate PPARy2 (98, 99) and promote differentiation of osteoblast-like cells into adipocyte-like cells (100). Thus, we hypothesize that diabetic associated hypertriglyceridemia can compound and further direct bone loss through promoting PPAR activity and adiposity in bone. In summary, we demonstrate that type I diabetic mice exhibit bone loss. 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J Endocrinol 159:297-306 MacDougald OA, Lane MD 1995 Transcriptional regulation of gene expression during adipocyte differentiation. Annu Rev Biochem 64:345-73 Lecka-Czernik B, Moerman EJ, Grant DF, Lehmann JM, Manolagas SC, Jilka RL 2002 Divergent effects of selective peroxisome proliferator- activated receptor-gamma 2 ligands on adipocyte versus osteoblast differentiation. Endocrinology 143:2376-84 Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans RM 1995 15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma. Cell 832803-12 Diascro DD, Jr., Vogel RL, Johnson TE, et al. 1998 High fatty acid content in rabbit serum is responsible for the differentiation of osteoblasts into adipocyte-like cells. J Bone Miner Res 13296-106. 79 Figure Legends Figure 1. Diabetes increases serum glucose, serum osmolality and serum triglycerides and is associated with decrease body weight. Serum from 5, 14, 21, and 28 days diabetic and control Balb/c mice was analyzed for glucose concentration, osmolality, and triglyceride levels. Body weight was recorded at every time point. Values are averages +/- SE obtained from 8-11 mice per condition. *p<0.05 Figure 2. Diabetes reduces trabecular bone volume. (A) Representative pCT lateral slices (20pm) through the proximal tibia from two control and diabetic mice are shown. (B) Representative pCT transverse slices (20pm) through the proximal tibia at a distance of 0.5 mm or 1 mm from the lowest region of the growth plate. Figure 3. Systemic and tibia specific measures of osteoclast activity in diabetic bone. (A) Diabetes decreases serum active TRAP5b levels and has no effect on urine DPD levels. Urine collected over a 24 hour period was obtained from diabetic and control mice in a metabolic cage at day 14. DPD was determined in control and diabetic urine samples and expressed relative to creatinine, to control for differences in urine volume. Serum was obtained from mice at 5, 14, 21 or 28 days after the induction of diabetes. Active TRAP5b levels were determined in control (C) and diabetic (D) mouse serum samples. 80 (B) Histomorphometry and TRAP5 mRNA analyses were preformed on tibias obtained from mice 28 days after the induction of diabetes. All values are averages +/- SE obtained from 4-9 mice per condition. *p<0.05. Figure 4. Osteocalcin mRNA and serum levels are decreased in diabetes, in contrast to runx-2 and alkaline phosphatase (Alk Phos) mRNA levels. Total RNA, extracted from tibia isolated from control and diabetic (2 week) adult Balb/c mice, was used for real time RT-PCR analysis with SYBR green dye. Levels of amplified genes were expressed relative to cyclophilin (a housekeeping gene) and relative to control levels, which were set to 1. Serum from 2 week diabetic and control mice was analyzed for osteocalcin levels. Values are averages +/- SE obtained from 8-11 mice per condition. *p<0.05 Figure 5. Adipocyte markers, PPARv2, aP2 and Resistin are increased in diabetic bone. Total RNA was extracted from tibia isolated from control and diabetic (2 week) adult Balb/c mice, and used for real time RT-PCR analysis with SYBR green dye. Levels of PPARv2, aP2 and resistin are expressed relative to cyclophilin (a housekeeping gene). Values are averages +/- SE obtained from 8- 11 mice per condition. *p<0.05. Figure 6. Adiposity is increased in diabetic tibia, in contrast to peripheral adipose tissue lipolysis. Tibia (bone) obtained from control and diabetic (4 weeks) mice were fixed and processed for plastic 81 embedding and sectioning. Representative Masson-Goldner trichrome stained sections demonstrate differences in the amount of mineralized bone (blue stain) and in the number of adipocytes (unstained large circular regions within the marrow) between control and diabetic animals. Representative fat pad and liver sections were obtained from control and diabetic mice. Sections were stained with hematoxylin 8 eosin and digitally photographed. Images in this dissertation are presented in color. Figure 7. Peripheral adipose tissue is decreased in diabetic mice. Transverse slices of mouse legs were obtained by pCT analysis. Representative images are shown, with medium grey (marked by black arrows) indicating regions of fat. In addition, dissected femoral fat depots were photographed and weighed. The later values were pooled from 5-9 mice per condition and expressed as averages +/- SE. *p<0.01. 82 wmmmwo wmwmmmmowmw coEE. cocoa—m 3350.55 3295 Eacow >u=£oEmo 325029.... £38m £325 2...... a. £925 >com 28 21 14 Days 83 Figure 2. Control Diabetic distance from growth plate: 0.5 mm 84 Table l Control Diabetes p-value Tibia Length 16.986 :l: 0.16 16.84 1 0.14 0.4 Cortical BMC 2.93 1 0.25 3.2 1: 0.12 0.25 BMD 1125114 1112112 0.48 MOI 0.141 1; .01 0.142 t .02 0.97 Trabec. BMC 1.4 :I: 0.12 0.79 :I: 0.09 * 0.001 BMD 295 :l: 12 223 i 9.6 *0.0002 BVF 13.4 i 1.9 4.2 :l: 0.72 *0.0001 BMC - bone mineral content, mg; BMD — bone mineral density, mg/cm3; MOI — moment of inertia, mm4; BVF — bone volume fraction, %; * statistically significant based on p-value. 85 TRAP5b (UIL) DPD/Creatinine (nmollmmol/24 hours) 50- 40- 30 10 0 Figure 3A. .3 N 1 .0 00 P a. I O Control I Diabeticfi 20~ I I * * " '1‘ 00' 607 CD CD day5 day14 day21 day28 86 Figure 3B. .4...4 . . . . . 4 00 75543210 3525.150 5 I 7. 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Diabetic Control 87 Table II Control Diabetes p-Value Bone Volume-% 14.5 :t 1.7 9.8 i 1.0 * 0.03 Trabecular Thickness (Plate)-pm 42.7 i 4.0 30.4 i 2.0 * 0.02 Trabecular Number (Plate)-/mm 3.4 i 0.14 3.2 1: 0.19 0.48 Trabecular Separation (Plate)-pm 258 :1: 15 293 i 23 0.23 Osteoid Volume/Bone Volume-% 0.55 i 0.10 0.48 i 0.11 0.68 Osteoid Surface/Bone Surface-% 3.9 :t 0.72 3.6 i 0.70 0.79 Osteoid Thickness-pm 2.3 1: 0.34 2.1 i 0.31 0.68 Osteoblast Surface/Bone Surface-% 2.0 1: 0.50 2.3 :t 0.73 0.73 Osteoblast Number/Bone Length/100mm 248 i 94 251 i 54 0.97 Mineral Apposition Rate/ Day- pm/Day 0.98 i 0.2 . 0.69 3: 0.11 0.20 Double labels/Bone Surface % 3.54 i 1.0 1.4 :I: 0.4 9: 0.04 Mineralizing surface/Bone Surface % 6.16 1: 1.2 4.9 :t 0.5 0.29 Bone formation rate/Bone Surface-mm3/cm2/y 2.5 t 0.4 1.43 i: 0.35 0.10 Mineralization Lag Time — Days 1.91 :I: 0.74 3.9 :1: 1.70 0.35 * statistically significant based on p-value. 88 Figure 4. 4213542 11 0000 5:58.03 Nxcam 0 _ _ ~ — fl _ _ u 3 5 4 0 2. 1 8. 6. 4. 0 0 0. 1 0 O 0 =___fio_2§ 5.28.03 8i 0__< £288.80 m. m w m w o 2585 52.30030 Escow Diabetic Control 89 Figure 5. 3 2 1 U 6 2 8 4 5.28.03 5.28.05 9.38.03 NEE; was 5.98”. ' Diabetic 0 Control 90 adipose bone liver Figure 6. Control 91 Diabetic Figure 7. Control Diabetic Fat pad wt (9) .0 9 .° 8 is a: P O .5 92 CHAPTER IV Inhibition of PPAR—v prevents type | diabetic bone marrow adiposity but not bone loss. Sergiu Botolin1 and Laura R McCabe1 Michigan State University1 Departments of Physiology and Radiology1 Molecular Imaging Research Center1 East Lansing, MI 48824 93 Micro-Abstract Type I diabetic mice exhibit 1) bone loss and 2) increased marrow adiposity. To examine the relationship between these characteristics marrow adiposity was prevented through PPARv inhibition. Interestingly, bone loss was not prevented. These findings suggest that marrow adiposity is not linked to bone density in diabetes and is not the cause of type | diabetic bone loss. Abstract Introduction: Diabetes type I is associated with bone loss and increased bone adiposity. Osteoblasts and adipocytes are both derived from mesenchymal stem cells located in the bone marrow, therefore it has been hypothesized that decreased osteoblast function and increased adiposity are directly and reciprocally linked. Methods: To test this relationship, we chronically treated control and insulin- deficient diabetic mice with a PPARy antagonist, bisphenol-A-diglycidyl ether (BADGE), to block PPARy activity, a known activator of adipocyte differentiation. Analyses of serum parameters demonstrate that BADGE treatment does not prevent diabetes-associated hyperglycemia or decreased body weight. Results: BADGE treatment successfully prevented diabetes-induced hyperlipidemia and effectively blocked diabetes type I induced bone adiposity. Despite the prevention of hyperlipidemia and bone adiposity, BADGE treatment 94 was unable to prevent diabetes type I suppression of bone formation markers (runx2 and osteocalcin) and bone loss (as determined by micro-computed tomography). Conclusion: These findings suggest that BADGE treatment is an effective approach to reduce serum triglyceride and free fatty acid levels as well as bone adiposity associated with type I diabetes. The inability of BADGE treatment to prevent bone loss in diabetic mice suggests the possibility that marrow adiposity is not necessarily directly linked to bone density status and is not the cause of type I diabetic bone loss. Key words: diabetes, osteoblast differentiation, adipocyte, bone marrow, PPARv 95 Introduction Diabetes type I is a metabolic disease marked by the inability of cells to take up glucose, as a result of the lack of serum insulin. As a consequence, cells must utilize other energy sources including lipids. Mobilization of free fatty acids and triglycerides, while providing needed energy, is thought to contribute to the secondary pathologies including beta-cell toxicity, retinopathy and nephropathy (1-3). In addition to being energy sources, serum lipids can also trigger and modulate cell signaling. Peroxisomal proliferator-activated receptor v2 (PPARyZ) is a critical transcription factor (of the nuclear receptor superfamily) whose activity is regulated by its binding to fatty acid ligands (4-6). Upon ligand binding, PPARvZ transcriptional activity is increased and gene promoters containing peroxisomal proliferator response elements are induced. Target genes are associated with adipocyte differentiation and include adipose-specific fatty acid binding protein (aP2; involved in intracellular fatty acid transport). Exogenous agonistic ligands for PPARs, in particular a class of drugs known as the thiazolidinedione family (ciglitazone, pioglitazone, troglitazone, rosiglitazone), are effective antidiabetic compounds that lower hyperglycemia, hyperinsulinemia and hypertriglyceridemia in patients with type II diabetes (4-6). A significant, but less well known, complication of type I diabetes is bone loss (7-19). Both men and women are vulnerable to this outcome (10). Bone loss, termed osteopenia or osteoporosis (between 1 and 2.5 or greater than 2.5 standard deviations below average bone mineral density, respectively) is associated increase in fracture risk and delayed fracture healing in the general 96 population as well as in type I diabetic patients (20-25). With effective therapeutics for type I diabetic patients, an increasing number of diabetic patients are undergoing age-related bone loss, but starting this process with lower bone density as a result of diabetes. This situation puts diabetic patients at an even greater risk for age-related bone fractures. To begin to understand the underlying mechanism for diabetes associated bone loss, we previously characterized the bone phenotype of diabetic (for 4 weeks) adult mice. We found that similar to type I diabetes in humans, streptozotocin-induced diabetic mice exhibit significant bone loss (26). Bone turnover markers indicate that osteoclast activity/bone resorption is not increased under diabetic conditions. In contrast, osteoblast activity and bone formation were reduced as determined histologically, by micro-computed tomography and by suppressed expression of markers of osteoblast maturation (such as osteocalcin) in bone. Interestingly, we also found a marked increase in bone marrow adiposity and adipocyte markers (PPARvZ and aP2) in type | diabetic mice. Both osteoblasts and adipocytes can be derived from mesenchymal stem cells located in the bone marrow, suggesting the possibility that diabetes type I conditions (including upregulation of PPARy2 (26) and hyperlipidemia) promote selection of the adipocyte over osteoblast lineage. This effect would cause a shift in skeletal composition marked by increased adipose storage and decreased mature osteoblasts, and would ultimately lead to bone loss. The reciprocal relationship between bone adiposity and bone mineral density has long been recognized. For example, age-related bone loss and 97 osteoporosis is accompanied by increased bone marrow adiposity (27, 28). Similarly, bone loss associated with the unloading of bone (limb disuse) is also accompanied by increased bone marrow adiposity (29). This relationship holds in vitro as well, where increasing adipocytic signals in cell culture systems reduces the number of mature bone producing osteoblast cells (30-33). The question remains, however, whether the induction of adiposity always drives the suppression of osteoblast function and bone loss or whether adiposity can be a secondary effect that does not influence the regulation of bone mineral density (27). If linked, the regulation of adiposity is a key therapeutic target (27, 34). Based on the above findings and the reciprocal relationship between adiposity and bone mineral density (35-38), we hypothesized that prevention of bone marrow adiposity would rescue bone loss in type I diabetic mice. We employed a PPARy antagonist, bisphenol-A—diglycidyl ether (BADGE), to block PPARy activity. BADGE was identified by Wright et al. (39), in a screen for PPARy ligands, as a PPARy antagonist that has no activity of its own, but prevented binding of PPARy agonists and PPARv transcriptional activity. BADGE antagonism of PPARv has been shown to prevent adipogenic cells such as 3T3-L1 cells from undergoing hormone-mediated differentiation (39) and to abolish the anti-inflammatory effects of rosiglitazone (under conditions of carrageenan-induced paw edema and lung pleurisy (40)). Correspondingly, we show that this treatment approach in vivo successfully inhibited diabetes-induced expression of adipocyte markers and marrow adiposity in diabetic bones. Interestingly, we were also able to prevent hyperlipidemia in BADGE treated 98 diabetic mice. However, to our surprise, suppression of PPARv activity, hyperlipidemia and bone adiposity did not prevent diabetes type l-induced bone loss. While this result potentially points to a role for PAPRy activity in osteoblast maturation, we did not see a suppression of osteoblast gene expression or bone mineral density in control animals treated with BADGE. Take together, these findings suggests that prevention of adipocyte differentiation is not sufficient to promote marrow stem cells toward the osteoblast lineage in type I diabetes. 