MECHANISM OF INTESTINAL AND SKE LE TAL MUSCLE GLUCOSE AND AMINO ACID UPTAKE IN PITUITARY PARS INTERMEDIA DYSFUNCTION By Jennifer L. De Vries A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Animal Science Master of Science 2015 ABSTRACT MECHANISM OF INTESTINAL AND SKELETAL MUSCLE GLUCOSE AND AMINO ACID UPTAKE IN PITUITARY PARS INTERMEDIA DYSFUNCTION By Jennifer L. De Vries Copyright by JENNIFER L. DE VRIES 2015 iv This thesis is dedicated to my loving fiancé and life partner, Ashley Brown. Without her unwavering support, encouragement, patience and most of all love, the completion of this work would not have been possible. I look forward to spending the rest of my life supporting and loving her in return. I love you, Ashley. v ACKNOWLEDGEMENTS Immeasurable appreciation and deepest gratitude for help and support throughout this process are extended to the following people who in one way or another have contributed in making my graduate research possible. Dr. Nathalie Trottier - for her support, advice, guidance, suggestions, provisions and constantly pushing me to grow as an academic member of the community throughout this entire graduate process. Dr. Cathy Ernst, Dr. Harold Schott, and Dr. Mike Allen - for their constant support, encouragement, constructive criticism and direction in helping me to accomplish this monumental task and goal . Dr. Steve Bursian - for his door always being open, constant support and words of encouragement at the times when I needed it the most. Janet Ireland, Jim Liesman, Patty Weber, Nancy Raney, and Adrienne Woodward - thank you all for your guidance in the lab, bei ng willing to help, and to teach. It was truly selfless and much appreciated and I am a better scientist and researcher because of each of you sharing your expertise with me. Lee - Anne Huber, Jenna Lubitz, and Celiné Diard - for their aid in sample collection, analysis, and in general keepi ng me sane and supported throughout my graduate process. Fellow graduate students/staff in the Department of Animal Science you have all aided in making this graduate process successful and memorable, and for this I thank you. Friends and family I could not have survived this journey without the unwavering support and patience from everyone in my personal life. It is because of your love I have had the motivation and determination to complete this process. vi TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ..................... viii LIST OF FIGURES ................................ ................................ ................................ ..................... i x KEY TO ABBREVIATIONS ................................ ................................ ................................ .. xiii CHAPTER ONE : INTRODUCTION ................................ ................................ ......................... 1 CHAPTER TWO : Evaluation of glucose tolerance and insulin sensitivity in horses with pituitary pars intermedia dysfunction ................................ ................................ ...................... 1 0 Abstract ................................ ................................ ................................ ................................ ..... 11 Introduction ................................ ................................ ................................ .............................. 13 Materials and Methods ................................ ................................ ................................ ............ 14 Animals and Housing ................................ ................................ ................................ ........... 14 Clinical Evaluation ................................ ................................ ................................ ............... 15 Glucose and Insulin Challenges ................................ ................................ .......................... 15 Pituitary Gland Collection ................................ ................................ ................................ .... 17 Analysis of Plasma Glucose and Serum Insulin Concentration ................................ ........ 17 Kinetic Analysis of Glucose and Insulin ................................ ................................ ............. 18 Statistical Analysis ................................ ................................ ................................ ................ 19 Results ................................ ................................ ................................ ................................ ....... 20 PPID assessment ................................ ................................ ................................ ................... 20 OGTT and FSIGTT ................................ ................................ ................................ .............. 20 Discussion ................................ ................................ ................................ ................................ .. 30 CHAPTER THREE : Skeletal muscle expression of GLUT4, INSR, and SNAT2 in horses with pituitary pars intermedia dysfunction ................................ ................................ .............. 35 Abstract ................................ ................................ ................................ ................................ ..... 3 6 Introduction ................................ ................................ ................................ .............................. 38 Materials and Methods ................................ ................................ ................................ ............ 40 Animals and Housing ................................ ................................ ................................ ........... 40 PPID and Insulin Status A ssessment ................................ ................................ .................. 40 Collection of Tissue ................................ ................................ ................................ .............. 41 Amino Acid Profile Analysis of Plasma ................................ ................................ ............... 41 Isolation of RNA and P reparation of cDNA ................................ ................................ ....... 41 Measurement of SLC2A4 , SLC38A2 and INSR Relative mRNA Abundance by Quantitative Reverse Transcription PCR ................................ ................................ ............ 42 Western Blotting Analysis ................................ ................................ ................................ .... 43 Statistical Analysis ................................ ................................ ................................ ................ 44 Results ................................ ................................ ................................ ................................ ....... 44 Amino Acid Profile in Plasma ................................ ................................ .............................. 44 SLC2A4 , INSR , and SLC38A2 m RNA Abundance in Gluteal Muscle ............................. 45 GLUT4 and INSR content in Gluteal Muscle ................................ ................................ ..... 45 vii Discussion ................................ ................................ ................................ ................................ .. 52 CHAPTER FOUR : Morphology of the small intestinal mucosa in horses with pituitary pars intermedia dysfunction ................................ ................................ ................................ ...... 59 Abstract ................................ ................................ ................................ ................................ ..... 60 Introduction ................................ ................................ ................................ .............................. 61 Materials and Methods ................................ ................................ ................................ ............ 62 Animals and Housing ................................ ................................ ................................ ........... 62 Clinical E valuation ................................ ................................ ................................ ............... 63 Collection of T issue ................................ ................................ ................................ .............. 63 Histological Examination of the Small I ntestine ................................ ................................ 64 Statistical Analysis ................................ ................................ ................................ ................ 65 Results ................................ ................................ ................................ ................................ ....... 65 Discussion ................................ ................................ ................................ ................................ .. 71 CHAPTER FIVE: Jejunal brush border membrane uptak e of D - glucose and L - glutamine and small intestinal mucosa SLC5A1 and SLC38A2 mRNA abundance in horses with pituitary pars intermedia dysfunction ................................ ................................ ...................... 77 Abstract ................................ ................................ ................................ ................................ ..... 78 Introduction ................................ ................................ ................................ .............................. 80 Materials and Methods ................................ ................................ ................................ ............ 81 Animals and Housing ................................ ................................ ................................ ........... 81 Collection of Tissue ................................ ................................ ................................ .............. 82 Preparation of Brush Border Membrane Vesicles ................................ .............................. 82 Measurement of Protein Concentration and Alkaline Phosphatase Enzyme Activity ...... 83 Measurement of D - Glucose and L - Gln Uptake into Jejunal Brush Border Membrane Vesicles ................................ ................................ ................................ ................................ .. 84 Estimation of D - Glucose and L - Gln Kinetics ................................ ................................ ....... 85 Real - t ime quantitative PCR (qRT - PCR) A nalysis ................................ ............................... 8 6 Statistical Analysis ................................ ................................ ................................ ................ 87 Results ................................ ................................ ................................ ................................ ....... 88 Membrane Purity ................................ ................................ ................................ .................. 88 Initial Time Course of D - Glucose and L - Gln Uptake into Brush Border Membrane Vesicles ................................ ................................ ................................ ................................ .. 88 D - Glucose and L - Gln Uptake into Brush Border Membrane Vesicles ............................... 88 SGLT1 and SNAT2 mRNA Abundance in Intestinal Mucosa Vesicles ............................. 89 Discussion ................................ ................................ ................................ ................................ .. 97 CHAPTER SIX : SUMMARY AND CONCLUSIONS ................................ .......................... 101 APPENDICES ................................ ................................ ................................ ........................... 104 APPENDIX A: CHAPTER TWO MATERIAL ................................ ................................ . 105 APPENDIX B : CHAPTER THREE MATERIAL ................................ ............................. 114 APPENDIX C : CHAPTER FIVE MATERIAL ................................ ................................ . 119 LITERATURE CITED ................................ ................................ ................................ ............ 122 viii LIST OF TABLES Table 2.1. Henneke body condition score, average body weight, pituitary gland characteristics, serum ACTH and cortisol concentrations of PPID (n=8) a Table 2.2. Demographics, History, Clinical Signs, and ODST of horses with PPID and horses without PPID. Table 2.3. OGTT and FSIGTT parameters of horses wi th PPID and horses without PPID ... . 25 Table 2.4 . Pearson correlation coefficients (R) between pituitary gland grade, pituitary gland weight and ACTH concentration and each of th ... .. 26 Table 3.1. Amino acid profile in the plasma of PPID and non - PPID horses ( µg/mL ... 46 Table 3.2. Reverse transcription quantitative real - time (RT - qPCR) assay primer information in the gluteal musc le of PPID and non - .... 48 Table 5.1 . Reverse transcription quantitative real - time (RT - qPCR) assay primer information in the intestinal mucosa of PPID and non - PPID horse . 90 Table 5.2. Kinetic parameters of glucose and glutamine uptake in equine small intestinal (jejunum) brush border membrane vesicles of control and PPID horses of similar ages .. ... 91 Table 5.3. Kinetic parameters of D - glucose and L - Gln uptake in several mammalian and chicken jejunal brush border membrane vesicles . .. .. .. 92 Table C.1 . Kinetics of glucose uptake rate into equine jejunal control and PPID brush border membrane vesicles. Each point is representative of the mean ± SEM from four control horse's . . .... 120 Table C.2 . Kinetics of Glutamine uptake rate into equine jejunal control and PPID brush border membrane vesicles. Each point is representative of the mean ± SEM fr om four control horse's ... 121 ix LIST OF FIGURES F igure 1.1 The anatomical structure of the equine pituitary. The pars distalis (1), pars intermedia (2), pars nervosa (3), hypophyseal stalk (4), and recess of third ventricle (5). From Dyce K.M, Sack W.O, and Wensing C.J. Textbook of Veterinary Anatomy. 2 nd ed., Philadelphia, PA; WB Sau .. 1 Figure 1.2 Hypothalamus - pituitary - adrenal axis. Hypothalamic release of corticotrophin releasing hormone (CRH) stimulates release of ACTH from the pituitary. Adrenocorticotropic hormone acts on the adrenal cortex to secrete cortisol, a glucocorticoid which acts on the hypothalamus and pituitary to inhibit release of CRH and ACTH, respectively. From www.biology.ucr.edu . ....................................................................... ........ ............................... . .. .... 3 Figure 1.3 Proopiomelanocortin prohormone produced in the pars intermedia is cleaved by PC - 1 to produce ACTH. Adrenocorticotropic hormone is cleaved by PC - - MSH and CLIP. From www.themedicalbi ochemistrypage.org and http: //slideplayer.com/slide/1496700/ . .. 4 Figure 2.1 . Plasma glucose (A) and serum insulin (B) concentrations during the OGTT in PPID (n=8) and control (n=4) horses following oral administration of 1 g/kg bwt 10% wt/vol dextrose ... 27 Figure 2.2 . Plasma glucose during the entire FSIGTT (A) and during the first 40 min (B) and serum insulin during the entire FSIGTT (C) and during the first 40 min (D) concentrations in PPID (n=8) and con trol (n=4) horses following i.v. administration of 300 mg/kg bwt 50% wt/vol dextrose solution at time 0 and 20 mU/kg bwt insu 28 Figure 3.1 . Fold change in mRNA abundance of SLC2A4, INSR, and SLC38A2 referred to by protein names, GLUT4, INSR, and SNAT2, in the gluteal muscle for PPID (n=8) relative to control (n=4) horses 49 Figure 3.2 . Western blot analysis of crude muscle membrane extracts p repared from gluteal biopsies for PPID (n=8) and control (n=4) horses treated with antibodies against GLUT4 depicted by (A) Western blotting showing protein expression level and (B) a graph indicati ng the relative expression level. Intensities of bands were estimated by using the program "ImageLab" from BioRad. Values were normalized to the GLUT4 control and expressed relative to the value of control horses .. 5 0 F igure 3. 3. Western blot analysis of crude muscle membrane extracts p repared from gluteal biopsies for PPID (n=8) and control (n=4) horses treated with antibodies against INSR depicted by (A) Western blotting showing protein expression level and (B) a graph indicating the relative expression level. Intensities of bands were estimated by using the program "ImageLab" from BioRad . Values were normalized to the INSR control and expressed relative to the value of control horses ... 5 1 x Figure 4.1. Villus height of the small intestine in PPID (n=8) and control (n=4) horses. Duo25, duodenum at 25 cm; Duo50, 50 cm distal to the pyloric sphincter; Jejunum, mid - jejunum; Ileum, mid - .. 67 Figure 4.2. Villus width of the small intestine in PPID (n=8) and control (n=4) horses. Duo25, duodenum at 25 cm; Duo50, 50 cm distal to the pyloric sphincter; Jejunum, mid - jejunum; Ileum, mid - .. 68 Figure 4.3. Villus crypt depth of the small intestine in PPID (n=8) and control (n=4) horses. Duo25, duodenum at 25 cm; Duo50, 50 cm distal to the pyloric sphincter; Jejunum, mid - jejunum; Ileum, mid - .. .. 69 Figure 4.4. Villus height:Crypt Depth (V:C) of the small intestine in PPID (n=8) and control (n=4) horses. Duo25, duodenum at 25 cm; Duo50, 50 cm distal to the pyloric sphincter; Jejunum, mid - jejunum; Ileum, mid - . .. 70 Figure 5.1 . Kinetics of D - g lucose uptake into equine jejunal Control (A) and PPID (B) brush border membrane vesicles. Brush border membrane vesi cles were loaded with a buffer containing 150 m M KSCN, 10 m M Mannitol, 5 m M HEPES ( pH 7.4) and incubated with another buffer containing 150 m M NaSCN and 5 m M HEPES ( pH 7.4) , 0.8 µM D [ 3 H] - g lucose at concentrations of 0, 0.1 0 , 0.25, 0.5 0 , 1 .00 , 2.5 0 , 5 .00 , and 7.5 0 m M D - g lucose. D - Mannitol was added to maintain osmolarity. Each point is representative of the LSMEAN ± SEM from four (control, A) and eight ( PPID , B) horses in triplicate uptake experiments .. ... ... 93 Figure 5.2. Kinetics of L - Gln uptake into equine jejunal Control (A) and PPID (B) brush border membrane vesicles. Brush border membrane vesicles were loaded with a buffer (150 mM KSCN, 10 m M Mannitol, 5 m M HEPES (pH 7.4) and incubated with another buffer containing 150 m M NaSCN an d 5 m M HEPES (pH 7.4), 0.8 µ M L [ 3 H] - Glutamine at concentrations of 0, 0.1, 0.25, 0.5, 1, 2.5, 5, and 7.5 m M L - Gln. D - Mannitol was added to maintain osmolarity. Each point is representative of the LSMEAN ± SEM from four (control, A) and eight (PPID, B) ho rses in triplicate uptake experiments . 94 Figure 5.3. Fold change in mRNA abundance of SLC5A1 in PPID horses relative to Control. Duo25, duodenum at 25 cm; Duo50, 50 cm distal to the pyloric sphincter; Jejunum, mid - jejunum; Ileum, mid - .. 95 Figure 5.4 Fold change in mRNA abundance of SLC38A2 in PPID horses relative to Control. Duo25, duodenum at 25 cm; Duo50, 50 cm distal to the pyloric sphincter; Jejunum, mid - jej unum; Ileum, mid - .. 96 Figure A.1 . Mean ± s.e acute response of insulin to glucose values derived for 8 PPID and 4 control horses following i.v. administration of 300 mg/kg bwt 50% dextrose solution followed later by the administration of 20 mu/kg bwt insulin in the FSGITT testing using the minimal 106 xi Figure A.2. Mean ± s.e insulin sensitivity values derived for 8 PPID and 4 control horses following i.v. administration of 300 mg/kg bwt 50% dextrose solution followed later by the administration of 20 mu/kg bwt insulin in the FSGITT testing using the minimal model for .. ,, 107 Figure A.3. Mean ± s.e glucos e sensitivity values derived for 8 PPID and 4 control horses following i.v. administration of 300 mg/kg bwt 50% dextrose solution followed later by the administration of 20 mu/kg bwt insulin in the FSGITT testing using the minimal model for 108 Figure A.4. Mean ± s.e disposition index values derived for 8 PPID and 4 control horses following i.v. administration of 300 mg/kg bwt 50% dextrose solution followed later by the administration of 20 mu/kg bwt insuli n in the FSGITT testing using the minimal model for .. . 109 Figure A.5 . Correlation of the Peak Insulin of OGT vs Acute Insulin Response to Glucose (AIRg) Correlation when comparing PPID horses to control horses of similar .. . 110 Figure A.6 . Correlation of the change in insulin OGT vs Insulin Sensitivity when comparing PPID horses to control horses of similar ... 111 Figure A.7 . Correlation of Insulin Sensitivity vs Acute Response of when comparing PPID horses to control horses of similar . ... 