99 Results Previously we demonstrated that streptozotocin-induced type I diabetes causes bone loss and increased bone adiposity (26). To test if the increase in bone adiposity is linked to the loss of bone in type I diabetes, we treated control and streptozotocin-induced type I diabetic adult mice with a PPARy antagonist, BADGE (39), to block maturation of adipocytes. Treatments began at the time that diabetes was confirmed and mouse phenotype parameters were measured at 5, 14, 21 and 28 days following treatment. Figure 1 demonstrates that type I diabetic mice lose weight and exhibit elevated blood glucose levels (above 30 mmol; ranging between 300-500 mg/dl) with or without BADGE treatment. Interestingly, examination of serum lipid levels, triglycerides and free fatty acids, demonstrated that BADGE treatment was very effective in preventing diabetic associated elevations in serum triglyceride and free fatty acid levels at 5, 21, and 28 days (Figure 1). Next, we examined the expression of adipocyte markers in control and diabetic bones with or without BADGE treatment. RNA levels were determined relative to cyclophilin RNA levels, which were not modulated at any time point by diabetes or BADGE treatment (data not shown). Figure 2 demonstrates that type I diabetes causes a significant and chronic induction of adipocyte markers. Treatment with BADGE suppressed diabetes-induced PPARy2 expression in mouse bone, consistent with BADGE antagonism of PPARy activity and the positive feedback of PPARyZ on its own expression. In fact, by day 28 PPARv2 levels were significantly lower in BADGE treated diabetic mice compared to 100 controls. Similarly, induced expression of a mature adipocyte marker, aP2, was also prevented by BADGE treatment (Figure 2). Effects were evident with 5 days treatment and were maintained out to 28 days. Consistent with gene expression analyses, histology demonstrated an increase in bone adiposity in diabetic mice that was prevented with BADGE treatment (Figure 3). To test the influence of BADGE inhibition of diabetes-induced bone marrow adiposity on osteoblast maturation, markers of osteoblast lineage and differentiation, (runx2 and osteocalcin) were examined. As shown in Figure 4, mRNA isolated from diabetic mouse bone exhibits significantly decreased levels of runx2 and osteocalcin at 5, 14 and 28 days after confirmation of diabetes. Treatment with BADGE was unable to prevent this suppression, even with 28 days of chronic treatment (Figure 4). Interestingly, at 28 days we were able to detect a significant enhancement of osteocalcin expression in BADGE treated controls compared to untreated controls. Bone pCT analyses confirm that the bone loss seen in diabetic mice is not prevented by BADGE treatment (Figure 5, Table I). To further examine the kinetics of BADGE treatment on the diabetic mouse phenotype, we acutely treated diabetic mice with BADGE from days 1-7 (early) or days 21-28 (late) and examined serum parameters and gene expression at day 28. Figure 6 demonstrates that any regimen of BADGE treatment was unable to restore serum glucose levels. Interestingly, treatment with BADGE just at the early onset of diabetes (days 1-7) was able to prevent hyperlipidemia in diabetic mice 21 days later (at day 28), similar to chronic 101 BADGE treatment (days 1-28). In contrast, late BADGE treatment (from 21 to 28 days) was unsuccessful at preventing diabetes-induced elevation in serum triglyceride levels. Early and late BADGE treatment did not influence osteocalcin or aP2 mRNA levels, although there was a trend toward reducing the later; this indicates that chronic treatment is most effective at suppressing markers of bone adiposity. One concern with our approach was that animals were being treated with BADGE at the time of confirmed diabetes (blood glucose levels greater than 300 mg/dl). Our data demonstrates that changes in gene expression are significant by only 5 days after confirmation of diabetes. This point is actually almost 2 weeks after the first injection of streptozotocin. Therefore, it is possible that key regulatory events are occurring prior to the point of confirmed diabetes and these events were not blocked. To address this possibility, we chronically treated mice with BADGE beginning at the time of the first streptozotocin injection, prior to any signs of diabetes (high glucose, polyuria, elevation in serum triglycerides). Figure 7 demonstrates that treatment with BADGE at the start of diabetes induction (prior to detection of hyperglycemia), during the induction of diabetes and throughout the time course caused a response similar to BADGE treatment beginning at the time of confirmation of diabetes. Specifically, diabetes-induced aP2 (Figure 7) and PPARv2 (data not shown) mRNA levels were prevented by BADGE treatment, while suppression of osteocalcin (Figure 7) and Runx2 (data not shown) mRNA levels were not blocked with BADGE treatment. 102 Discussion To test the role of bone adiposity in mediating bone loss in diabetic mice, we used a PPARy antagonist, BADGE, to block adipocyte differentiation. PPARv was targeted because of its key role in the progression of adipocyte differentiation and its association with bone mineral density parameters. Activation of PPARy by elevated serum lipids or by synthetic agonists, such as rosiglitazone (used in the treatment of type II diabetes), stimulates bone marrow adiposity and bone loss through PPARy—dependent and PPARy-independent pathways (35, 36). Consistent with PPARvZ function in adipocyte differentiation, null mutation of PPARy in embryonic stem cells prevents troglitazone induced adipogenesis, but interestingly also enhanced osteogenesis (37). Heterozygous PPARy deficient mice exhibit enhanced bone mineral density that became prominent with aging; these animals exhibit normal osteoblast and osteoclast functions, but osteoblastogenesis in the bone marrow appears enhanced (37). Similarly, lipodystrophic PPARyhyp’hyp mice, which congenitally lack PPARy in white adipose tissue, exhibit an increase in bone mineral density and bone area (38). Suppression of PPARy activity with BADGE treatment in our studies effectively blocks type I diabetes-induced expression of PPARy2 target genes (PPARy2, aP2) and bone marrow adiposity but not bone loss. In contrast to the above reports, our findings suggest that adiposity (and serum lipid levels) is not linked to bone density in type I diabetes. Possible differences between these studies include the fact that we are focusing on the influence of a PPARy antagonist in a diseased state, which unlike control or aging animals is 103 associated with immediate decreases in osteoblast maturation markers and increases in adipocyte markers (within 5 days of confirmed diabetes). Two separate mechanisms could be at play: one directed at stem cell lineage selection and another directed at osteoblast maturation. Our data suggests that the former plays less of a role in causing bone loss in type I diabetes than the later. Unlike previous reports, suppression of PPARV in these studies was not congenital and was induced in adult mice (15 weeks old). Early suppression of PPARv prior to reaching an adult stage could effectively prevent pre-adipocytes from forming in the marrow and truly block the adipocyte lineage. In our studies, it is possible that pre-adipocytes are present in the marrow, although not loaded with fat and not visibly evident, and they respond to type, I diabetes by storing fat and becoming visible. Thus, the increased numbers of adipocytes are not a result of changes in lineage selection, but changes in adipocyte maturation. It is also possible that suppression of PPARV with BADGE affects osteoblast maturation thereby contributing to an inability to prevent bone loss. It is known that osteoblasts express PPARy1 and other PPAR members (41) and that treatment of osteoblasts with PPAR agonists can enhance or suppress osteoblast maturation in vitro, depending upon the concentration (41). Studies in mice with PPARy haploinsufficiency suggest that systemically reduced PPARy levels enhance bone mineral density (most prominent in aging mice) through changes in marrow cell lineage selection (37). The increase in bone mineral density suggests that full PPARv activity is not needed for osteoblast maturation to occur. Examination of our BADGE treated control animals indicates that 104 BADGE treatment did not actively suppress bone density and actually trended toward increasing bone mineral density and markers of osteoblast maturation (with long term treatment) similar to previous reports. Interestingly, ovariectomized mice with PPARy haploinsufficiency still exhibited a decrease in bone volume when compared to haploinsufficeint shams 4 weeks after surgery (37). This finding further suggests the possibility that PPARV and adiposity may not be linked to all forms of bone loss. Our findings also demonstrate an increase in serum triglycerides and free fatty acids, which were normalized with BADGE treatment. Our finding of hyperlipidemia in type I diabetes is consistent with reports in other type I diabetic rodent models including CS7BL/6J mice (42) and rats (43). While dyslipidemia is not always present in well controlled patients, when metabolic control is poor, serum lipid levels are usually elevated and linked to diabetic complications (1). Increased plasma fatty acid levels are associated with beta-cell lipotoxicity (2), and elevated triglyceride levels are predictive factors for the development of renal and retinal complications in type I diabetic patients (3). Hyperlipidemia is also thought to influence bone. Marrow cells isolated from mice fed high fat, atherogenic diets fail to undergo osteoblast differentiation in vitro (44), suggesting a key role for serum lipids in the activation of PPARv and marrow cell lineage selection. Our results suggest that despite maintenance of normal serum triglyceride and free fatty acid levels, type I diabetic animals still lose bone. This further indicates that fat metabolism and adiposity are not linkedto the bone loss seen in type I diabetes. 105 In addition to changes in total lipid levels, it is clear that diabetes also causes changes in lipid profiles. In mouse models of diabetes (streptozotocin injection or non-obese diabetes) increased levels of certain di- and tri- unsaturated fatty acids are found (6). The composition of non-esterified fatty acids in diabetic serum changes under type I diabetes conditions, such that levels of 18:1n-9 (oleic) and 18:2n-6 (linoleic acid) are significantly increased (6). The diabetes-induced changes in lipid profiles, many of which are ligands (or their metabolites are ligands) for PPARy , may play a key role in modulating PPARy activity and marrow adiposity. Treatment of osteoblast-like cells (ROS 17/2.8) and SaOS-2/B1O cells) with factors such as rabbit serum, which contains high levels of palmitic, oleic and linoleic fatty acids, can activate PPARs and induce adipocyte-like differentiation (31). While we did not directly examine lipid profiles, we were able to suppress overall levels and prevent induction of PPARy target genes in diabetic bone. However, we cannot distinguish whether BADGE directly affects PPARy activity in bone to cause the block in adiposity, or if BADGE prevents bone adiposity through effective suppression of hyperlipidemia. In summary, systemic treatment with a PPARy antagonist effectively blocks type | diabetes-induced hyperlipidemia and bone adiposity. Both of these conditions are thought to be involved in bone loss, yet their prevention by BADGE treatment was ineffective in blocking type I diabetes-induced bone loss. 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Dev Cell 61483-495 Ahdjoudj S, Fromigue O, Marie PJ 2004 Plasticity and regulation of human bone marrow stromal osteoprogenitor cells: potential implication in the treatment of age-related bone loss. Histol Histopathol 19:151-157 Duque G 2003 Will reducing adopogenesis in bone increase bone mass?: PPARgamma2 as a key target in the treatment of age-related bone loss. Drug News Perspect 16:341-346 Ali AA, Weinstein RS, Stewart SA, Parfitt AM, Manolagas SC, Jilka RL 2005 Rosiglitazone causes bone loss in mice by suppressing osteoblast differentiation and bone formation. Endocrinology 146:1226-1235 110 36. 37. 38. 39. 40. 41. 42. 43. 44. Picard F, Auwerx J 2002 PPAR(gamma) and glucose homeostasis. Annu Rev Nutr 22:167-197 Akune T, Ohba S, Kamekura S, Yamaguchi M, Chung Ul, Kubota N, Terauchi Y, Harada Y, Azuma Y, Nakamura K, Kadowaki T, Kawaguchi H 2004 PPARgamma insufficiency enhances osteogenesis through osteoblast formation from bone marrow progenitors. J Clin Invest 113:846- 855 Cock TA, Back J, Elefteriou F, Karsenty G, Kastner P, Chan S, Auwerx J 2004 Enhanced bone formation in lipodystrophic PPARgamma(hyp/hyp) mice relocates haematopoiesis to the spleen. EMBO Rep 5:1007-1012 I; Wright HM, Clish CB, Mikami T, Hauser S, Yanagi K, Hiramatsu R, Serhan CN, Spiegelman BM 2000 A synthetic antagonist for the peroxisome proliferator-activated receptor gamma inhibits adipocyte differentiation. J Biol Chem 275:1873-1877 Cuzzocrea S, Pisano B, Dugo L, lanaro A, Maffia P, Patel NS, Di Paola R, Ialenti A, Genovese T, Chatterjee PK, Di Rosa M, Caputi AP, Thiemermann C 2004 Rosiglitazone, a ligand of the peroxisome proliferator-activated receptor-gamma, reduces acute inflammation. Eur J Pharmacol 483279-93 Jackson SM, Demer LL 2000 Peroxisome proliferator-activated receptor activators modulate the osteoblastic maturation of MC3T3-E1 preosteoblasts. FEBS Lett 471 :1 19-124 Pighin D, Karabatas L, Pastorale C, Dascal E, Carbone C, Chicco A, Lombardo YB, Basabe JC 2005 Role of lipids in the early developmental stages of experimental immune diabetes induced by multiple low-dose streptozotocin. J Appl Physiol 98:1064-1069 Zhang W, Lu D, Kawazu S, Komeda K, Takeuchi T 2002 Adenoviral insulin gene therapy prolongs survival of IDDM model BB rats by improving hyperlipidemia. Horm Metab Res 342577-582 Parhami F, Jackson SM, Tintut Y, Le V, Balucan JP, Territo M, Demer LL 1999 Atherogenic diet and minimally oxidized low density lipoprotein inhibit osteogenic and promote adipogenic differentiation of marrow stromal cells. J Bone Miner Res 14:2067-2078 lll Figure Legends Figure 8. BADGE treatment prevents hyperlipidemia in diabetic mice, but does not correct hyperglycemia or prevent weight loss. Type I diabetes was induced in Balb/c mice by streptozotocin-injection. Body weight and serum parameters were monitored at 5, 14, 21 and 28 days after the confirmation of diabetes (a blood glucose greater than 300 mg/dl) in control (circles), diabetic (triangle), control + BADGE (squares) and diabetic +BADGE treatment (diamond). BADGE treatment began at the time of confirmed diabetes. Values represent averages obtained from 8-11 mice per condition +/- SE. * p<0.05. Figure 9. BADGE treatment blocks induction of adipogenic gene expression in diabetic bones. Total RNA was extracted from tibia isolated from control (C), diabetic (D) and BADGE treated mice at 5, 14, 21, and 28 days after the confirmation of diabetes. Levels of PPARv2 and aP2 mRNAs were determined by realtime RT-PCR and are expressed relative to cyclophilin. Values are averages +/- SE obtained from 8-11 mice per condition. *p<0.05. Figure 10. PPARy2 antagonist treatment prevents type | diabetes-induced bone adiposity. Representative images of histological sections of mouse proximal tibias 28 days after the confirmation of diabetes. Adipoctye number was determined in control, diabetic and 112 BADGE treated mice. Values are averages +/- SE obtained from 8-11 mice per condition. *p<0.05. Figure 11. BADGE treatment is unable to rescue osteogenic gene expression in diabetic bone. Total RNA was extracted from tibia isolated from control (C), diabetic (D) and BADGE treated mice at 5, 14, 21, and 28 days after the confirmation of diabetes. Levels of OC and Runx-2 mRNAs were determined by realtime RT-PCR and are expressed relative to cyclophilin. Values are averages +/- SE obtained from 8-11 mice per condition. *p<0.05. Figure 12. BADGE treatment does not prevent type I diabetes- induced bone loss. Representative pCT lateral slices (20pm) through the proximal tibia from untreated or treated control and diabetic mice obtained 28 days after the confirmation of diabetes and after BADGE treatment (which began at the time of confirmation of diabetes) are shown. Trabecular and cortical bone density measurements (obtained from 8-11 mice per condition) are shown in Table I. Figure 13. Chronic BADGE injection is the most effective treatment regime in preventing type I diabetes-induced hypertriglyceridemia and aP2 expression. Type I diabetes was induced in Balb/c mice by streptozotocin-injection. Upon confirmation of 113 diabetes (a blood glucose greater than 300 mg/dl), termed day 0, mice were treated with daily with BADGE either chronically (days 1-28), early (days 1-7; not treated from days 8-28) or late (days 21-28; not treated from days 1-20) in the progression of diabetes. Serum parameters (glucose and triglycerides) and tibial mRNA levels of aP2 and osteocalcin were determined for all animals 28 days after confirmation of diabetes and are expressed relative to cyclophilin levels. Values represent averages obtained from 8-11 mice per condition +/- SE. * p<0.05. Figure 14. BADGE treatment beginning at the time of streptozotocin injection is unable to prevent osteocalcin mRNA suppression in diabetic mice. Type I diabetes was induced in Balb/c mice by streptozotocin-injection. At the time of the first injection daily BADGE treatment was begun. Mouse tibias were obtained from control (C), diabetic (D) and BADGE treated mice 5 and 28 days after the confirmation of diabetes. Tibial mRNA levels of aP2 and osteocalcin were determined for all animals by real time RT-PCR and are expressed relative to cyclophilin. Values represent averages obtained from 8-11 mice per condition +/- SE. * p<0.05. 114 Figure 8. 0 coEEv $0033 .555 :29... 82.8029; 53.—Om $3.5. 0 ... L. ... ..1 some. din. Escow 0 28 21 4 1 Days 115 5 DAYS 28 DAYS 21 DAYS 14 DAYS PPAR-372 2.5 - 21 ’f 1.1 1.54 wee i—-—4* Figure 9. aP2 6 i 4 1 1 rm r-H-x- I—H-x. J_ a: O N & a: c ...s N a.) .5 o J 1 l 1 L 1 I -l , 4 3]: T 4- "1 2.. o[ If ‘- C D BADGE 116 ‘— , W ’ j ._ _ Figure 10. Control ..‘-. i . . ~1- + BADGE 80 60 20 at a: ‘3. o o .9 'o < 117 5 DAYS 14 DAYS 28 DAYS 21 DAYS 118 0.5 o , + BADGE Figure 12. Control 119 Diabetic Table III Trabecular region: BMC(mg) BMD(mg/cm3) BVF(%) C 1.6210.09 379111 4112 C+B 1.7610.10 403127 4412 D 1.1710.07* 27517.0* 2111* D+B 1.2410.02* 272 15.2* 2211* Cortical region: BMC(mg) BMD(mg/cm3) BVF(%) C 43010.16 1183121 1110.2 C+B 4.47 1 0.17 1204 114 11 1 0.4 D 3.94 1 0.26 1142 1 19 9.2 1 0.2* D+B 3.64 1 0.22 1135 1 27 9.9 1 0.3 BMC - bone mineral content; BMD - bone mineral density; BVF - bone volume fraction; * statistically different (relative to controls) based on p-value < 0.05. 120 Figure 13. 2255 omooam Escom 2295 325039.... Eacow on; 4 2 0 5.28.0? Nam 2° 00° swinger: £28860 ‘ Early, Late Chronic D BADGE 121 0C Figure 14. *JI I1 —‘ aP2 .5. 1 .5. fl 1 o .11. . 2 J q 2 96b m got em D BADGE C . D C 122 CHAPTER V Diabetic Adiposity and DMSO Treatment in Streptozotocin Diabetic Mice Sergiu Botolin1 and Laura R McCabe1 Michigan State University1 Departments of Physiology and Radiology1 Molecular Imaging Research Center1 East Lansing, MI 48824 123 Introduction Diabetes type I is a disease resulting from a lack or insufficiency in insulin secretion. It is associated with multiple long term complications among which is bone loss. Diabetic bone loss has been associated with impaired osteoblast maturation based on the fact that markers of osteoblast maturation such as osteocalcin are reduced in diabetic patients as well as in experimental models of diabetes(1),(2),(3). Previous studies from our laboratory also demonstrate a decrease in both serum osteocalcin levels and osteocalcin mRNA levels in bones from diabetic mice, further supporting the concept of impaired osteoblast maturation in diabetes. Our work also provided evidence of unchanged or even decreased bone resorption in diabetes type I, excluding increased bone resorption and increased osteoclast activity as pathophysiological mechanism of diabetes associated bone loss. We also demonstrate an increase in bone marrow adiposity associated with diabetic status. Increase in adiposity is expressed as an increase in number of adipocyte in bone marrow as well as an increase in adipocyte specific gene markers such as aP2 and PPARy2. In order to investigate the role of increased adiposity in diabetic bone loss, we subjected diabetic mice to pharmacological PPARy inhibition with a PPARy antagonist, bisphenoI-A-diglycidyl ether (BADGE) (4). BADGE was shown previously to have a PPARy antagonist properties by preventing adipogenic cells such as 3T3-L1 cells from undergoing hormone-mediated differentiation and by reducing anti- inflammatory effects of rosiglitazone (under conditions of carrageenan-induced 124 paw edema and lung pleurisy) (5). We found that treatment of diabetic mice with BADGE for different periods of time inhibited diabetes-induced expression of adipocyte markers and marrow adiposity in diabetic bones. Interestingly, we were also able to prevent hyperlipidemia in BADGE treated diabetic mice. To our surprise, suppression of PPARy activity, hyperlipidemia and bone adiposity did not prevent diabetes type I-induced bone loss, suggesting that prevention of adipocyte differentiation is not sufficient to promote marrow stem cells toward the osteoblast lineage in type I diabetes. In the above mentioned study, we compared the effects of BADGE treatment between BADGE treated diabetic animals and BADGE treated controls and non-injected control and diabetic mice, leaving a place for a potential action of vehicle alone (DMSO). To further address this issue, we investigated the role of the vehicle treatment on bone gene expression patterns in diabetic and control mice. 125 Results Previously we demonstrated that daily intraperitoneal treatment with BADGE (30 mg/kg in 15% DMSO), reduced bone mRNA levels of adipocyte markers such as aP2 and PPARv2 in streptozotocin induced diabetic mice, but lacked the ability to rescue osteoblast markers osteocalcin and Runx-2. Since in our previous studies BADGE was administered in 15% DMSO, we subjected a group of control and streptozotocin-induced diabetic mice to 5 day, intraperitoneal treatment with 100 pl of 15% DMSO for a total of 5 days. This treatment interval was chosen because we demonstrated that 5 days of BADGE administration can affect bone parameters in control mice. Figure 1 demonstrates the effects of DMSO treatment on osteoblastic and adipocytic markers gene expression. Interestingly, DMSO alone lowered the expression of aP2 mRNA in diabetic animals from 5 fold, seen In diabetic untreated animals, to 3 fold. However, 5 days BADGE treatment lowered expression of aP2 to less than one fold difference when compared with BADGE treated controls. PPARv2 mRNA levels are not different in both BADGE and DMSO treated controls and diabetics, whereas they are elevated in non-treated diabetics. Osteocalcin shows a marked decrease in expression levels in diabetics from DMSO treated group and untreated animals, with no difference in the BADGE treated group. Runx-2 expression is significantly decreased in diabetic animals only in untreated group, with no significant change in DMSO and BADGE treated groups. Figure 2 shows the relationship between untreated, BADGE and DMSO treated controls after 5 days of respective treatments. BADGE treatment 126 significantly decreases osteocalcin mRNA levels, with no change in the expression of aP2, PPARy2 and runx-2. DMSO treatment significantly reduced aP2 mRNA level in treated compared with not treated controls, whereas levels of PPARv2, osteocalcin and runx-2 remained unchanged. 