112 Figure A.8 . Correlation of the change in insulin OGT vs Insulin Sensitivity whe n comparing PPID horses to control horses of similar . .. ... 113 Figure B.1 . Western blot analysis of crude muscle membrane extracts prepared from gluteal biopsies of control horses and PPID horses and treated with antibodies against INSR showing .. .. 115 Figure B.2. Western blot analysis of crude muscle membrane extracts prepared from gluteal biopsies of control horses and PPID horses and treated with antibodies aga inst INSR depicted by a graph indicating the relative expression level. Intensities of bands were estimated by using the program "ImageLab" from BioRad. Values were normalized to the INSR control and expressed relative to the value of control horses . The d ata are - ... . 116 Figure B.3 . Western blot analysis of crude muscle membrane extracts prepared from gluteal biopsies of control horses and PPID horses and treated with antibodies against GLUT4 showing .... 117 xii Figure B.4 . Western blot analysis of crude muscle membrane extracts prepared from gluteal biopsies of control horses and PPID horses and treated with antibodies against GLUT4 depicted by a graph indicating the relative expression level. Intensities of bands were esti mated by using the program "ImageLab" from BioRad. Values were normalized to the GLUT4 control and expressed relative to the value of control horses. The data are expressed as means ± s.e. using - test ... 118 xiii KEY TO ABBREVIATIONS xiv 1 CHAPTER ONE : INTRODUCTION the aged horse (Schott, 2006). It is estimated that 15 to 30% of horses older than 15 years of age are affected (McGowan et al., 2013; Brosnahan and Paradis, 2003), with an overwhelming 50% of the affected horses euthanized due to the deteriorating physical appearance and laminitis. Geriatric horses play a key role in the equestrian industry where they serve as experienced horses prospering until their maximum life expectancy. Although two drugs, pergolide mesylate a nd cyproheptadine, are commonly used to treat the PPID condition, they are very costly and their effectiveness decreases over time. I n addition, the daily management of horses with PPID is tedious. There appears to be no specific breed (McGowan, 2008) or sex (Schott et al., 2001) more prone to developing PPID. The known physiological mechanisms behind the pathology is represented in Figure 1 .1 . Figure 1.1. The anatomical structure of the equine pituitary. The pars distalis (1), pars intermedia (2), par s nervosa (3), hypophyseal stalk (4), and recess of third ventricle (5). From Dyce K.M, Sack W.O, and Wensing C.J. Textbook of Veterinary Anatomy. 2 nd ed., Philadelphia, PA; WB Saunders, 1987. 2 The condition is progressive and characterized by hypertrophy, hyperplasia, adenoma and loss of dopaminergic inhibition of the pars intermedia of the pituitary gland resulting in increased circulating concentration of pro - opiomelanocortin peptides (POMC) as illustrated in Figure 1.2 and Figure 1.3, respectively . Hors es with PPID are reported to have as much as one - eighth the serum concentration of dopamine (Millington et al., 1988) and one - sixth the dopaminergic nerve terminals (McFarlane et al., 2005). The increase in POMC from corticotropes and melanotropes increase - lipotropin from the pars distalis by stimulating the activity of prohormone convertase 1 (PC1) (Hillyer et al., 1992; Love, 1993; van der Kolk et al., 1993; Dybdal et al., 1994; van der Kolk et al., 1 995; Eiler et al., 1997; SM Reed, 1998). In non - - melanocyte - MSH) and corticotropin - like intermediate lobe peptide - - END), lipo trophin and other small peptides (McFarlane, 2011). Horses without PPID almost exclusively produced ACTH in the pars distalis however horses affected with PPID produce increased concentrations of ACTH from the pars intermedia at a rate faster than its conv ersion into MSH and CLIP. Horses diagnosed with PPID characteristically have increased blood concentration of cortisol, although t he mechanism remains largely not understood (Cartmill et al., 2003). 3 Figure 1.2. Hypothalamus - pituitary - adrenal axis. Hy pothalamic release of corticotrophin releasing hormone (CRH) stimulates release of ACTH from the pituitary. Adrenocorticotropic hormone acts on the adrenal cortex to secrete cortisol, a glucocorticoid which acts on the hypothalamus and pituitary to inhibit release of CRH and ACTH, respectively. From www.biology.ucr.edu . 4 Figure 1.3. Proopiomelanocortin prohormone produced in the pars intermedia is cleaved by PC - 1 to produce ACTH. Adrenocorticotropic hormone is cleaved by PC - 2 to produce - MSH and CLIP. From www.themedicalbiochemistrypage.org and http://slideplayer.com/slide/1496700/ . Horses diagnosed with PPID exhibit a variety of clinical signs, most commonly including poor body condition, appearance of muscle wasting, abnormal fat dep osits, hirsutism, hyperhidrosis, abnormal fat deposits, and a heightened risk for secondary infections (Innerá et al., 2013; Klinkhamer et al., 2011; McFarlane et al., 2010; Aleman et al., 2006; Donaldson et al., 2004). Hirsutism is the incomplete, delayed , and lightening of the coat and the most obvious clinical sign of PPID with a 90% prediction for the disease (Frank et al., 2006). It has been proposed that hirsutism results from the chronically high blood concentration of MSH (Van Synthesis of ACTH Synthesis of Pro - Opiomelanocortin (POMC) 5 der Kolk et al., 1990 - 1992). Hyperhidrosis or excessive sweating was originally thought to be a result of hirsutism, but recent studies have shown PPID horses to excessively sweat in cool conditions and after being body clipped (Schott, 2002). Muscle wasting of epaxial and gluteal muscles in horses with PPID possibly could be attributed to the increased glucocorticoid production, most notably cortisol, which is known in other species to be a contributing factor to type 2 muscle fiber atrophy, sa rcoplasmic lipid accumulation, greater myofiber size variation, and increased proteolysis (Aleman and Nieto, 2010; Aleman et al., 2006). Inhibition of lipolysis due to hyperinsulinemia has been postulated as a reason why horses with PPID develop abnormal f at deposits over the neck, tail head, and eyes (Schott, 2002). Human studies have shown visceral adiposity stores to be highly correlated with higher plasma leptin concentrations (Davis et al., 2009). The concentration of leptin in many species, including hamsters (Dodson and Steiner, 1998) and sheep (Wilcox, 2005), has also shown to fluctuate with season (Fazio et al., 2008; McFarlane, 2007; Frank et al., 2006; Couetil et al., 1996; McFarlane et al., 2010; Beech et al, 2009; Fazio et al, 2008; Dybdal et al , 1994; McFarlane et al., 2012; Donaldson et al., - MSH secretion in both the pars distalis and the pars intermedia (Fazio et al., 2008). Leptin levels are lowest in horses d uring the day and peak at night, with a greater effect of season in the spring and summer (Pørksen et al., 2002). The effect on the endocrine system due to seasonal changes is another characteristic feature of the PPID condition. Aged horses with PPID were shown to exhibit seasonal changes in the circadian peripheral plasma concentrations of MSH serotonin, dopamine, and cortisol (Jeffcot et al., 1986). Dopamine release from the pars intermedia neurons inhibits POMC - derived peptide secretion (Schmidt et a l., 1992). 6 Despite their poor physical appearance, horses with PPID have characteristically high appetite (Mountjoy, 2010). Thus poor nutrient absorption and peripheral nutrient utilization may contribute to the pathophysiology of the condition. The over arching goal of this thesis was to assess nutrient absorption and peripheral muscle utilization in PPID and age - matched non PPID horses. In the first chapter of this thesis, insulin sensitivity and glucose tolerance was measured in a total of 14 horses donated for this study. The aim was to classify horses into 3 groups: aged control horses without PPID, aged PPID horses without insulin resistance, and aged PPID horses with insulin resistance. This goal was based on the available literature with an es timated insulin resistance rate of at least 50% in the PPID population. In 57 - 94% (Keen et al., 2004; van der Kolk et al., 1995; Hillyer et al., 1992) and 45 to 90% of the PPID - afflicted horse population (Keen et al., 2004; van der Kolk et al., 1995), abno rmalities in glucose homeostasis or glucose intolerance and insulin resistance have shown to persist. Also, horses with PPID have been reported to have both hyperinsulinemia at rest and a reduced hypoglycaemic response to exogenous insulin ( Freestone et al ., 1992; Garcia and Beech, 1986; Jeffcott et al., 1986). Studies have shown differing results in regards to whether insulin resistance is directly (Keen et al., 2004; van der Kolk et al., 1995; Donaldson et al., 2004) or indirectly (Mastro et al., 2015; va n den Berg et al., 2013; Vick et al., 2007) associated with the PPID condition. Insulin resistance and central fat deposit in men is associated with excess blood cortisol concentration and cortisol production rates (Purnell et al., 2009; Phillips et al., 1998; Filipovsky et al., 1996; Stolk et al., 1996). Pituitary pars intermedia dysfunction has also been associated with an increased risk for laminitis (Donaldson et al., 2004; Carter et al., 2009; Geor, 2009) and laminitis in PPID horses has been thought to be a result of high serum insulin concentrations (Treiber et al., 2006; Asplin 7 et al., 2007). Many of the studies reported above however lacked in adequate control subjects that were age - matched control, or included ponies. In Keen et al. (2004) cited above, the majority of test subjects were ponies who are known to be more susceptible to insulin resistance than horses (Schott, 2002; van der Kolk, 1997). Also, in Klinkhamer et al. (2011) and Garcia and Beech (1986), the increased insulin resistance sta tus of horses diagnosed with PPID was made in comparison to younger non - PPID horses. Older horses however have been shown to be more prone to insulin resistance than younger horses (Malinowski et al., 2002), with similar observations in humans (Barbieri e t al., 2001; Paolisso et al., 1999). In a recent study by Mastro et al. (2015), PPID - affected horses compared to age - matched controls did not differ in insulin sensitivity or glucose tolerance when assessed using the reciprocal of the square root of insul in (RISQI), the modified insulin to glucose ratio (MIRG), and isoglycemic hyperinsulinemic clamp approaches. Therefore, in the first chapter of this thesis, geriatric horses were evaluated for PPID based on both endocrinological testing and post - mortem ex amination of the PG, and assessed for insulin resistance and glucose intolerance using oral glucose tolerance and frequently sampled intravenous glucose tests. In the second chapter, molecular entities involved in the regulation of intracellular muscle glucose and glutamine utilization were determined to further confirm the insulin status determined in this cohort of horses. Thus, transcript abundance of SLC2A4 , INSR and SLC38A2, respectively encoding GLUT4, insulin receptor and SNAT2, and protein abun dance of GLUT4 and insulin receptor were measured in muscle tissue. In adipocytes and muscle cells, insulin stimulates the translocation of GLUT4 to the plasma membrane (Huang, 2007). The first determinant of insulin - dependent peripheral glucose uptake i s the availability on the cell membrane of the GLUT4 transporter (Huang, 2007). With respect to AA transport, insulin 8 activation involves in part the recruitment of the Na + - coupled neutral AA transporter 2 (SNAT2) from an endosomal compartment (Evans et al ., 2008). Molecules associated with recruitment and fusion of SNAT2 - containing vesicles to the surface membrane are also implicated in the regulation of GLUT4 translocation (Evans et al., 2008). Insulin - mediated neutral AA uptake via SNAT2 is down - regulat ed in metabolic acidosis and insulin resistance, causing proteolysis and muscle wasting in humans (Evans et al., 2008). Therefore, characterizing the abundance of SNAT2 in muscle tissue of PPID and non - PPID horses provided additional insight into the poss ible impact of PPID on insulin - dependent nutrient transport. The second part of the thesis is focused on the link between PPID and intestinal function based on the notion that gastrointestinal health status is a key determinant of nutrient availability. It is proposed that reduced nutrient absorptive capacity by the small intestinal mucosa contribute to the apparent poor nutritional status of geriatric PPID horses. First, nutrient absorptive capacity was assessed by histomorphometry analysis of the small intestinal mucosa in Chapter 3. Histomorphological changes to the small intestinal mucosa such as villus atrophy and increased crypt depth are associated with poor nutrient absorption and malnutrition in animals (Stevenson et al., 2013) and humans (Dewar and Ciclitira, 2005; and Ventura et al., 2013). To date, no study has addressed whether PPID is associated with h istomorphometric changes of the small intestinal mucosa. Second, in Chapter 4, glucose and Gln transport across brush border membrane of the j ejunal mucosa was measured ex vivo, along with the molecular entities responsible for their transport. Brush border membrane vesicles (BBMV) preparation from small intestinal mucosa is a useful ex vivo tool to precisely characterize transport kinetics of nutrients in response to changes in physiological or dietary processes. Dietary AA are a major fuel for the small intestinal mucosa and are necessary for maintaining the intestinal mucosal morphology and 9 function in neonatal piglets (Wu, 1998). Glutamine i s a preferred nutrient of intestinal enterocytes for mucosal growth and function (Ganapathy et al, 1994; Salloum et al., 1994; Windmueller and Spaeth, 1980) . Glutamine is transported by SNAT2 , which is encoded by SLC38A2 (solute carrier family 38, member 2 ). In horses, glucose is transported across the apical membrane of small intestinal epithelial cells (Dyer et al., 2002) by the high - affinity, Na + - glucose cotransporter SGLT1, encoded by SLC5A1 (solute carrier family 5, member 1). In chapter 4, it is hy pothesized that the transport capacity (V max ) of D - glucose and L - Gln across the apical membrane is reduced in PPID compared to non - PPID horses, and that reduced V max is related to lower mRNA abundance of SLC38A2 and SLC5A1. The overarching hypothesis of th is thesis is that glucose and glutamine utilization by geriatric horses suffering from PPID is reduced in the intestine and peripheral muscle tissue. The objectives of this study were to: (1) measure glucose tolerance and insulin sensitivity in horses wi th PPID and horses with no PPID (control) ; (2) quantify mRNA abundance of SLC2A4 , INSR and SLC38A2, and quantify protein abundance of INSR and GLUT4 in muscle tissue of PPID and non - PPID horses (control) ; (3) evaluate the morphology of the mucosa in the pr oximal, mid and distal regions of the small intestine in horses clinically confirmed with PPID and no PPID ; (4) measure transport capacity of D - glucose and L - Gln across jejunal BBMV of PPID and non - PPID horses, and quantify the mRNA abundance of SLC5A1 an d SLC38A2 in duodenal, jejunal and ileal mucosa of PPID and non - PPID horses . The following chapters are organized into individual manuscripts which respectively address the above objectives. Chapter 5 presents a summary of these results and overall concl usions. 10 C HAPTER TWO : Evaluation of glucose tolerance and insulin sensitivity in horses with pituitary pars intermedia dysfunction J.L. De Vries a , H.C. Schott II b , S. Pratt - Phillips c , R.J. Geor b , and N.L. Trottier a,* a Department of Animal Science, C ollege of Agriculture and Natural Resources, Michigan State University, 474 S. Shaw Lane, Anthony Hall, East Lansing MI 48824 USA b Department of Large Animal Clinical Sciences, College of Veterinary Medicine, 736 Wilson Rd., Veterinary Medical Center, East Lansing MI 48824 USA c Department of Animal Science, College of Agriculture and Life Science, North Carolina State University, Raleigh NC 27695 USA * Corresponding author. Tel.: +1 517 432 5140. E - mail address: trottier@msu.edu (N.L. Trottier). Work was completed at Michigan State University in the Department of Animal Science and Large Animal Clinical Sciences. This study was supported by the Morris Animal Foundation. Work from this study was in part prese nted at the American Society of Animal Science Midwest meeting , the European Workshop on Equine Nutrition, and the Havemeyer Foundation Meeting . We acknowledge Sue Wismer, Jenna Lubitz, Lee - Anne Huber, and Dave Chamberlin for their help in sample collecti on. We would also like to acknowledge Janet Ireland for assistance with insulin analysis of serum samples. 11 Abstract Pituitary pars intermedia dysfunction (PPID) is characterized by a progressing loss of dopaminergic inhibition of the pituitary pars interme dia. It commonly causes lethargy, poor physical appearance, abnormal sweating, hirsutism and muscle loss in the geriatric horse, and therefore is of welfare concern. We hypothesized that horses with PPID have reduced tolerance to glucose and sensitivity t o insulin compared to horses with no PPID. The objective was to measure glucose tolerance and insulin sensitivity in horses with PPID and horses with no PPID (control). Eight PPID and 4 age - matched control geriatric horses (> 19 yr) were donated for this study. Grade of PPID was determined based on an overnight dexamethasone suppression test (DST), ACTH testing and histopathology evaluation of the pituitary gland (PG). Glucose tolerance and insulin sensitivity were measured based on an oral glucose tolera nce test (OGTT) and a frequently sampled intravenous glucose tolerance test (FSGITT). Compared to control horses, PG grade and ACTH concentration of PPID horses were greater ( P to be greater ( P = 0.079), respectively. Glucose concentration of PPID horses tended to be greater than that of control at 90 min ( P = 0.099). Compared to control, insulin concentration of PPID horses was greater or tended to be greate r at times 3 ( P = 0.091), 23 ( P = 0.083), 24 ( P = 0.056), 25 ( P = 0.027) and 27 min ( P = 0.048) following i.v. infusion of insulin during FSIGTT. The OGTT parameters IB OGTT , Peak Ins OGTT , GB OGTT , Peak Gluc OGTT , dGLUC OGTT, and dINS OGTT did not differ betwe en control and PPID horses. Compared to control horses, the OGTT parameter dGLUC OGTT tended to be greater ( P = 0.078) in PPID horses. The FSIGTT parameters AIRg, DI, SI, Sg, IB FSGITT , GB FSGITT , dGLUC FSIGTT , dINS FSIGTT , FSIGTTAUC I , and FSIGTTAUC G did not d iffer between control and PPID horses. The disposition index (DI) correlated with ACTH concentration ( P = 0.04). In this study, geriatric horses with PPID did not 12 have lower insulin sensitivity and glucose tolerance compared to non PPID, geriatric horses. The positive correlation between DI and ACTH concentration is indicative of some degree of insulin resistance related to PPID, and a tendency for greater FSIGTTAUC I in PPID horses may be indicative of some degree of hyperinsulinemia. Neither PPID nor co ntrol horses however could be classified as insulin resistant based on the estimated SI parameter. During the OGTT, there was an equal number of horses in both PPID and control cohorts that exhibited signs of glucose intolerance. Geriatric horses and th ose with PPID in particular should be carefully managed with feeding diets containing low concentration of soluble carbohydrates in an effort to reduce the risk for laminitis . 