127 Discussion To test the role of vehicle (DMSO) on osteoblast and adipocyte gene profiles, we subjected control and diabetic mice to treatment with DMSO. We found that DMSO treatment reduced the fold increase In expression of adipocyte markers. In non treated diabetic mice aP2 mRNA levels are 5 fold increased when compared with non treated controls. DMSO treated diabetic mice showed a 3 fold increase in aP2 expression when compared with DMSO treated controls. However, BADGE treatment was able to reduce aP2 mRNA level expression to less than one fold difference when compared with BADGE treated controls, thus placing BADGE as the most effective aP2 mRNA level lowering drug when compared with DMSO. Interestingly, 5 day DMSO treatment had an inhibitory effect on aP2 expression in DMSO treated controls when compared with non treated controls. This in part could be the reason for the 3 fold difference between DMSO treated controls and diabetics. We previously reported that increase in bone marrow adiposity is associated with increase in mRNA levels of PPARy2. The fold increase for PPARv2 was always more modest than that for aP2 and so it was in this experiment. Non treated diabetic mice showed a 60% increase in PPARy2 mRNA levels. Interestingly, both BADGE and DMSO treatments reduced PPARv2 mRNA levels in treated diabetic mice to their respective control levels, suggesting an inhibitory effect of DMSO on adipocyte differentiation. This is consistent with studies by Wang et al., which demonstrated that DMSO exercises 128 inhibitory effects in multiple steps of adipocyte differentiation, including the predifferentiation growth arrest state and terminal step of differentiation (6). In contrast, DMSO treatment has little or no effect on osteoblasts gene expression. Both untreated and DMSO treated diabetic animals exhibited significant decrease in osteocalcin gene expression when compared to respective controls. Interestingly, BADGE treated mice did not show a difference between diabetic and control groups regarding osteocalcin mRNA levels. However, our data demonstrates that BADGE blocks diabetes-induced osteocalcin suppression. It also appears that DMSO is responsible for suppressed basal osteocalcin levels since levels are down in both BADGE and DMSO treated controls. As previously reported by us, runx-2 mRNA level is significantly decreased at 5 days of confirmed diabetes when compared with controls. BADGE and DMSO treated mice did not show significant differences in runx-2 expression between treated controls and diabetic animals, however the control animals showed significant variability. We did observe a trend toward runx2 suppression in diabetic DMSO treated animals compared to their corresponding controls. BADGE treated diabetic animals, however, showed the least amount of runx 2 suppression (similar to our findings with osteocalcin expression), suggesting that BADGE itself may help to prevent diabetes induced suppression of the osteoblast phenotype. Woodbury et al., reported that bone marrow stromal cells (MSC), undergo rapid and robust transformation into neuron-like phenotypes in vitro following 129 treatment with chemical induction medium including dimethylsulfoxide (DMSO) (7). Neuhuber et al., demonstrated that upon addition to bone marrow stromal cells, DMSO causes a disruption of the actin cytoskeleton, retraction of the cytoplasm and formation of long processes that strikingly resemble neurites, but with no motility and no further elaboration during time-lapse studies (8). In summary, we report that previously described adipose-inhibitory effects of BADGE treatment in diabetic mice can be in part due to dimethylsulfoxide (DMSO). Future studies using different PPARV antagonists and different vehicles will contribute to a better understanding of the diabetic induced adiposity and its role in diabetic bone loss. 130 References 1. Herrero S, Calvo OM, Garcia-Moreno C, Martin E, San Roman Jl, Martin M, Garcia-Talavera JR, Calvo JJ, del Pino-Montes J 1998 Low bone density with normal bone turnover in ovariectomized and streptozotocin- induced diabetic rats. Calcif Tissue Int 622260-265 2. Verhaeghe J, van Herck E, Visser WJ, Suiker AM, Thomasset M, Einhorn TA, Faierman E, Bouillon R 1990 Bone and mineral metabolism in BB rats with long-term diabetes. Decreased bone turnover and osteoporosis. Diabetes 39:477-482 3. Bouillon R, Bex M, Van Herck E, Laureys J, Dooms L, Lesaffre E, Ravussin E 1995 Influence of age, sex, and insulin on osteoblast function: osteoblast dysfunction in diabetes mellitus. J Clin Endocrinol Metab 80:1194-1202 4. Wright HM, Clish CB, Mikami T, Hauser S, Yanagi K, Hiramatsu R, Serhan CN, Spiegelman BM 2000 A synthetic antagonist for the peroxisome proliferator-activated receptor gamma inhibits adipocyte differentiation. J Biol Chem 275:1873-1877 5. Cuzzocrea S, Pisano B, Dugo L, lanaro A, Maffia P, Patel NS, Di Paola R, Ialenti A, Genovese T, Chatterjee PK, Di Rosa M, Caputi AP, Thiemermann C 2004 Rosiglitazone, a ligand of the peroxisome proliferator-activated receptor-gamma, reduces acute inflammation. Eur J Pharmacol 483279-93 6. Wang H, Scott RE 1993 Inhibition of distinct steps in the adipocyte differentiation pathway in 3T3 T mesenchymal stem cells by dimethyl sulphoxide (DMSO). Cell Prolif 26:55-66 7. Woodbury D, Schwarz EJ, Prockop DJ, Black lB 2000 Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 612364-370 8. Neuhuber B, Gallo G, Howard L, Kostura L, Mackay A, Fischer I 2004 Reevaluation of in vitro differentiation protocols for bone marrow stromal cells: disruption of actin cytoskeleton induces rapid morphological changes and mimics neuronal phenotype. J Neurosci Res 772192-204 131 Figure Legends Figure 15. Diabetic bone response to BADGE and DMSO treatment. Total RNA was extracted from tibia isolated from non treated and BADGE or DMSO treated control (C) and diabetic (D) mice at 5 days after the confirmation of diabetes. Levels of PPARv2 and aP2 mRNAs and osteocalcin and runx-2 were determined by real-time RT-PCR and are expressed relative to cyclophilin. Values represent averages obtained from 3-5 mice per condition +/- SE. * p<0.05. Figure 16. Bone effects of BADGE and DMSO treatment in control mice. Total RNA was extracted from tibia isolated from control (C) mice after 5 days of no treatment or treatment with BADGE or DMSO. Levels of PPARv2 and aP2 mRNAs and osteocalcin and runx-2 were determined by real-time RT-PCR and are expressed relative to cyclophilin. Values represent averages obtained from 3-5 mice per condition +/- SE. * p<0.05. 132 Figure 15. gLiL-iLi En * 3 H :1. lira 11 0., l 2. __ I 2 z 1.5 I 1.5] I 1 8' ‘ I I 0.31 V * ”'3‘ 1 * 1 film x 1 I C 30.5 a: o. . C 133 oc PPARyZ aP2 Runx-2 0.9 < 0.6 4 0.3 - Figure 16. t-—t BADGE B4 DMSO CHAPTER VI Bone responds immediately to streptozotocin-induced diabetes Sergiu Botolin1 and Laura R McCabe1 Michigan State University1 Departments of Physiology and Radiology1 Molecular Imaging Research Center1 East Lansing, MI 48824 135 Abstract Type I diabetes is associated with significant bone loss, bone marrow adiposity, and decreased expression of osteogenic genes and increased expression of adipogenic genes. Type I diabetic bone loss is thought to result from suppressed bone formation rather than increased resorption. All studies to date have examined the bone phenotype changes after confirmation of diabetes, which can be anywhere from 2 to greater than 7 weeks after the first injection of the diabetes inducing agent. To understand early events in the etiology of diabetes associated bone loss, we induced diabetes in BALB/c mice by streptozotocin injection and examined serum markers of bone metabolism and gene expression in tibias at 1, 3, 5, 7, 9, 11, and 17 days after the first injection. Within 3 days body mass, fat pad mass and muscle mass was significantly decreased in diabetic compared to control mice. Blood glucose levels were also significantly elevated at this time point. Decreased levels of serum markers of bone resorption and formation were evident 5 days post injection and were maintained throughout the time course, indicating that bone resorption is not increased at any point in the etiology of diabetic bone loss. Suppression of osteogenic genes (runx2 and osteocalcin) and induction of adipogenic genes (PPARy2 and aP2), changes that we have observed at later stages of diabetes (2-7 weeks post injection), were clearly and significantly evident 5 days after the first injection of streptozotocin. These findings indicate that responses by bone to diabetes occur immediately with the onset of diabetes. 136 Introduction Insulin-dependent diabetes mellitus (IDDM) is a complex disorder associated with multiple long term complications. It evolves with progression to nearly complete beta cell destruction and establishment of a hyperglycemic state. Unfortunately, at the time of clinical symptoms presentation, pancreatic B-cells are irreversibly and almost completely damaged. This makes it difficult to monitor and understand the early events and their timing in diabetic patients. Data on the timing of early changes is scarce and comes from experimental models of induced diabetes such as streptozotocin injected diabetic rodent. In this model, animals are injected daily with streptozotocin, for 5 consecutive days. Streptozotocin is a nitrosourea derived from Streptomyces achromogenes which causes pancreatic B-islet infiltaration of T cells and macrophages in rodents, similar to the histology of human type I pancreatic biopsies [1, 2]. Monitoring of the glycemic profile in fasted rats following streptozotocin (80 mg/kg. i.p.) injection demonstrates a rapid response with evidence of hyperglycemia and reduced plasma insulin levels within two hours [3]. This response is followed by a drop in blood glucose levels and high plasma insulin levels at 6 hours, but ultimately a progressive state of chronic hyperglycemia ensues. It is known that on of the complications of IDDM is bone loss and ongoing population studies continue to support these observations in humans [4-13]. Similarly rodent IDDM models also exhibit significant bone loss [14-22]. Examination of bone metabolism markers in humans and rodent models of IDDM suggest that the bone loss is primarily due to a defect in bone formation rather 137 than resorption [17, 18, 23-25]. In fact, analyses of serum markers of bone resorption indicate that if anything, resorption is actually decreased in parallel with the decrease in osteoblast activity [18, 25, 26]. However, it remains unknown if osteoclast activation occurs early in the disease progression and contributes to the significant bone loss seen at later time points. The above findings support that IDDM promotes a true suppression of bone formation most likely through a reduction of osteoblast numbers and/or maturation. Previously we demonstrated that streptozotocin-induced diabetes mellitus in BaIb/C male mice is associated with bone loss and suppression of serum osteocalcin and osteocalcin mRNA levels in bone [18]. In addition, visible changes in bone histology marked by increased marrow adiposity were observed and further confirmed by increased mRNA levels of markers of adipocyte maturation, PPARyZ and aP2 [18]. The elevated expression of adipogenic markers and decreased expression of osteogenic markers was apparent as early as 17 and 19 days after the first injection of streptozotocin [18, 27]. Considering the present literature on the timing of early changes in streptozotocin induced diabetes, and our previous data, we hypothesize that the decision for suppressed osteoblast phenotype and increased bone marrow adiposity in diabetes occurs at the immediate onset of the diabetic state. Our studies examining the immediate time course of changes within the bone and serum of streptozotocin-injected animals demonstrates that osteoclast activity is not increased at any time during the progression of diabetes and is actually significantly reduced by 5 days after the first streptozotocin injection. We also found a reduction in serum osteocalcin 138 and mRNA levels of osteogenic markers and an increase in adipocyte markers within 5 and 7 days after the first streptozotocin injection [18, 27]. These findings suggest that streptozotocin-induced changes in bone phenotype occur rapidly and in parallel with marked changes in metabolic status, marked by reduced body weight and fat pad mass and by elevations in serum glucose levels. 139 ‘1. Results Previously we demonstrated that streptozotocin-induced diabetes induces changes in body weight, serum parameters and bone histology and gene expression resulting in bone loss. We also reported that these changes are evident five days after confirmation of diabetes. Based on the protocol for diabetes induction: daily injections of streptozotocin for 5 days followed by a 7 day period prior to confirmation of diabetes, the day 5 time point in our studies is actually 17 days after the animals receive their first injection of streptozotocin. The marked changes that we observe at this time suggest that the events involved in the changes in bone and serum parameters must occur much earlier. To identify the events involved in streptozotocin-induced diabetic bone loss, we injected mice with streptozotocin and examined serum, muscle and bone parameters at 1, 3, 5, 7, 9, 11, and 17 days after the first streptozotocin or citrate buffer (vehicle control) injection. Figure 1 demonstrates that 3 days after the first injection of streptozotocin the mice lose a significant amount of weight, which is maximum five days post- injection. This indicates that animals lose their body mass early in the process and reach a state of equilibrium. Similarly, blood glucose levels are significantly increased 3 days after the first injection of streptozotocin and continue to rise over the course of diabetes induction (Figure 1). The immediate change in body mass was not associated with elevated serum free fatty acids (Figure 2), but was directly associated with the loss of femoral fat pad weight and size (Figure 2), which were significantly reduced 3 days following the first injection of 140 streptozotocin and reached maximum reduction in size at 5 days after injection. The muscle mass of the tibialis anterior and heart were also significantly reduced with the onset of diabetes (Figure 3), indicating an overall degeneration/reduction of mesenchymal derived tissues. Previously we demonstrated that at 17 days post-injection, animals exhibit increased expression of adipocyte markers and decreased expression of mature osteoblast markers. To determine when the onset of these changes first occur, we isolated RNA from the tibias of 8-11 mice per condition at 1, 3, 5, 7, 9, 11, and 17 days after the first streptozotocin or citrate buffer (vehicle control) injection. Figure 4 demonstrates that induction of diabetes significantly induces aP2 and PPARyZ mRNA levels at 5 and 7 days post-injection, respectively. This correlates with the elevation of blood glucose and other early events of diabetes that we observed. Next, we examined the onset of diabetes and its effects on serum parameters of bone metabolism and on levels of osteoblast gene expression in bone. Decreased serum PYD and osteocalcin levels indicate that both bone resorption and formation are suppressed immediately with the onset of diabetes, 5 days post-injection (Figure 5). Consistent with the suppression of serum bone formation markers, Figure 6 demonstrates that osteocalcin mRNA levels, a marker of mature osteoblasts, are significantly reduced in diabetic tibias 5 days post-injection. Unlike other adipocyte gene-, serum marker-, and tissue-related changes, however, there was a trend toward reduced osteocalcin mRNA levels within 24 hours of the first streptozotocin-injection. Similar changes were also 141 seen with runx2 mRNA levels. Specifically, runx2 expression was significantly suppressed in streptozotocin injected animals at 5 days post-injection and beyond, but was also significantly suppressed 24 hours after the first injection. This finding potentially implicates streptozotocin itself in the early modulation of gene expression in osteoblasts. 