13 Introduction Pituitary pars intermedia dysfunction (PPID) also known as Cu progressive endocrine disorder affecting the aging horse population (Schott, 2002; van der Kolk, 1997). The disorder is characterized by hypertrophy, hyperplasia, adenoma and loss of dopaminergic inhibition of the pars intermedia of th e pituitary gland resulting in increased circulating concentration of pro - opiomelanocortin peptides (POMC), including ACTH (Reed, 1998; Eiler et al., 1997; van der Kolk et al., 1995; Dybdal et al., 1994; Love, 1993; van der Kolk et al., 1993; Hillyer et a l., 1992). Horses diagnosed with PPID exhibit a variety of clinical signs, most commonly including poor body condition, appearance of muscle wasting, abnormal fat deposits, hirsutism, and heightened risk for secondary infections (Innerá et al., 2013; Klink hamer et al., 2011; McFarlane et al., 2010; Aleman et al., 2006; Donaldson et al., 2004). Pituitary pars intermedia dysfunction has also been associated with an increased risk for laminitis (Carter et al., 2009; Geor, 2009; Donaldson et al., 2004). Earl ier studies reported horses with PPID to have resting hyperinsulinemia and reduced hypoglycaemic response to exogenous insulin post glucose tolerance test ( Freestone et al., 1992; Garcia and Beech, 1986; Jeffcott et al., 1986). Abnormalities in glucose hom eostasis or glucose intolerance have been reported in 57 to 94% (Keen et al., 2004; van der Kolk et al., 1995; Hillyer et al., 1992) and insulin resistance in 45 to 90% of the PPID - afflicted horse population (Keen et al., 2004; van der Kolk et al., 1995). In the work reported by Keen et al. (2004) and van der Kolk et al. (1995) however half to 100% of the candidates were ponies, which are typically at higher risk for insulin resistance (Donaldson et al., 2004). Conversely, several more recent studies have reported absence of insulin resistance in horses with PPID (Mastro et al., 2015; van den Berg et al., 2013; Vick et al., 2007). Reeves et al. (2001) and more recently McGowan et al. (2013) 14 suggested that insulin insensitivity may not be specific to PPID pe r se and Malinowski et al. (2002) proposed that insulin resistance in horses with PPID is related to aging. So far, only one of the studies cited above (Mastro et al., 2015) included matched - age controls. Furthermore, there have been no studies examining the incidence of insulin resistance and glucose intolerance in horses confirmed with PPID based on both endocrinological testing and post - mortem examination of the PG. Continued assessment of glucose metabolism in the aging, PPID horse is critical in ord er to design dietary and management strategies targeted to this segment of the equine population and maintain their welfare. We hypothesized that geriatric horses with PPID have a lower sensitivity to insulin and tolerance to glucose compared to geriatric horses without PPID. The objective of this study was to determine insulin sensitivity and glucose tolerance in geriatric horses with PPID and without PPID condition. Materials and Methods All methods were approved by the Institutional Animal Care and U se Committee at Michigan State University (03 - 12 - 064 - 001) . Animals and Housing Fourteen horses, 10 presenting clinical symptoms of PPID and 4 presenting no clinical symptoms of PPID were donated for this study between September and December of 2012 follo PPID horses was to obtain three experimental groups including non PPID horses, PPID horses with insulin resistance and glucose intolerance, and PPID horses with no insu lin resistance and glucose intolerance. This specific study was part of a larger study aimed as assessing intestinal and peripheral muscle nutrient utilization of PPID horses with and without insulin insensitivity 15 and glucose intolerance. Horses were of multiple breeds and genders (mare and gelding) ranging from 19 to 39 years of age. No ponies were admitted. Average age between control (25.3 ± 2.1 years) and PPID (28.6 ± 1.5 years) did not differ (Table 1). Following admission, all horses were housed i Dactylis glomerata L. ) and bromegrass hay ( Bromus mollis L.) , and a commercial grain concen trate a containing 14% crude protein, 1.8% fat and 18% crude fiber, at 1.8 and 0.5% of their body weight, respectively. Clinical Evaluation Horses were monitored for medical stability for a period of 5 days prior to being assessed for PPID. Horses were s ubjected to ACTH testing as described in Beech et al. (2009) and an overnight dexamethasone suppression test (ODST) as described by Dybdal et al. (1994). Plasma ACTH concentration was measured using a commercially available radioimmunoassay kit according c . Serum cortisol concentration was measured using a commercially available kit according to the gamma counter. Information including history of clinical signs associated with PPID, treatments and diet was collected for each horse. Body condition scoring (BCS) was performed based on the Henneke scoring method (Henneke et al., 1983) and body weight (BW) recorded. G lucose and Insulin Challenges Three days post ODST, horses were fitted with jugular catheters in the evening as described in Marc et al. (2000). The following morning (i.e., day 4), prior to feeding, horses 16 were subjected to an oral glucose tolerance tes t (OGTT) per Pratt et al. (2006). Briefly, following a baseline blood sample at time 0 min, Karo Syrup Light b was administered orally, with blood samples taken at 60, 75, 90 and 120 min after administration. On day 5, prior to feeding, a frequently sampl ed intravenous glucose tolerance test (FSGITT) was performed as described in Tóth et al. (2009) by administering 100 mg/kg BW dextrose (50% wt/vol) at time zero min and 20 mU/kg BW insulin 1 at time 20 min. Blood samples were collected for determination of plasma glucose and serum insulin concentrations at the following times: - 15, - 1, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 19, 22, 23, 24, 25, 27, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, and 180 min. Horses were loosely restrained in a stall during t he entire procedure. Blood was transferred to evacuated tubes containing EDTA c or no additive d for separation of plasma and serum, respectively. Tubes containing EDTA were placed on ice and centrifuged (1,500 × g ction for plasma and or allowed to clot at room temperature for 2 h prior to centrifugation for separation of serum. Plasma and serum aliquots were harvested and stored at - a (Equine Senior, Purina, St. Louis, MO) b (Karo Syrup L ight, ACH Food Companies Inc., Summit, IL) c ( BD Vacutainer®, Franklin Lakes, NJ) d (BD Vacutainer®, Franklin Lakes, NJ) e (Fatal - Plus, Vortech Pharmaceuticals, Dearborn, MI) f (Pureview® Hematoxylin and Eosin Stain System, Cancer Diagnostics Inc. Durham , NC) g (Wako Autokit Glucose,Wako Life Sciences, Inc., Mountain View, CA) h (Spectramax Plus 384 Microplate Reader, Molecular Devices LLC, Sunnyvale, CA) i (Insulin Coat - a - Count®, Siemens, Munich, BAV) j (Cobra II Auto Gamma, Perkin - Elmer Inc., Waltham, M A) k (WinSAAM, Raymond Boston, University of Pennsylvania, Kennet Square, PA and SEM, Stata Corp., College Station, TX) l (SAS version 9.3, SAS Institute Inc., Cary, NC) 17 Pituitary Gland Collection Horses were euthanized on the fol lowing day (i.e., day 6) with pentobarbital e (95 mg/kg BW) administered IV using the jugular catheter already in place. Following confirmation of death, the skull was opened on the dorsal aspect to excise the brain and access the pituitary gland (PG). The PG was gently dissected out and the weight, length, width, and height were recorded. The gland was cut in half, and one half was flash - frozen in liquid N and stored other half transferred into 10% formalin solution (10 mL formalin/100 mL H 2 0) for subsequent preparation for hematoxylin and eosin f staining and grading as described in Miller et al. (2008). The following numerical grading system was use d: 1 (within normal limits), 2 (focal or multifocal PI hypertrophy or hyperplasia), 3 (diffuse PI adenomatous hyperplasia), 4 (PI adenomatous hyperplasia with microadenomas [1 - to 5 - mm diameter]), or 5 (adenoma [>5 - mm diameter] in PI or pars anterior). Oth er tissues were also harvested, including muscle, tail fat, and the small intestinal mucosa. These tissues were flash frozen and stored analyses as part of a subsequent study. The liver was removed and weighed. Analysis of Plasma Glucose and Serum Insulin C oncentration Glucose concentration in plasma was measured using a colorimetric assay kit g according to the manufa reader h . Serum insulin concentrations were measured using a commercially available radioimmunoassay kit i wit h a gamma counter j . Use of this kit for horses has been previously validated (Tinworth et al., 2011; Freestone et al., 1991). Both assays were performed in duplicate. The assay was repeated for any duplicate samples with values differing by more than 5% f or glucose and 10% for 18 insulin. The intra assay coefficient of variation was 4.12 and 8.14%, and inter assay coefficient of variation was 7.79 and 12.61%, for glucose and insulin, respectively. Kinetic Analysis of Glucose and I nsulin Glucose and insulin values from FSIGTT were analyzed based on the minimal model developed by Bergman et al. (1981) with a commercially available software k as described by Hoffman et al. (2003). The following parameters were estimated for each horse: acute insulin response to glucose (AIRg), insulin sensitivity (SI), disposition index (DI), glucose - dependent glucose disposal (SG), baseline insulin (IB FSIGTT ), baseline glucose (GB FSIGTT ), area under the glucose curve (FSIGTTAUC G ) and area under the insulin curve (FSIGTTAUC I ). D isposition index was determined according to the following equation: DI = AIRg × SI The difference between peak glucose or insulin level and baseline glucose or insulin level at time zero min (dGLUC OGTT and dINS OGTT , respectively) were calculated from the OGTT. The reference ranges for glucose and insulin values used to interpret the OGTT were based upon criteria established by Frank (2013) as followed: plasma glucose concentration values following oral administration of glucose higher than 125 mg/dL were considered as indicative of an excessive glucose response and glucose intolerance; insulin concentrations values less than 45 µIU/mL were considered as normal insulin responses, values ranging from 45 to 60 µIU/mL as equivocal and values exceeding 60 mIU/m L as indicative of an exaggerated response to insulin characteristic of insulin resistance. 19 Statistical Analysis Commercially available statistical software l was used to perform analysis of data using the mixed procedure with significance at P P Glucose tolerance and insulin sensitivity were determined using a one - way analysis of variance with the fixed effect of treatment (PPID and control) and the random effect of horse nested within treatment . T he Shapiro - Wilk test was used to test the ANOVA assumption of normally distributed errors using the studentized residuals in the mixed procedure was used to test the assumption of homogeniety of variance. Differences between treatm ents t - test for all data collected: cortisol concentration, ACTH concentration, BCS, body weight, and pituitary gland grade, weight, length, and diameter, OGTT parameters (IB OGTT , Peak Ins OGTT , dINS OGTT , GB OGTT , Peak Gluc OGTT , and dGLUC OGTT ), and FSIGTT parameters (AIRg, DI, SI, Sg, IB FSIGTT , GB FSIGTT , dGLUC FSIGTT , dINS FSIGTT FSIGTTAUC G and FSIGTTAUC I ). Spearman correlation coefficients were determined between the PG grade, PG weight and ACTH concentration and each of the gluc ose metabolism parameters from the OGTT, i.e., IB OGTT , Peak Ins OGTT , dINS OGTT , GB OGTT , Peak Gluc OGTT , and dGluc OGTT , and from the FSGITT, i.e., AIRg, DI, SI, Sg, IB FSIGTT and GB FSIGTT . Spearman correlation coefficients were also determined between IB FSIGT T and SI, and between IB OGTT and Peak Ins OGTT . 20 Results PPID A ssessment Of the ten horses presenting clinical signs associated with PPID and thus admitted as possible PPID candidates, eight were confirmed with PPID ( horses 7 - 14 in Table 2 ). Criteria for confirming presence and assessing severity of PPID included the ODST results combined with the PG histologic scores and weights. The other 2 potential PPID horse candidates ( horses 1 and 2 in Table 2 ) could not be clearly classified as PPID and thus were excluded from the study. The four horses admitted as potential non - PPID candidates, i.e., control group ( horses 3 - 6 in Table 2 ), clearly suppressed the oral dexamethasone administration, and had PG weighing less than 3 g and histological grades of 3 and le ss. Variables BCS and BW, PG grade, PG L , PG W , and PG H , plasma ACTH concentration and cortisol concentrations pre and post - dexamethasone are presented in Table 1. The BCS and BW did not differ between control and PPID. Compared to control, the PG of PPID h orses was heavier (P = 0.012) and greater in length ( P = 0.035), width and height ( P = 0.002 and P = 0.001, respectively). Histological PG grades were greater ( P < 0.001) for horses with PPID compared to control horses. Compared to control horses, ACTH con centration tended to be greater ( P = 0.079) and post - dexamethasone plasma cortisol concentration was greater ( P = 0.008) in PPID afflicted horses. Results from the ODST (Table 2) revealed complete suppression in all 4 control horse s (horses 3 - 6), and parti al (horses 7 and 13) and failed suppression (horses 8, 9, 10, 11, 12 and 14) in the 8 PPID afflicted horses. OGTT and FS I G TT Glucose and insulin concentrations during the OGTT are presented in Figures 1 A and B, respectively . Glucose and insulin concen trations did not differ between control and PPID horses 21 at any time points of the OGTT. Four horses out of the PPID group and two of the control group had glucose values > 125 mg/dL at times 60, 75 and 90 min (data not shown). Plasma glucose and serum insu lin concentrations during the FSIGTT are presented in Figure 2 A though D. Glucose concentration of PPID horses tended to be greater than that of control at 90 min ( P = 0.099) (Figure 2 A and B). Insulin concentration tended to be greater at 3 min ( P = 0 .091), was greater at 8 min ( P = 0.068), tended to be greater at 23 ( P = 0.083) and 24 min ( P = 0.056), and greater at 25 ( P = 0.027) and 27 min ( P = 0.048). At one time point (16 min) prior to insulin infusion, insulin concentration of PPID horses was lo wer (P = 0.023) than that of control. Glucose and insulin concentrations during FSIGTT did not differ between PPID and control horses at any other ime points. Table 3 presents calculated and estimated parameters derived from glucose and insulin responses to the OGTT and FSIGTT. Baseline insulin OGTT , Peak Ins OGTT , GB OGTT , Peak Gluc OGTT , and dINS OGTT did not differ between PPID and control horses, and dGLUC OGTT tended to be lower ( P = 0.078) in PPID horses. The FSIGTT estimated parameters including AIRg, D I, SI, Sg, IB FSIGTT , GB FSIGTT , dGLUC FSIGTT , dINS FSIGTT , FSIGTTAUC I , or FSIGTTAUC G did not differ between PPID and control horses. Correlation coefficients between the PG grade, PG weight and ACTH concentration and each of the OGTT and FSGITT parameters are presented in Table 4. The PG grade and weight were poorly associated with most of the OGTT and FSIGTT parameters (0 < r < 0.5; P > 0.1). The PG weight correlated with Peak Gluc OGTT P < 0.01) and the PG grade and weight correlated with dGluc OGTT P P = 0.02, respectively). The ACTH concentration correlated with DI (r = 0.60, P = 0.04), Peak Gluc OGTT P < 0.01) and dGLUC OGTT (r = P = 0.02). The SI correlated with IB FSIGTT (r = 0.60, P < 0.04) and PeakIns OGTT correlated with IB OGTT (r = 0.72, P = 0.01). The IB OGTT correlated with IB FSIGTT (r 22 = 0.64, P =0.03), whereas, GB OGTT did not correlate with GB FSIGTT (r = 0.25, P = 0 .44). 23 Table 2.1 . Body condition score, body weight, pituitary gland characteristics, serum ACTH and cortisol concentrations of horses with PPID and horses without PPID. Variable Control PPID P - value BCS 4.75 ± 0.80 5.13 ± 0.57 0.710 BW 463.3 ± 21.6 433.8 ± 15.3 0.291 Liver Wt 5.236 ± 0.495 5.020 ± 0.350 0.730 Liver Wt/BW (%) 1.10 ± 0.10 1.20 ± 0.10 0.679 PG Grade Wt L W H ACTH Cortisol Pre - dex Post - dex 2.50 ± 0.268 2.28 ± 0.767 8.50 ± 1.63 19.50 ± 1.23 19.50 ± 0.87 9 23.00 ± 26.12 158.0 ± 36.7 10.0 ± 21.9 4.38 ± 0.19 5.18 ± 0.54 13.40 ± 1.56 25.75 ± 0.87 24.38 ± 0.62 85.63 ± 18.47 156.0 ± 25.9 99.3 ± 14.5 <0.001 0.012 0.035 0.002 0.001 0.079 0.972 0.008 BCS, body condition score; BW, body weight; PG, pituit ary gland; Wt, weight (g); L, length (µm); W, width (µm); H, height (µm); ACTH, adrenocorticotropic hormone (pg/mL); Cortisol, cortisol concentration samples (nmol/L pre and post dexamethasone administration; Pre - dex, pre - dexamethasone plasma cortisol conc entration sample (nmol/L); Post - dex, post - dexamethasone plasma cortisol concentration sample (nmol/L) . Two groups were included in this study: horses with PPID and non - PPID horses of > 19 years of age. 24 Table 2.2 Demographics, History, Clinical Signs, and ODST of horses with PPID and horses without PPID. Horse Treatment Breed Sex Age ODST 1 2 3 4 5 6 7 8 9 10 11 12 13 14 None None Control Control Control Control PPID PPID PPID PPID PPID PPID PPID PPID QH Pinto TWH TB TB Morgan Pinto Arabian TB Arabian Arabian Arabian QH QH M G M G G G G M M M M M M G 32 27 26 27 29 19 30 31 29 31 19 30 32 27 Partial Fail Suppressed Suppressed Suppressed Suppressed Partial Fail Fail Fail Fail Fail Partial Fail QH, quarter horse; TWH, tennessee walking h orse; TB, thoroughbred; G, gelding; M, mare; ODST, overnight dexamethasone suppression test. Two groups were included in this study: horses with PPID and non - PPID horses of > 19 years of age. 25 Table 2.3 OGTT and FSIGTT parameters of horses with PPID and h orses without PPID. Variable Control PPID P - value OGTT IB Peak Ins GB Peak Gluc dGLUC dINS FSIGTT AIRg DI SI Sg IB GB dGLUC dINS AUC I AUC G 8.45 ± 5.33 24.68 ± 10.49 96.20 ± 5.56 134.48 ± 8.62 39.47 ± 6.14 15.55 ± 6.42 185.9 ± 85.7 290 ± 278 2.86 ± 0.97 1.66 ± 0.35 9.24 ± 3.63 86.65 ± 3.83 47.08 ± 7.13 186 ± 23.0 508 ± 140 2515 ± 131 13.56 ±3.77 27.93 ± 7.42 94.43 ± 3.93 120.19 ± 6.09 24.73 ± 4.34 12.16 ± 4.54 333.3 ± 60.6 805 ± 196 2.55 ± 0.69 1.76 ± 0.25 12.09 ± 2. 57 89.18 ± 2.71 45.58 ± 5.04 203 ± 16.2 795 ± 98.9 2306 ± 95.6 0.452 0.805 0.800 0.206 0.078 0.676 0.190 0.161 0.803 0.821 0.536 0.602 0.867 0.552 0.124 0.221 OGTT, oral glucose test; IB, baseline insulin (mU/L); Peak Ins, peak insulin (mU/L); GB, bas eline glucose (mg/dL); Peak Gluc, peak glucose (mg/dL); OGT dGLUC, difference between peak glucose concentration at time sixty - five min and baseline glucose concentration at time zero min (mg/dL); OGT dINS, difference between peak insulin concentration at time sixty - five min and baseline insulin concentration at time zero min (mU/L); FSIGTT, frequently sampled intravenous glucose tolerance test; AIRg, acute insulin response to glucose (mU min L - 1 ); DI, disposition index (× 10 - 2 ); SI, insulin sensitivity (×1 0 - 4 mU/min/L); Sg, glucose effectiveness (% min - 1 ); FSIGTT dGLUC, difference between peak glucose concentration and baseline glucose concentration at time zero min (mg/dL); FSIGTT dINS, difference between peak insulin concentration and baseline insulin con centration at time zero min (mU/L); AUC Gluc , area under the glucose curve (mmol/L/180 min); AUC Ins , area under the insulin curve (mmol/L/180 min). Two groups were included in this study: horses with PPID and non - PPID horses of > 19 years of age. 26 Ta ble 2.4 . Pearson correlation coefficients (R) between pituitary gland grade, pituitary gland weight and ACTH concentration and each of the OGTT and FSIGTT parameters. FSIGTT and OGTT parameters Pituitary gland grade Pituitary gland weight ACTH R P - v alue R P - value R P - value OGTT IB 0.13 0.69 0.17 0.59 0.14 0.65 Peak Ins 0.00 0.99 0.01 0.97 0.07 0.83 dINS 0.11 0.74 0.17 0.60 0.24 0.46 GB 0.05 0.87 0.19 0.56 0.48 0.11 Peak Gluc 0.32 0.31 0.58 0.05 0.77 < 0.01 dGLUC 0.50 0.10 0.66 0.02 0.68 0.02 FSIGTT AIRg 0.32 0.31 0.37 0.23 0.40 0.20 DI 0.46 0.13 0.47 0.12 0.60 0.04 SI 0.03 0.93 0.12 0.71 0.31 0.32 Sg 0.10 0.76 0.13 0.69 0.14 0.67 IB 0.17 0.59 0.05 0.87 0.10 0.77 GB 0.01 0.98 0.12 0.70 0.36 0.26 OGTT, oral glucose test; IB, baseline insulin (mU/L); Peak Ins, peak insulin (mU/L); dINS, difference between peak insulin level and baseline insulin level at time zero min (mU/L); GB, baseline glucose ( mg/dL); Peak Gluc, peak glucose (mg/dL); dGLUC, difference between peak glucose concentration at time sixty - five min and baseline glucose concentration at time zero min (mg/dL); FSIGTT, frequently sampled intravenous glucose tolerance test; OGTT, oral gluc ose test; AIRg, acute insulin response to glucose (mU min L - 1 ); DI, disposition index (× 10 - 2 ); SI, insulin sensitivity (×10 - 4 mU/min/L); Sg, glucose effectiveness (% min - 1 ); AUC Gluc , area under the glucose curve (mmol/L/180 min); AUC Ins , area under the in sulin curve (mmol/L/180 min). Two groups were included in this study: horses with PPID and non - PPID horses of > 19 years of age. 27 Figure 2.1 . Plasma glucose (A) and serum insulin (B) concentrations during the OGTT. 28 Figure 2.2 . Plasma glucose during full FSIGTT and first 40 min (A and B) and serum insulin during full FSIGTT and first 40 min (C and D) concentrations during the FSIGTT. B A 29 Figure 2.2 . (con t * D * * 30 Discussion The concentr ation of basal