142 Discussion Type I diabetes is associated with significant bone loss [8, 18, 22], increased bone marrow adiposity [18] and increased fracture risk [9, 38]. The changes in bone phenotype are usually examined at 2 or 4 weeks post- confirmation of diabetes and in our previous study we looked as early as 5-7 days after the confirmation of diabetes, which amounts to 17 days after the first injection of streptozotocin [18, 27]. Here we utilized the induced suppression of pancreatic B-cell function by streptozotocin to allow an even earlier analysis of the time course of diabetic changes in bone. Our findings demonstrate that changes in body mass, blood glucose levels, fat and muscle mass and bone phenotype occur before or by 5 days after the first injection of streptozotocin. Pighin et al. [39] also report reduced fat pad mass in mice 6 days after injection of streptozotocin. The adipose tissue lipolysis that we see suggests that free fatty acids are being mobilized as an energy source during the onset of insulin deficiency [39, 40]. However, we were unable to detect an early increase in serum free fatty acids as has been reported by others [39]. But, by 17 days after the first streptozotocin injection we were able to see an increase and have previously reported elevated blood lipid levels at 17, 19, 26, 33, and 40 days after the first injection of streptozotocin (corresponding with days 5, 7, 14, 21 and 28 days after diabetes confirmation) [18, 27]. It may be that at early time points there is a rapid uptake of mobilized fats by tissues in our animal model. Also, we saw a rapid increase in blood glucose levels within 3 days of streptozotocin injection, in contrast to Pighin et al. [39] who found that 143 blood glucose levels were not significantly elevated until day 12. The metabolic differences between our studies, glucose and lipid levels in the blood, most likely stem from differences between mouse strains, C57BL/6J versus BALB/c, and their ability to compensate for early pancreatic B-cell destruction. To our knowledge, only a handful of studies have examined the early effects of streptozotocin-induced diabetes on gene expression. Wang et al. [41] report some of the earliest changes: a significant reduction of both GLUT2 protein and mRNA expression in pancreatic islets isolated from streptozotocin- injected mice 4 days after the first streptozotocin injection. In our studies, we identified significant changes in tibial mRNA profiles at 5 days after the first streptozotocin injection. The changes that we saw, decreased runx2 and osteocalcin mRNA levels and increased PPARy2 and aP2, were maintained out to day 17 and are consistent with differences we see at later stages (days 17, 19, 26, 33, and 40 days after the first injection of streptozotocin - corresponding to days 5, 7, 14, 21 and 28 days after diabetes confirmation) [18, 27]. This suggests that the immediate changes in mouse blood glucose levels and overall metabolic state may in turn dramatically, immediately, and chronically influence osteoblast and adipocyte maturation. Of additional interest is the suppression of runx2 24 hours after the initiation of diabetes by streptozotocin. This effect occurs prior to any observable hyperglycemia or weight loss. Potential mediators of runx2 suppression at this time include: 1) aspects of the immediate metabolic adaptation to reducing insulin levels, 2) a rise in systemic inflammatory cytokines 144 as a result of pancreatic inflammation and/or 3) the immediate hyperglycemia for a short period following streptozotocin, which subsequently followed by hypoglycemia as reported by West et al. [3]. The fact that the levels of runx2 mRNA return to control levels at day 3 suggests that indeed the suppression of runx2 at the early time point is mediated by something specific to the event of the first injection. However, by day 5, the suppression of mRNA levels of runx 2 and osteocalcin are evident and maintained throughout the extended time course of diabetes. In these studies, streptozotocin was used to induce diabetes and allow identification of the early onset of IDDM associated changes in bone phenotype, something that cannot be done in models of spontaneous IDDM occurrence. One cannot exclude that secondary effects of streptozotocin could contribute to our findings. However, studies in other models of diabetes including non-obese type I diabetic mice (NOD) [22] and in virally induced diabetic animals [23] demonstrate that chronic diabetes leads to bone loss marked by decreased bone formation. We have also found that NOD mice not only exhibit decreased bone formation but also increased marrow adiposity similar to our streptozotocin model (Botolin et al. manuscript in preparation), further confirming that the effects seen in the streptozotocin model reproduce those seen in spontaneous cases of IDDM. An additional question that has been a thorn in studying bone metabolism under IDDM conditions has been “does bone resorption/osteoclast activation occur during the early onset of diabetes prior to the time points that have been 145 studied In the literature. To answer this question we monitored serum PYD levels and found that they were significantly decreased in diabetic animals 5 days after the first streptozotocin injection and remained reduced through day 17. Our past studies also demonstrate reduced osteoclast activity at day 17 (day 5 after confirmation of diabetes) and at later time points extending to day 40 (day 28 after confirmation of diabetes) [18]. Thus, at all time points studied (except 3 days after the first streptozotocin injection) bone resorption is suppressed along with formation. In summary, in the streptozotocin-induced diabetes mouse model of bone loss, which is marked by decreased bone formation, changes in bone phenotype/gene expression are evident within 5 days of the first injection of streptozotocin. These changes include suppression of osteogenic genes and induction of adipogenic genes. Understanding the factors present at these early stages of diabetes development will allow us to further understand the cause of diabetic bone loss. 146 Acknowledgements This work was funded by a grant from NIH (DK061184) to LRM. 147 References 10. 11. 12. Maksimovic-lvanic, D., et al., Down-regulation of multiple low dose streptozotocin-induced diabetes by mycophenolate mofetil. Clin Exp Immunol, 2002. 129(2): p. 214-23. 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Trogan, E., et al., Laser capture microdissection analysis of gene expression in macrophages from atherosclerotic lesions of apolipoprotein E-deficient mice. Proc Natl Acad Sci U S A, 2002. 99(4): p. 2234-9. Miao, J., et al., Elevated hip fracture risk in type 1 diabetic patients: a population-based cohort study in Sweden. Diabetes Care, 2005. 28(12): p. 2850-5. Pighin, D., et al., Role of lipids in the early developmental stages of experimental immune diabetes induced by multiple low-dose streptozotocin. J Appl Physiol, 2005. 98(3): p. 1064-9. Solomon, S.S., et al., Hormonal control of lipolysis in perifused adipocytes from diabetic rats. Endocrinology, 1985. 117(4): p. 1350-4. Wang, Z. and H. Gleichmann, GLUT2 in pancreatic islets: crucial target molecule in diabetes induced with multiple low doses of streptozotocin in mice. Diabetes, 1998. 47(1): p. 50-6. 151 Figure Legends Figure 17. STZ injections induce hyperglycemia and body weight loss 3 days after the first injection. Type I diabetes was induced in Balb/c mice by 5 daily streptozotocin-injection. Body weight and serum parameters were monitored at 1, 3, 5, 7, 9, 11, and 17 days after the first Streptozotocin or Citrate injection. Citrate Buffer (controls) injected mice are squares and Streptozotocin injected mice are triangle. Values represent averages obtained from 8-11 mice per condition +/- SE. * p<0.05. Figure 18. Early loss of femoral fat depots is associated with normal serum free fatty acids levels. Serum free Fatty Acid concentration was determined in serum from mice injected with Citrate buffer (controls) and Streptozotocin at 1, 3, 5, 7, 9, 11, and 17 days after the first Streptozotocin or Citrate buffer injection. Femoral Fat depots were dissected from surrounding tissues and were weighted and photograph. Values represent averages obtained from 8-11 mice per condition +/- SE. * p<0.05. Figure 19. Muscle tissue weight loss in result to STZ injections. Tibialis Anterior muscles were dissected from the insertion points and weighted. Hearts were dissected from the central vasculature and weighted. Both muscle tissues were taken from mice injected with Citrate buffer (controls) and Streptozotocin at 1, 5, 11, and 17 days after the first Streptozotocin or Citrate 152 . am , buffer injection. Values represent averages obtained from 8-11 mice per condition +/- SE. * p<0.05. Figure 20. Early increase in adipocyte markers PPARv2 and aP2 in bones from STZ injected mice. Total RNA was extracted from tibia isolated from Citrate buffer (controls) and Streptozotocin injected mice at 1, 3, 5, 7, 9, 11, and 17 days after the first Streptozotocin or Citrate buffer injection. Levels of PPARy2 and aP2 mRNAs were determined by realtime RT-PCR and are expressed relative to cyclophilin. Values are averages +/- SE obtained from 8-11 mice per condition. *p<0.05. Figure 21. Serum osteoblast maturation and bone resorption markers are decreased in STZ injected animals 5 days post injection. Serum Osteocalcin levels were determined in serum from mice injected with Citrate buffer (controls) and Streptozotocin at 1, 3, 5, 7, 9, 11, and 17 days after the first Streptozotocin or Citrate buffer injection. Serum PYD levels were determined in serum from mice injected with Citrate buffer (controls) and Streptozotocin at 1, 5, 11, and 17 days after the first Streptozotocin or Citrate buffer injection. Figure 22. Immediate decrease in Runx-2 mRNA is followed by a decrease in osteocalcin mRNA in STZ injected animals. Total RNA was extracted from tibia isolated from Citrate buffer (controls) and Streptozotocin injected mice at 1, 3, 5, 7, 9, 11, and 17 days after the first Streptozotocin or 153 Citrate buffer injection. Levels of Osteocalcin and RUNX-2 mRNAs were determined by realtime RT-PCR and are expressed relative to cyclophilin. Values are averages +/- SE obtained from 8-11 mice per condition. *p<0.05. 154 “—Tms'm—_ -m'n.'u-7 Figure 17. ._x 0 Body mass (9) mama 0100100 44 *1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I .- U" .3 O Glucose (mmol) 001 4 7 .fir i 7 1de 3dpi 5de 7dpi 9dpi 11de 155 .3 a) I Serum FFA (mEq/L) .9 O ,—‘ #3 a: N .0 .5 O) Femoral fat pad mass (9) Figure 18. L. N l b on . '9 17dpi 156 17dpi Figure 19. ’39.. . infill-(fl In + 6 0. 0 m m. m m. m. 0 0 0 0 O .9mmmE 6:35.. 9.39:. O 4.... 0.25 . * .x. 2 m. 1. .._. 0 M 0. M .932: :8... 17de 11dpi 5dpi 1dpi 157 aP2] Cyclophilin PPARgZ I Cyclophilin Figure 20. a]: 34 2 1 a: * * 1 _ \ o =I< 21 * =l< a]: 1- a. ‘7 l o . . . 1dpi 3dpi 5dpi 7dpi 9dpi 11dpi 17dpi 158 y "1.. Serum OC Serum PYD 600 - 500 « 400 ~ 300 ~ 200 ~ 100 ~ Figure 21. 1.1.. 1dpi 3dpi 5dpi 7dpi 9de 11dpi 159 17dpi CC I Cyclophilin Figure 22. Runx2 I Cyclophilin A 1 =l< * a 31‘ T * r * I * 1dpi 3dpi 5dpi 7dpi 9dpi 11dpi 17dpi 160 CHAPTER VII Chronic hyperglycemia modulates osteoblast gene expression through osmotic and non-osmotic pathways Sergiu Botolin1 and Laura R McCabe1 Michigan State University1 Departments of Physiology and Radiology1 Molecular Imaging Research Center1 East Lansing, MI 48824 161 Abstract lnsulin dependent diabetes mellitus (IDDM; type I) is a chronic disease stemming from little or no insulin production and elevated blood glucose levels. IDDM is associated with osteoporosis and increased fracture rates. The mechanisms underlying IDDM associated bone loss are not known. Previously we demonstrated that osteoblasts exhibit a response to acute (1 and 24 hours) hyperglycemia and hyperosmolality. Here we examined the influence of chronic hyperglycemia (30 mM) and its associated hyperosmolality on osteoblast phenotype. Our findings demonstrate that osteoblasts respond to chronic hyperglycemia through modulated gene expression. Specifically, chronic hyperglycemia increases alkaline phosphatase activity and expression and decreases osteocalcin, MMP-13, VEGF and GAPDH expression. Of these genes, only MMP-13 mRNA levels exhibit a similar suppression in response to hyperosmotic conditions (mannitol treatment). Acute hyperglycemia for a 48-hour period was also capable of inducing alkaline phosphatase and suppressing osteocalcin, MMP-13, VEGF and GAPDH expression in differentiated osteoblasts. This suggests that acute responses in differentiated cells are maintained chronically. In addition, hyperglycemic and hyperosmotic conditions increased PPARy2 expression, although this increase reached significance only in 21 day chronic glucose treated cultures. Given that osteocalcin is suppressed and PPARy2 expression is increased in type I diabetic mouse model bones, these 162 -g-J'T I.