plasma ACTH and dexamethasone suppression testing are accepted as the most sensitive and specific diagnostic tests for PPID (Perkins et al., 2002; Couëtil et al., 1996; van der Kolk et al, 1995; Dybdal et al., 1994), assuming careful interpr etation given the seasonal variation in cortisol and ACTH concentrations (Donaldson et al., 2005; McFarlane et al., 2004). In this study, all samples were collected between September and December of the same year. Only a handful of published studies howe ver have confirmed the sensitivity and specificity of the disorder histopathologically in addition to endocrine testing (Klinkhamer et al., 2011). McFarlane et al. (2005) reported a 79% agreement between post - and ante - mortem assessment. In our study, of the horses clearly classified as PPID following post - mortem examination of the PG, 75 % failed and 25 % partially failed to suppress the dexamethasone. The PG of these horses was one - fifth the size (H, L, and W) and weight relative to the horses classifie d as controls who had clearly all suppressed dexamethasone. This study reports on the relationship between PPID condition and insulin and glucose dynamics. Insulin resistance and central fat deposit in men has been well established to be associated wit h excess blood cortisol concentration and cortisol production rates (Purnell et al., 2009; Phillips et al., 1998; Filipovsky et al., 1996; Stolk et al., 1996) however the mechanis ms of action remains largely not understood (Rosmond, 2005) and poorly docume nted in horses. Our study is the first to use both the OGTT and FSIGTT to assess insulin sensitivity and glucose tolerance in PPID horses. The PPID and control horses had comparable body weights and condition scores, thus any difference in glucose metabol ism parameters would not be attributed to body fatness per se. 31 Our mean AIRg values fell within the reported range from 206 ± 88 to 973 ± 393 by Carter et al. (2009) and 67 to 805 mU min L - 1 by Bamford et al. (2014). However despite a near two - fold grea ter AIRg and FSIGTTAUC i for the PPID group relative to control (333.25 ± 60.58 vs. 185.85 ± 85.67 mU min L - 1 and 908 ± 136 vs. 518 ± 192 mU min L - 1 , respectively), these values did not differ, presumably due to the large variation. This variation was more attributed to the control group in part because of the smaller sample size but also because this was a more heterogeneous group by nature. The AIRg represents the pancreatic insulin secretion in response to an acute intravenous dose of glucose (Huffman e t al., 2009) and thus greater AIRg reflects an increased pancreatic response to glucose challenge (Treiber et al., 2005). The latter may be related to reduced insulin tissue sensitivity (Tiley et al., 2007), interference from other hormones (Huffman et al ., 2009) or delayed insulin clearance by the liver (Mittleman and Bergman, 2000). Both PPID and non - PPID horses in our study did not differ in their ability to effectively use glucose based on the estimated Sg values, which closely aligned with those of Pratt et al. (2005) (i.e.,1.77 ± 0.90 % min - 1 ). Similarly, parameters of glucose dynamics from the OGTT did not differ either between PPID and control horses. Half of the horses out of the PPID cohort and control group however presented some degree of glucose intolerance when basing glucose results on the reference values suggested by Frank et al. (2013). Frank et al. (2013) proposed that glucose concentration values exceeding 125 mg/dL during the OGTT are indicative of excessive glucose responses. Wh en based on the SI values, neither PPID nor control horses appeared to be insulin resistant, with values falling in the reported range of 0.471 to 5.43 ± 0.94 (×10 - 4 mU/min/L) (Pratt et al., 2005; Bamford et al., 2014). There was a noticeable 4 - fold great er DI for PPID relative to control horses despite a lack of statistical significance. The DI provides an indication of whether pancreatic insulin response is compensating for any degree of insulin 32 resistance (Boston et al., 2013). Thus this raises an impo rtant question as to whether our sample size limited our ability to clearly rule out an association between PPID and altered insulin metabolism. A similar remark can be made regarding the AIRg and in particular the FSIGTTAUC I . Indeed, the relatively stro ng positive correlation in particular between DI and ACTH concentration, and the higher insulin concentration following insulin administration may be further indicative of some degree of altered glucose metabolism in the PPID group. The results from the O GTT however ind icated that neither PPID nor control horses exhibited hyperinsulinemia, since insulin concentrations remained well below 45 mU/ mL (Frank et al., 2013). On the other hand, the FSIGTT represent an acute response to intravenous glucose and i s more sensitive compared to the traditional OGTT (Pratt et al., 2005). Prior studies have reported on the increased insulin resistance status of horses diagnosed with PPID in comparison to younger non - PPID controls (Klinkhamer et al., 2011; Garcia and Beech, 1986). Another study by Keen et al. (2004) reported insulin resistance in the PPID horse population as well, yet the majority of the test subjects in these studies were ponies who are known to be more susceptible to PPID than horses (Schott, 2002; van der Kolk, 1997). The inclusion of ponies (Couëtil et al., 1996; van der Kolk et al., 1993; Hillyer et al., 1992; Schott et al., 1992) is common in part due to the difficulty with recruitment of horses with PPID. Older horses have been shown to be mor e prone to insulin resistance than younger horses (Malinowski et al., 2002), with similar observations in humans (Barbieri et al., 2001; Paolisso et al., 1999). In a recent study by Mastro et al. (2015), PPID affected horses (no ponies) compared to age - ma tched controls did not differ in insulin sensitivity or glucose tolerance when assessed by the reciprocal of the square root of insulin (RISQI), the modified insulin to glucose ratio (MIRG), and isoglycemic hyperinsulinemic clamp approaches. Finally, PG w eight and PG grade 33 correlated poorly with the majority of parameters of glucose metabolism from the OGTT and FSIGTT. The negative correlation between ACTH and Peak Gluc OGTT , and ACTH and dGluc OGTT is puzzling. However, we have found that PPID horses have a reduced uptake of glucose across the small intestinal mu cosa as well as shorter villi (Chapter 4 ) and therefore may have reduced capacity for glucose absorption. This might also explain the lower glucose response to the OGTT in the PPID horses compare d to the controls . Consequently, the FSIGTT instead of the OGGT may likely be a better tool to assess glucose metabolism in PPID horses. Due to the lack of difference in insulin response in the OGTT and FSGITT between treatments , we were unable to classify any PPID horses as IR. One limitation of this study was the recruitment of geriatric horses that were otherwise healthy. Despite a clear homogeneity of variance across both treatment groups and normal distribution of all of the data, additional control h orses would have decreased variability, in particular that associated with DI, AIRg and FSIGTTAUC I . In conclusion, no difference in insulin sensitivity and glucose tolerance were found during either OGTT or FSGITT when comparing horses with PPID to their age - matched non - PPID control. Both PPID and control horses in this study had comparable body weight and condition score. Equal number of PPID and non - PPID horses exhibited signs of glucose intolerance during OGTT when evaluated with reference values est ablished by Frank et al. (2013). Although the DI was noticeably greater in PPID than control horses, the large variation in particular in the control group precluded detecting a statistical difference. Nonetheless, the positive and relatively strong cor relation between ACTH concentration and the DI, and the sustained higher insulin concentration following the i.v. insulin administration is indicative that there was compensation to some degree of insulin resistance in the PPID group. Again however 34 neithe r PPID nor control horses however could be classified as insulin resistant based on the estimated SI values alone. Nonetheless, our results suggest that geriatric horses and those in particular with PPID should be carefully managed with feeding diets cont aining low concentration of soluble carbohydrates. 35 CHAPTER THREE : Skeletal muscle expression of GLUT4, INSR, and SNAT2 in horses with pituitary pars intermedia dysfunction J.L. De Vries a , H.C. Schott II b , C.W. Ernst a , P.S. Weber b , J.P Steibel a , and N. L. Trottier a,* a Department of Animal Science, College of Agriculture and Natural Resources, Michigan State University, 474 S. Shaw Lane, Anthony Hall, East Lansing MI 48824 USA b Department of Large Animal Clinical Sciences, College of Veterinary Medi cine, 736 Wilson Rd., Veterinary Medical Center, East Lansing MI 48824 USA * Corresponding author. Tel.: +1 517 432 5140. E - mail address: trottier@msu.edu (N.L. Trottier). Work was completed at Michigan State U niversity in the Department of Animal Science in conjunction with the Department of Large Animal Clinical Sciences. This study was supported by the Morris Animal Foundation. Work from this study was in part presented at the American Society of Animal Scien ce Midwest meeting, Des Moines, Iowa, March 17 th , 2014 and at the European Workshop on Equine Nutrition, Leipzig Germany , September 29 th , 2014. We acknowledge Sue Wismer, Jenna Lubitz, Lee - Anne Huber, and Dave Chamberlin for their help in sample collectio n. We would also like to acknowledge Nancy Raney for assistance with gene expression analysis. 