- findings suggest that diabetes-associated hyperglycemia may modulate osteoblast gene expression, function and bone formation and thereby contribute to type I diabetic bone loss. 163 Introduction Insulin dependent diabetes mellitus (IDDM; type I) is a chronic disease stemming from little or no insulin production and elevated blood glucose levels. IDDM is associated with many complications including neuropathy, nephropathy, retinopathy and decreased bone mineral density. While the later complication has not received much attention, it is clear that IDDM is associated with decreased bone mass (1-10), osteoporosis (4, 11-13) and increased fracture rates (14-17). A recent clinical study found that 67% of male and 57% of female patients with IDDM suffered from osteopenia of the femoral neck and/or lumbar spine (4) and 14-20% of IDDM patients age 20-56 met the criteria for more extensive bone loss, osteoporosis (4, 13). Clinical and basic research indicates that modulation of osteoblast rather than osteoclast activity is involved in diabetic bone loss. For example, in the streptozotocin induced mouse model of IDDM, we demonstrated through serum marker analyses and histology that osteoclast number and activity is not influenced by IDDM (18) similar to reports by others (19, 20). In contrast, osteoblast function/maturation is suppressed and marrow adiposity is increased in IDDM mice. Studies by Bouillon et al. (21) and others support an osteoblast maturation defect based on serum levels of early osteoblast markers (peptide of procollagen, PICP) which remain normal while later stage markers such as osteocalcin are decreased in diabetes. Similarly, we also found a decrease in serum levels of osteocalcin and osteocalcin mRNA levels in bones of IDDM mice. Our additional finding of increased expression of PPARy2, aP2 and resistin in 164 streptozotocin-induced diabetic mice corresponded with increased adipocyte maturation and suggested the possibility that IDDM may also affect lineage selection of mesenchymal stem cells, leading to adipocyte rather than osteoblast maturation. The mechanism accounting for IDDM associated bone loss is unknown. Many studies focus on the anabolic role of insulin and IGF-1 (12, 22-26). It is clear that insulin is involved in the complications of diabetes based on the finding that insulin treatment can reduce diabetic complications (10, 27, 28). However, whether this is a direct effect or secondary to insulin’s restoration of other serum factors is not known. One major consequence of insulin treatment is maintenance of euglycemia which in itself could be important for avoiding complications (29). Reports in other systems and in bone have suggested that elevated glucose can contribute to diabetic complications through a variety of mechanism including increasing reactive oxygen species (30, 31), polyol-pathway activity (32-34), protein kinase C activity (35-38), and/or nonenzymatic glycosylation of key proteins such as collagen I or IGF-1 (25, 29, 39-42). In addition, cells can also respond to hyperglycemia through an osmotic response. Because osteoblasts express glucose transporters, GLUT1 and GLUT 3 (43, 44), with low Km (1-2 mM and <1 mM, respectively), glucose transport is maximal at euglycemic state (glucose concentration of 3-5.5 mM) so an increase in extracellular glucose could be an osmotic stress. During osmoadaptation to extracellular hyperosmotic conditions, virtually all cells undergo a volume change and shrink (45, 46). Adaptive responses 165 include modulation of membrane ion transporters (short-term) and/or metabolic pathways (long-term) that draw water back into the cell to partially restore cell volume and intracellular solute concentration (45, 47-65). Previously we demonstrated that acute (24 hour) hyperglycemia and its associated hyperosmolality are capable of modulating osteoblast signaling pathways, gene expression and phenotype (66, 67). Specifically, osteocalcin expression in day 14 osteoblasts was decreased, while expression of collagen I and c-jun was increased. Given that diabetes is a chronic disease associated with chronic extracellular glucose elevation, we asked the question “Does chronic hyperglycemia and or hyperosmolarity lead to chronic changes in osteoblast gene expression?” We focused on genes that we have demonstrated to be modulated in IDDM mice and we also compared our response to short-term treatments (48 hours) to distinguish between acute versus long-term responses, with the thought that perhaps chronic treatment leads to an adaptive response in osteoblasts that results in normalization of gene expression. Our results demonstrate that chronic hyperglycemia significantly influences osteoblast gene expression causing increased alkaline phosphatase expression and decreased osteocalcin, lVIMP-13, VEGF and GAPDH expression. Of these genes, only MMP-13 mRNA levels exhibit a similar suppression in response to hyperosmotic conditions (mannitol treatment). For most genes, effects were seen early on (day 5 or 14) and were maintained. Acute hyperglycemia treatment (48 hours) caused a similar modulation of gene expression but effects were predominantly seen 166 only in fully differentiated osteoblasts. The changes in gene expression suggest that later stages of osteoblast differentiation are suppressed by hyperglycemia and suggest a trend toward increased expression of early markers of adipocyte phenotype. In combination with the finding that osteoblast marker genes are suppressed in diabetic mouse bones (18), our in vitro findings suggest that diabetic hyperglycemia may contribute to the suppression of osteoblast differentiation and bone loss in diabetic mice. 167 Results Prior to beginning our in vitro studies we directly measured blood glucose levels and plasma osmolality in normal and diabetic Balb/c mice. As expected, diabetic animals exhibited elevated blood glucose levels of 300-500 mg/dl (18). In terms molar quantities, non-fasting control mice had glucose levels of 9.7 i 0.2 mM (levels higher than standard alpha-MEM cell culture medium, 5.5 mM) while diabetic mice had glucose levels of 39.2 i 3.5 mM; this corresponds to an average increase of 30 mM glucose. Similarly, diabetic mice exhibit an elevation in blood osmolality (335.8 1 3.3 mmol/kg) compared to controls (305.2 1 2.2 mmol/kg); this represents an increase of 30 i 5 mmol/kg (18). Based on these findings, osteoblasts were treated in vitro with 30 mM glucose to attain a final concentration of 35.5 mM glucose and a final osmolality of 322 mmol/kg (compared to 295 in control cultures). To control for the effects of glucose- associated hyperosmolarity, osteoblasts were treated with 30 mM mannitol to attain a final media concentration of glucose equal to control cultures (5.5 mM glucose) but with an osmolarity of 322 mOsm. First, we examined if chronic glucose treatment has a long-term influence on cell number, since changes in cell number could ultimately affect osteoblast phenotype and gene expression. DNA levels per tissue culture plate were determined at 7, 14, 21 or 28 days after plating. Total DNA levels (an indicator of cell number) did not significantly differ between control, glucose and mannitol treated cultures at day 7 (1.0 :I: 0.2, 1.1 1 0.1, 1.4 t 0.1, relative to day 7 controls, 168 respectively) but by day 14 (1.9 :I: 0.3, 1.7 :t 0.2, 1.7 1: 0.2 relative to day 7 controls, respectively) and 21 to 28 days (2.7 :I: 0.2, 2.2 :I: 0.2, 2.2 :I: 0.1) a gradual decrease in cell number was noted in glucose and mannitol treated cultures. This decrease did not reach statistical significance. Visual examination of the cells under bright field did not demonstrate any remarkable differences in osteoblast morphology or confluency under hyperglycemic and/or hyperosmotic conditions (Figure 1). However, examination of histological staining demonstrated that alkaline phosphatase activity was increased in glucose treated cultures (Figure 1). Staining for mineralization did not appear to differ between conditions (data not shown). Next, analyses of gene expression were carried out. Four subsets of genes were examined and included those associated with early osteoblast maturation, Hate osteoblast maturation, adipocyte maturation and metabolic stress/hypoxia. For each set of genes, chronic and 48 hour treatments were examined. Cyclophilin mRNA levels were not significantly modulated by glucose or mannitol treatments and were used as a housekeeping control gene. Figure 2 depicts the time course of early osteoblast maturation markers (runx2, alkaline phosphatase and collagen l mRNA levels) under control, chronic glucose (30 mM) or chronic mannitol (30 mM) treatments. While runx 2 mRNA levels were elevated at 7, 14 and 29 days under hyperglycemic conditions, the change was not significant compared to untreated osteoblasts. However, the marked induction of alkaline phosphatase expression by hyperglycemia was significant at all time points and was consistent with the observed elevation in alkaline 169 phosphatase staining (Figure 1). This effect was not osmotic in nature since chronic treatment with mannitol did not affect alkaline phosphatase expression. Expression of collagen I was not markedly modulated by any treatment, but at day 29 glucose its levels reached statistical significance in glucose compared to control cultures. To determine if these changes with long-term treatment could be contributed to a short-term response to the continual glucose addition, we examined if alkaline phosphatase or collagen I mRNA levels were altered by 48 hours of glucose or mannitol treatment. The short-term responses were variable and did not exactly follow long-term responses (Figure 3). Again, collagen l expression was not markedly modulated by any treatment, but its levels were modestly but significantly affected in day 7 mannitol treated cells. Alkaline phosphatase expression was decreased by short-term treatment at 14 days (unlike any response seen in chronic treatment), but tended to increase under short-term hyperglycemic conditions at 21 days and was significantly increased at 29 days. As expected, examination of late stage markers of osteoblast differentiation, osteocalcin and collagenase 3/MMP-13 (68, 69), demonstrated an increase in expression in control cells with increasing time in culture. However under hyperglycemic conditions osteocalcin and MMP-13 mRNA levels were decreased by hyperglycemia; this is in contrast to hyperglycemia-induced elevation of early stage osteoblast maturation (Figure 2). Specifically, at 14 and 21 days post-seeding osteocalcin expression was decreased in glucose treated cultures and by 29 days the decrease was statistically significant. Mannitol 170 treated cells also showed a trend toward decreased osteocalcin expression at days 14 and 21, however the difference was never statistically significant. In contrast, MMP-13 expression was significantly suppressed by both glucose and mannitol treatments at 7, 14, 21, and 29 days, suggesting that chronic hyperglycemia-associated hyperosmotic stress is responsible for the suppression in MMP-13 expression in osteoblasts. When short-term (48 hour) responses were examined, both osteocalcin and mannitol mRNA levels were significantly suppressed by glucose treatment in day 21 and 29 cells. This reflects the suppression seen with chronic treatment. However, acute mannitol treatment did not affect MMP-13 mRNA levels, but did suppress osteocalcin mRNA levels in day 29 cells. Also, similar to collagen I regulation, a modest but significant induction in MMP-13 expression was seen in mannitol treated day 7 cells. Previously we have shown that diabetes type I is associated with increased bone PPARy2 mRNA levels and marrow adiposity (18). Therefore, we next asked the question: can chronic hyperglycemia or hyperosmotic stress directly influence PPARy2 mRNA expression, a marker of early stage adipocyte differentiation? Figure 6 demonstrates that hyperglycemia and its associated hyperosmotic stress can increase mRNA levels of PPARy2. Specifically, chronic glucose treatment and mannitol treatment for 14 and 21 days lead to nearly a 2- fold induction in PPARy2 mRNA levels. However, the response was variable and reached statistical significance only in chronically glucose treated day 21 cultures; lipid staining at this time point did not differ between conditions (data not 171 shown). The PPARy2 response to 48 hour glucose or mannitol treatment did not demonstrate a significant difference between conditions (data not shown). Given that diabetes type I is associated with decreased circulation, we next examined the expression of genes associated with vascularization and hypoxic Iglycolytic responses: vascular endothelial growth factor (VEGF) and glyceraldehydes phosphate dehydrogenase (GAPDH). Figure 7 demonstrates that basal expression of VEGF and GAPDH is Increased with osteoblast differentiation and that chronic hyperglycemia is effective at suppressing the expression of both of these genes at days 21 and 29. Chronic hyperosmolarity did not cause a significant suppression, but did trend toward decreasing VEGF and GAPDH and gave an intermediate response between control and glucose treated cells. Acute hyperglycemic treatment at day 29 was also effective at suppressing both VEGF and GAPDH mRNA levels (Figure 8). 172 Discussion Elevated blood glucose level is a key characteristic of type I diabetes. Chronic hyperglycemia itself is thought to contribute to diabetic complications. To examine the influence of chronic hyperglycemia and its associated hyperosmotic stress on osteoblast phenotype, mouse osteoblasts were cultured in vitro under pathologic conditions of hyperglycemia as seen in streptozotocin- induced type I diabetic mice. In addition, cells were exposed to pathologic hyperosmolality at levels also seen in diabetic mice. This approach allows the distinction between responses directly related to hyperglycemia and hyperosmolality versus those that are secondary to other diabetes-modulated factors (serum hormones and nutrients) or involve other cell types (immune, hematopoetic, neural, osteocyte, etc...). Our findings demonstrate that indeed osteoblasts respond to chronic hyperglycemia through modulated gene expression. Specifically, chronic hyperglycemia increases alkaline phosphatase activity and expression and decreases osteocalcin, MMP-13, VEGF and GAPDH expression. Changes were apparent by day 7 for alkaline phosphatase and MMP-13 and were maintained throughout the rest of the time course. Of these genes, only MMP-13 mRNA levels exhibit a similar suppression in response to hyperosmolarity (mannitol treatment). Examination of 48-hour treatment indicates that induction of alkaline phosphatase and suppression of osteocalcin, MMP-‘l3, VEGF and GAPDH can be mediated acutely in day 29 osteoblasts (and for osteocalcin and MMP-13, also in day 21 osteoblasts). In addition, similar to in 173 vivo gene analyses (18), osteoblasts exhibit a trend toward increased PPARy2 expression, suggesting a potential contribution by hyperglycemic and hyperosmotic conditions toward this phenomenon. Interestingly, chronic treatment tended to influence osteoblast gene expression at significantly earlier time points compared to acute treatments. For example, acute hyperglycemia suppresses osteocalcin and MMP-13 in day 21 and 29 cells, but not in day 7 or 14 cells (as seen for MMP-13 expression in chronically treated osteoblasts). Alkaline phosphatase, GAPDH and VEGF changes are only seen with acute treatment of day 29 cells, despite chronic treatment causing a significant changes day 7, 14, 21 osteoblasts, respectively. These findings suggest that chronic elevation of blood glucose levels in vivo could impact osteoblasts at all stages of maturation, whereas periodic bouts of hyperglycemia will predominantly influence mature osteoblast function. Similar to our previous studies examining 1- and 24-hour glucose treatments (66, 67), we also found that longer incubations (48-hour and chronic 29 day treatments) suppress osteocalcin mRNA levels. Unlike our previous studies, long-term suppression of osteocalcin expression was not hyperosmolality dependent. In fact, modulation of alkaline phosphatase, VEGF and GAPDH mRNA levels by hyperglycemia was also not associated hyperosmolality. This suggests that osteoblasts exhibit at least two hyperglycemia responses: an early acute response that is hyperosmolality driven and a late acute and chronic response that is predominantly hyperglycemia driven. These findings are 174 consistent with those of Balint et al. (70) who reported that chronic hyperglycemia but not hyperosmolality influences calcium uptake in osteoblast cultures in vivo. Mechanisms accounting for long-term responses to hyperglycemia include activation of PKC signaling pathway, nonenzymatic glycosylation, modulation of redox state, increased polyol pathway activity, increased glucose metabolism (29, 32-42). The later possibility is of interest since it is related to an observed decrease in medium pH (ranging from 7.4 to 7.1), 48 hours after hyperglycemic treatment. The drop did not occur in mannitol treated cultures, which retained a pH of 7.4 through out the experiment. An increase in glucose metabolism would contribute to the suppression of pH by increasing extracellular lactic acid production or another byproduct of glucose metabolism. Previous studies have demonstrated that osteoblasts express acid-sensitive channels (71, 72) and respond to decreased extracellular pH by decreasing mineralization and gene expression (73). However, acid pH is shown to suppress alkaline phosphatase mRNA levels while we see alkaline phosphatase mRNA levels increase in glucose treated cells. This suggests that extracellular pH changes may contribute to some but not all of the changes that we see in osteoblast gene expression under high glucose conditions. Reduced hypoxia inducible factor (HIF) activity may also contribute to suppression of VEGF and GAPDH by chronic elevated glucose conditions. Hypoxia and its corresponding induction of HIFs are known to upregulate VEGF and GAPDH (74-77). Recent studies suggest that hyperglycemia and hyperosmolality can reduce HIF transcriptional activity and hypoxia 175 responsiveness in primary dermal fibroblasts and endothelial cells (78). A similar response may be occurring in osteoblasts, and is in the process of being tested. While elevated extracellular glucose plays a key role in osteoblast gene regulation, MMP-13 also responds significantly to elevated extracellular mannitol (hyperosmolality) at all time points studied (days 7, 14, 21, 28). This suggests that under chronic hyperglycemia conditions osteoblasts are actively mediating a hyperosmotic response that modulates expression of a subset of genes. Other cells, such as mesangial and endothelial, also mount an osmotic response to pathologic elevations (22 mM) in extracellular glucose (79). In addition, human retinal corneal epithelial cells exhibit changes in WIMP-13 (as well as other MMPs) expression in response to hyperosmolar conditions (80). However the retinal osmotic-response causes an induction of MMP-13 whereas we see a suppression of MMP-13. These differences may stem from differences in specific transcription factors present in the two different cell types, retinal epithelial versus osteoblast. For example runx2 is expressed in osteoblasts and binds the MMP-13 promoter (along with ubiquitous AP-1 members) to regulate its transcription (81 ). Taken together, we hypothesize that osteoblasts exhibit immediate/early acute changes in gene expression in response to hyperglycemia that are predominantly driven by hyperosmolality. During this period osteoblasts undergo immediate volumetric changes (cell shrinking) induced by hyperglycemia- associated hyperosmolality. With longer incubations, osteoblasts develop adaptive measures to compensate for the hyperosmotic stress, which no longer 176 influence expression of osteoblast marker genes, except for a subset that includes MMP-13. Under these conditions, elevated levels of glucose and its consequences (other than hyperosmolality) are responsible for changes in osteoblast gene expression. The changes that we observed, suppression of markers of late stage osteoblast differentiation, in combination with the finding that osteoblast marker genes are suppressed in diabetic mouse bones (18), suggest that diabetic hyperglycemia may contribute to the suppression of osteoblast differentiation and bone loss in diabetic mice. 177 Acknowledgements We thank Regina Irwin for her technical assistance and insightful suggestions. 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Kidney Int 46:105-12 Li DO, Chen Z, Song XJ, Luo L, Pflugfelder SC 2004 Stimulation of matrix metalloproteinases by hyperosmolarity via a JNK pathway in human corneal epithelial cells. Invest Ophthalmol Vis Sci 45:4302-11 Selvamurugan N, Chou WY, Pearman AT, Pulumati MR, Partridge NC 1998 Parathyroid hormone regulates the rat collagenase-3 promoter in osteoblastic cells through the cooperative interaction of the activator protein-1 site and the runt domain binding sequence. J Biol Chem 273:10647-57 185 Figure Legends Figure 23. Osteoblast morphology and number does not change under chronic hyperglycemia, but alkaline phosphatase activity is increased. Mouse osteoblasts (MC3T3-E1 cells) were treated with 30 mM glucose or mannitol or remained untreated (control). Sugars were added with each feeding. Shown is a brightfield image of confluent cultures taken at day 14 and a representative photo of alkaline phosphatase activity staining. Figure 24. Expression of early and mid stage markers of osteoblast differentiation is modulated by chronic hyperglycemia. Osteoblasts were chronically treated with 30 mM glucose (triangles) 0r mannitol (diamonds) or were maintained under standard 5.5 mM glucose conditions (control, squares). Cells were harvested at 7, 14, 21 or 29 days and expression of runx2, alkaline phosphatase and collagen l was determined and expressed relative to cyclophilin (housekeeping gene) levels. Each point represents an average of 3 separate experiments :1: SE, *p<0.05. Figure 25. Expression of early and mid stage markers of osteoblast differentiation is modulated by 48-hour hyperglycemic conditions. Osteoblasts were treated for 48 hours with 30 mM glucose (black bars) or mannitol (grey bars) or were maintained under standard 5.5 mM glucose conditions (control, white bars). Cells were harvested at 7, 14, 21 or 29 days and expression of alkaline phosphatase and collagen I was determined and 186 expressed relative to cyclophilin (housekeeping gene) levels. Each point represents an average of 3 separate experiments :t SE, *p<0.05. Figure 26. Expression of late stage markers of osteoblast differentiation is modulated by chronic hyperglycemia and, in the case of MMP-13, hyperosmolarity. Osteoblasts were chronically treated with 30 mM glucose (triangles) or mannitol (diamonds) or were maintained under standard 5.5 mM glucose conditions (control, squares). Cells were harvested at 7, 14, 21 or 29 days and expression of osteocalcin and MMP-13 was determined and expressed relative to cyclophilin (housekeeping gene) levels. Each point represents an average of 3 separate experiments 1: SE, *p<0.05. Figure 27. Expression of late stage markers of osteoblast differentiation is modulated by 48-hour hyperglycemic conditions. Osteoblasts were treated for 48 hours with 30 mM glucose (black bars) or mannitol (grey bars) or were maintained under standard 5.5 mM glucose conditions (control, white bars). Cells were harvested at 7, 14, 21 or 29 days and expression of osteocalcin and MMP-13 was determined and expressed relative to cyclophilin (housekeeping gene) levels. Each point represents an average of 3 separate experiments 1 SE, *p<0.05. 187 Figure 28. Expression of adipocyte-phenotype marker, PPARv2, is increased under chronic hyperglycemic conditions. Osteoblasts were chronically treated with 30 mM glucose (triangles) or mannitol (diamonds) or were maintained under standard 5.5 mM glucose conditions (control, squares). Cells were harvested at 7, 14, 21 or 29 days and PPARvZ expression was determined and expressed relative to cyclophilin (housekeeping gene) levels. Each point represents an average of 3 separate experiments 1 SE, *p<0.05. Figure 29. Expression of VEGF and GAPDH is modulated by chronic hyperglycemia. Osteoblasts were chronically treated with 30 mM glucose (triangles) or mannitol (diamonds) or were maintained under standard 5.5 mM glucose conditions (control, squares). Cells were harvested at 7, 14, 21 or 29 days and expression of VEGF and GAPDH was determined and expressed relative to cyclophilin (housekeeping gene) levels. Each point represents an average of 3 separate experiments 1 SE, *p<0.05. Figure 30. Expression of late VEGF and GAPDH in differentiated osteoblasts is reduced under 48-hour hyperglycemic conditions. Osteoblasts were treated for 48 hours with 30 mM glucose (black bars) or mannitol (grey bars) or were maintained under standard 5.5 mM glucose conditions (control, white bars). Cells were harvested at 7, 14, 21 or 29 days and expression of VEGF and GAPDH was determined and expressed relative to cyclophilin (housekeeping gene) levels. Each point represents an average of 3 separate experiments 1 SE, *p<0.05. 188 Figure 23. :4 "'-'."$t-5".-‘-‘:4¢1-‘ . PM» 6 . .1 ”9.: “$35? _;.' \- . .-' control glucose mannitol 189 Figure 24. 5.28.85 5:56.03 5.28.3831 «5.3... 8i 3.2 _ _oo 29 21 14 Days 190 Figure 25. Col I Alk Phos N 5. 1 25 15. 9.2... 5 What": .. u 936 ...N 1 o 2 “mama ww. C 48 hour 48 hour 191 Figure 26. ...q1 000000 208642 1 5.2863 Eofioooumo 140 * o — o 0 o o o 0 o o o 4 3 2 1 5:; 2&2 ”Was: Days 192 Figure 27. MMP-13 Osteocalcin < q < 2 5555 a 2 II .1. * I. M C 1‘1 mxmuox. 1‘1 51... o mxmu w; 9.8 mm 98.3 48 hour 48 hour 193 x .x. a 2 e r U .m. F £28.05 NEE“. 29 21 14 Days 194 Figure 29. 5.28.2.2 5.28.9.2 mom> zonl< IL c'G'M‘ 48 hour GAPDH 11 ,1. G C 48 hour 196 CHAPTER VIII Discussion. Bone loss is a well accepted long-term complication of diabetes type I. Diabetes being a multisystemic chronic disease, makes difficult finding pathophysiological mechanisms for diabetic osteoporosis. Association of diabetes with such pathological findings as hypoinsulinemia, decreased IGF-1, hyperglycemia, increased blood osmolality, microvasculature alteration and nutritional disturbances make this task even more difficult. Streptozotocin induced diabetic mouse model is widely used in studying pathophysiology of diabetes and diabetic complications. Reported data regarding mechanisms of bone loss and the bone loss status itself in this mouse model is scarce and not enough to make conclusions and therapeutic management recommendations. Therefore, we set out to characterize bone phenotype, pathophysiological mechanisms and potential treatment tactics of diabetic bone loss in wild type, Balb/c streptozotocin-induced diabetic mice. As our first aim we characterized systemic and bone morphological and molecular changes in response to streptozotocin-induced diabetes in a mouse model. Osteocalcin and Runx-2 are decreased as early as 5 days of confirmed diabetes, indicating a suppression of osteoblasts differentiation and maturation. This was accompanied by a reduction of serum osteocalcin levels, which was 197 consistently reported in diabetic patients and experimental models (1 ),(2),(3), and by bone loss as determined by uCT and histology analyses. Diabetes-associated suppression of osteoblast maturation is also suggested in bone implant/healing studies done in rats, which indicate that diabetic animals exhibit decreased calvarial defect healing (4), and decreased bone formation around titanium (5) or hydroxyapatite bone implants (6). In marrow ablated mice, both runx2 and osteocalcin mRNA levels were found to be decreased in diabetic relative to control bones at 4 and 6 days after ablation, a time at which bone formation was dramatically induced in controls (7). Because the changes in bone density occurred within 1 month, we assessed the role of osteoclast activity in IDDM bone loss. Several resorption- indicative markers such as urine deoxypyridinoline (8) and serum tartrate resistant acid phosphatase (TRAP5) (9) where not changed in diabetic mice at any time point throughout the experiment. Moreover, at day 14 and 21 we observed a significant decrease in active serum TRAP5b levels. Our results are consistent with previous reports on bone resorptive activity in diabetes. Herrero et al., demonstrate decreased pyridinium crosslinks after 12 weeks of diabetes in rats (1). Similarly, Verhaeghe et al., demonstrate a decrease in urinary deoxypyridinoline secretion in rats diabetic for 8 weeks (2). Besides impaired bone status, we are the first to report an increase in adipose tissue in bone marrow. We found that the number of adipocytes and expression of adipocyte markers (PPARy2, resistin and aP2) is increased in diabetic type I bone. Selection of adipogenesis over osteoblastogenesis is a 198 common theme that has been reported in other conditions of bone loss, including age-related osteoporosis and disuse (10), (11),(12), (13). PPARy is the number one candidate for playing a role in this selection based on studies demonstrating that PPARy insufficiency results in the enhancement of osteogenesis and suppression of adipogenesis in mice (14) and on studies demonstrating that elevation of PPARy2 levels can promote adipogenesis in pluripotent mesenchymal cells in vitro (11). Activation of PPARy by elevated serum lipids or by synthetic agonists, such as rosiglitazone (used in the treatment of type II diabetes), was shown to stimulate bone marrow adiposity and bone loss through PPARy-dependent and PPARy-independent pathways (15), (16). Interestingly, we found an increase in serum triglycerides and free. fatty acids in diabetic mice, which can be in part responsible for elevated PPARyZ levels and subsequent increase in adipocyte proliferation and maturation. To test the role of PPARy2 in diabetic bone loss we set our goals to pharmacologically inhibit PPARyZ and investigate bone status in diabetic mice. Null mutation of PPARy in embryonic stem cells prevents troglitazone induced adipogenesis, but interestingly also enhanced ontogenesis (14). Heterozygous PPARy deficient mice exhibit enhanced bone mineral density that became prominent with aging; these animals exhibit normal osteoblast and osteoclast functions, but osteoblastogenesis in the bone marrow appears enhanced (14). Similarly, lipodystrophic PPARy'W‘”hyp mice, which congenitally lack PPARv in white adipose tissue, exhibit an increase in bone mineral density and bone area (17). Thus, PPARy2 levels have a dominant suppressive influence on osteogenesis. 199 Our results indicate that inhibition of PPARy2 with BADGE, which is a PPARy antagonist (18), effectively blocks type I diabetes-induced expression of PPARv2 target genes (PPARv2, aP2) and bone marrow adiposity but not bone loss. This is in contradiction with the above reports, suggesting that adiposity (and serum lipid levels) is not linked to bone density in type I diabetes. It is possible that we detected two independent diabetic-related mechanisms. The first Is characterized by an increase in adipocyte proliferation and differentiation and the second is manifested with reduction in osteoblasts maturation under diabetic conditions. Interestingly, when we set our goal to investigate the role of vehicle (DMSO) alone on diabetic bone loss, we found that it behaved very much similar to BADGE regarding adipocyte gene marker expression. Literature reviewed on this topic reveals that DMSO was previously reported to have an action on differentiation of different cell types (19), (20). Moreover, Wang et al., demonstrated that DMSO inhibits multiple steps of adipocyte differentiation, including the predifferentiation growth arrest state and terminal step of differentiation (21). In contrast, DMSO treatment has little or no effect on osteoblasts gene expression, suggesting that effects of BADGE on osteoblast gene expression most likely stem from BADGE itself. Given that we observed that some of the effects attributed to the PPARy antagonist BADGE could stem from partial agonist effects in osteoblasts and vehicle effects in adipocytes, the accuracy and specificity of PPARg2 inhibition by BADGE was compromised. Classically, PPARyZ is viewed to be activated by members of C/EBP family CEBPB and CEBP6 (22). Preliminary studies from our 200 laboratory indicate a biphasic response of CEBPB with the onset of diabetes. Specifically, CEBPB expression is increased immediately after the first streptozotocin injection, remains elevated for about 7 days, goes back to normal expression, and then is raised again at day 17 post injection. To mechanistically test the role of CEBPB upregulation in the development of diabetic bone loss, our laboratory is preparing for studies on CEBPB knockout mice. It would be very interesting to investigate adipose tissue development and bone quality status in these mice under normal conditions and streptozotocin induced diabetes. Based on the classical association between adipose and bone tissue, I would hypothesize that CEBPB knockout animals would not be susceptible to diabetic boneloss. Since we found alterations in adipogenic and osteogenic gene expression as early as day 5 after confirmation of diabetes, we set our goal to investigate the immediate response of adipose and bone markers to streptozotocin induced diabetes. Interestingly, differences in body mass, serum glucose concentration and femoral fat pads weight were evident since 3rd day post injection. Pighin et al., also report reduced fat pad mass and an increase in serum free fatty acids levels in mice 6 days after injection of streptozotocin (23). However, in our experiments, serum free fatty acids levels were increased starting with day 17 and were maintained increased at 17, 19, 26, 33, and 40 days after the first injection of streptozotocin (24). Tibialis anterior muscle and heart weights were significantly decreased in STZ injected animals 5 days after the first injection. At this time point tibial mRNA levels of aP2 showed a significant increase where 201 osteocalcin mRNA level was decreased. PPARy2 was detected to be increased starting with day 7 after the first injection. Serum osteocalcin followed tibial osteocalcin mRNA expression pattern, and was significantly decrease starting with day 5 post injection. Examination of serum levels of PYD (pyridinoline crosslinks), which is another quantitative measure of bone resorption, demonstrated a significant decrease in STZ injected animals starting with day 5 post injection. This again suggests an inhibitory effect of diabetes on bone resorptive activity. We were very surprised to detect these immediate changes following streptozotocin injections. Interestingly, literature review revealed that O’Brien et al., using the same injection protocol, detected apoptotic cells within the islets of Langerhans of treated animals from day 2 until day 17 post injection (25). Karabatas et al., investigated the effect of the transfer of mononuclear spleen cells (MSs) from STZ injected mice to normal syngeneic recipients and on cultured dispersed rat islet cells. They found that MSs from multiple low doses of streptozotocin (mId-SZ) mice were able to diminish insulin secretion when transferred to normal syngeneic recipients and presented anti-beta-cell immune aggression when cocultured with dispersed rat islet cells as early as day 4 after mld-SZ administration (26). Of special interest is the suppression of runx2 24 hours after the initiation of diabetes by streptozotocin. West et al., reported an immediate hyperglycemic and hypoinsulinemic status 2 hours after the injection with STZ. This was followed by hypoglycaemia associated with high levels of plasma insulin after six after the injection (27). It is also possible that the immediate reduction in Runx-2 202 mRNA levels were associated with either immediate metabolic adaptation to reducing insulin levels, or a rise in systemic inflammatory cytokines as a result of pancreatic inflammation. Runx-2 mRNA levels normalized at day 3 after the first injection but were decreased at day 5 and were maintained so throughout the time course, suggesting a special effect of the first injection of STZ on Runx-2 and bone in general. Since West et al. observed immediate significant fluctuations in serum insulin and glucose levels, and we report a decrease in runx-2 expression 24 hours after the first STZ injection, we can hypothesize that these two processes are linked together and are at the foundation of the very early diabetic bone changes. A future time course study examining gene expression and metabolic status during first 24 hours after STZ injection would further develop relationships between immediate systemic changes and runx2 expression. Since elevated blood glucose is a key characteristic of type I diabetes and one of the primary studied pathological finding of diabetes mellitus, we set to investigate the contribution of chronic hyperglycemia itself on osteoblasts status. Mouse MC3T3-E1 osteoblasts were cultured in vitro under pathologic conditions of hyperglycemia and pathologic hyperosmolality as seen in streptozotocin- induced type I diabetic mice. This approach allows us to distinguish between responses directly related to hyperglycemia and hyperosmolality. Our findings demonstrate that osteoblasts respond to chronic hyperglycemia by increasing alkaline phosphatase activity and expression and decreasing osteocalcin, MMP- 13, VEGF and GAPDH expression. Of these genes, only MMP-13 mRNA levels 203 exhibit a similar suppression in response to hyperosmolarity (mannitol treatment). Similar to in vivo gene analyses (24), osteoblasts exhibit a trend toward increased PPARyZ expression, suggesting a potential contribution by hyperglycemic and hyperosmotic conditions toward this phenomenon. Interestingly, our in vitro studies did not show an alteration of runx-2 expression in response to chronic and subacute hyperglycemia or hyperosmolarity. However, these studies did not investigate runx-2 expression 24 hours after establishment of hyperglycemic/hyperosmotic state, a time point that we see significant changes in vivo. An in vitro time course study during first 24 hours of hyperglycemia would be of significant value in answering this question. To summarize, we are the first to extensively characterize bone status in streptozotocin induced diabetic mouse model. To our knowledge, we carried out the most extensive and multi-approach oriented study of diabetic bone loss development. We demonstrate that streptozotocin induced diabetes is associated with significant bone loss and is characterized by increase in bone marrow adiposity. Increased bone adiposity but not bone loss was rescued by inhibition of PPARy with BADGE treatment, suggesting a concomitant and independent occurrence of these two phenomena. Changes in bone phenotype/gene expression are evident in early streptozotocin induced diabetes, employing early stages of diabetes as very crucial regarding normal bone homeostasis. There was no evidence in literature that diabetes can affect bone at such early stages. As a result of our work, we provide a full time sheet of diabetic related bone changes. 204 We are also the first to report a novel finding associated with diabetes such as increase in bone marrow adiposity. This pathological phenomenon was strongly associated with many proostopenic and proostoporotic situations but never with diabetes. We were able to detect an increase in proadipogenic gene markers such as aP2 and PPARgZ as early as five days after the first injection with streptozotocin. While increase in bone adiposity is believed to be causative of bone loss we provide evidence that inhibition of PPARg2 and subsequent inhibition of adipocyte differentiation and bone adiposity may not be linked with osteopenic/osteoporotic diabetic bone changes. At the same time we are the first to report that administration of a PPARg antagonist has a serum triglycerides and free fatty acids lowering effect as we described in our work. We also provide evidence that chronic hyperglycemia in vitro has a suppressive action on osteocalcin mRNA expression in osteoblasts. Together with suppression of osteocalcin in hyperglycemic diabetic mice, in vitro chronic hyperglycemia places itself as a serious candidate in suppression of OC gene expression. Taken together, present work provides detailed characterization of bone status in steptozotocin-induced diabetic mouse model. 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