36 Abstract Pituitary pars intermedia dysfunction (PPID) is the most common endocrinopathy of the aging horse . Loss of dopaminergic inhibition of the pituitary par s intermedia results in an increase in blood concentration of adrenocorticotropic hormone (ACTH) and cortisol resulting in a range of clinical symptoms. Horses affected with PPID exhibit a range of clinical symptoms that may include lethargy, abnormal swea ting, hirsutism , and muscle loss. Peripheral nutrient utilization appears to be limiting based upon these clinical symptoms. Expression level of glucose and amino acid transport er and the insulin receptor in the gluteal muscle of horses with PPID were qua ntified and plasma amino acid profile was measured. We hypothesized that horses with PPID have a reduced mRNA abundance of SLC2A4 , INSR , and SLC38A2 encoding GLUT4, INSR, and SNAT2, as well as reduced protein expression of GLUT4 and insulin receptor ( INSR) in muscle tissue compared to non - PPID horses (control). The objectives were to 1) quantify mRNA abundance of SLC2A4 , INSR and SLC38A2 and 2) quantify protein abundance of INSR and GLUT4 in muscle tissue of PPID and non - PPID horses (control). Twelve horse s of multiple breeds and genders, and of similar ages ( 28.6 ± 1.5 and 25.3 ± 2.1 years for PPID and control, respectively) were donated for this study. Following admission to the study, horses were maintained on second cutting mixed orchardgrass and bromeg rass hay and senior feed at 1.8 and 0.5% of their BW, respectively for a period of 5 d while being monitored for medical stability. Presence and grade of PPID was determined based on basal ACTH concentration, overnight dexamethasone suppression test (ODST) and post mortem histopathological evaluation of the pituitary gland. Plasma samples were collected to measure the amino acid profile. Gluteal muscle samples were collected and flash frozen in liquid nitrogen , and stored at - 80°C. Total RNA was isolated an d assessed for purity followed by cDNA synthesis. Primers for target genes 37 SLC2A4 , INSR , and SLC38A2 (respectively encoding GLUT4, INSR, and SNAT2 ), and for the reference genes GAPDH, ACTB, and SDHA ( respectively encoding glyceraldehyde - 3 - phosphate dehyd - actin and succinate dehydrogenase complex, subunit A, flavoprotein ) were designed using equine gene sequences from GenBank. Quantification was done using q RT - PCR and abundance of each target gene expressed as fold change relative to control . Th e content of glucose transporter (GLUT4) and insulin receptor (INSR) was determined in muscle biopsies using Western blot analysis. Differences between treatments were determined using a mixed model in SAS, i ncluding treatment as a fixed effect and hors e n ested within treatment as the random term . Significant differences were evaluated at P 0.05 and tendency at P 0.1. Plasma aspartic acid w as greater ( P = 0.01) and ornithine tended to be greater ( P =0.07) in PPID compared to control horses . Plasma threonine ( P = 0.07) and carnosine ( P = 0.08) tended to be lower, and glutamine ( P = 0.05), m ethionine ( P = 0.04), 3 methyl histidine ( P = 0.02) were lower in PPID compared to control horses. Compared to control horses, mRNA abundance of SLC2A4 , INSR , and SLC38A2 , and protein abundance of GLUT4 and INSR in the gluteal muscle of PPID did not differ . The results of this study show that the muscle wasting associated with the pathology of the PPID condition is not due to a reduced utilization of glucose and amino acid at the systemic level in regards to SLC2A4 , INSR , and SLC38A2 abundance and content. 38 Introduction endocrine disorder affecting the aging horse population (van der Kolk., 1997; Schott, 2002). The disorder is characterized by hypertrophy, hyperplas ia, and adenoma of the pars intermedia of the pituitary gland resulting in increased circulating concentration of pro - opiomelanocortin peptides (POMC) including ACTH (Hillyer et al., 1992; van der Kolk et al., 1993; Love, 1993; Dybdal et al., 1994; van der Kolk et al., 1995; Eiler et al., 1997; Reed, 1998) . Horses diagnosed with PPID exhibit a variety of clinical signs most commonly including poor body condition, appearance of muscle wasting, abnormal fat deposits, hirsutism, and heightened risk for second ary infections ( Donaldson et al., 2004; Aleman et al., 2006; McFarlane, 2011; Klinkhamer et al., 2011; Inner å et al., 2013 ) . Despite their poor nutritional status, PPID - affected horses demonstrate increased a ppetite indicating that changes in intermediary metabolism are present and that peripheral nutrient util ization may be compromised. Abnormalities in glucose homeostasis or glucose intolerance have been reported in 57 to 94% (Hiller et al., 1992; van der Kolk et al., 1995; Keen et al., 2004) and insulin resistance in 45 to 90% of the PPID - afflicted horse population (van der Kolk et al., 1995; Keen et al., 2004) . In the work presented by Keen et al. (2004) and van der Kolk et al . (1995) h owever , half to 100% of the candidates were ponies, which are typical ly at higher risk for insulin resistance (Donaldson et al., 2004) . Conversely, more recent studies have reported absence of insulin resistance in horses with PPID (Vick et al., 2007; van den Berg et al., 2013; Mastro et al., 2015) . Reeves et al. (2001) an d more recently McGowan et al. (2013) suggested that insulin insensitivity may not be specific to PPID per se and Malinowski et al . (2002) proposed that insulin resistance in horses with PPID may be related to aging. So far, only one of the studies cited above (Mastro et al., 2015) included matched - age controls. We have also 39 shown (Chapter 1) that horses with PPID were not insulin resistant nor glucose intolerant compared to age - matched non - PPID horses based on an oral glucose tolerance test (OGTT) or fre quently sampled intravenous glucose tolerance test (FSIGTT). The data was indicative nonetheless of compensation to some degree of insulin resistance in the PPID group based on a relatively strong positive correlation between ACTH concentration and insuli n disposition index, and a sustained higher insulin concentration following the intravenous insulin administration. In adipocytes and muscle cells, insulin stimulates the translocation of GLUT4 to the plasma membrane (Huang and Czech, 2007) . The first determinant of insulin - dependent peripheral glucose uptake is the availability on the cell membrane of the GLUT4 tra nsporter (Huang and Czech, 2007) . With respect to AA transport, insulin activation involves in part recruitment of the Na + - coupled neutral AA transporter 2 (SNAT2) from an endosomal compartment (Dyer et al., 2009) . Molecules associated with recruitment and fusion of SNAT2 - containing ve sicles to the surface membrane are also implicated in the regulation of GLUT4 translocation (Dyer et al., 200 9). Insulin - mediated neutral AA uptake via SNAT2 is down - regulated in metabolic acidosis and insulin resistance, causing proteolysi s and muscle wasting in humans (Evans et al., 2008) . Based on these notions, the objectives of this study were to 1) quanti fy the mRNA abundance of SLC2A4 and INSR , an d protein abundance of GLUT4 and I NSR in the gluteal muscle tissue to further evaluate the insulin and glycemic status in the cohort of PPID and non PPID horses available for this study; 2) quantify the mRNA abu ndance of SLC38A2 in gluteal muscle tissue to assess whether neutral amino acid transport is compromised in PPID horses; and 3) determine the basal plasma amino acid profile to assess muscle protein metabolism. 40 We hypothesized that geriatric horses with PP ID have reduced mRNA abundance of SLC2A4 , SLC38A2 , and INSR , an d reduced protein abundance of INSR and GLUT4 compared to geriatric non - PPID horses. Materials and Methods All methods were approved by the Institutional Animal Care and Use Committee at Mich igan State University (03 - 12 - 064 - 001) . Animals and H ousing Eight horses confirmed with PPID and 4 non PPID (control) horses were donated for this and between the months of September and December as described earlier (Chapt er 1) . Horses were of multiple breeds and genders (mare and gelding) across groups with an average age of 25.3 ± 2.11 years for control and 28.6 ± 1.49 years for PPID horse. Following admission for the study, a ll horses were housed indoor, in a temperatur (3.05m × 3.05m or 3.66m × 3.66m ). All horses were fed second cutting mixed o rchard grass ( Dactylis glomerata L. ) and b rome - grass hay ( Bromus mollis L.) , and a commercial grain concentrate ( Equine Senio r, Purina) containing 14% crude protein , 1.8% fat and 18% crude fiber at 1.8 and 0.5% of their body weight, respectively. PPID and Insulin Status A ssessment Horses were evaluated for presence and grade of PPID, and insulin status as described elsewhere (Chapter 1). In brief, horses were subjected to ACTH testing (Beech et al., 2009) and an overnight dexamethasone suppression test (ODST ) ( Innerå et al., 2003) . Insulin sensitivity and glucose tolerance was assessed following an OGTT (Pratt et al., 2006) and a FSGITT ( Tóth et al., 2009) . 41 Collection of T issue Horses were euthanized on the day following the frequently sampled intravenous glucose tolerance test (FSGITT) (i.e., day 6) with pentobarbital (Fatal - Plus, Vortech Pharmaceuticals) (95 mg/kg BW) admi nistered intravenously using the jugular catheter already i n place. Immediately following confirmation of death, samples from various tissues were collected and archived. Middle gluteal muscle tissue was sampled and any fat and fascia was removed. The m uscle sample was cut into smaller pieces , transferred to conical tubes (~5 g) and immediately flash frozen in liquid nitrogen, and stored at 80°C . Amino Acid Profile Analysis of P lasma Plasma was collected and shipped to Agricultural Experiment Station C hemical Laboratories on dry ice (University of Missouri - Columbia, Columbia, MO) for free AA analysis according to Spackman et al. (1958) and Deyl et al. (1986 ). Plasma samples were analyzed for analysis of complete free AA profile concentration according to AOAC method ( AOAC Official Method , Agricultural Experiment S tation Chemical Laboratories, University of Missouri - Columbia College of Agriculture, Food and Natural Resources). Isolation of RNA and P reparation of cDNA Total RNA was isolated from the glut instructions using the mir Vana TM miRNA Isolation Kit (Ambion ® , LifeTechnologies Inc. TM ). Approximately 0.1 mg of tissue was weighed out and homogenized (Kinematica TM Polytron TM ) on ice for 3 - s intervals in 10 volu mes of lysis/binding buffer per tissue mass. The isolated RNA concentration was measured using a spectrophotometer (Nanodrop 1000, Thermo Scientific) and further assessed for both purity and confirmation of concentration (Agilent Bioanyalyzer 2100). 42 Compli mentary DNA was manufactured using a High - Capacity cDNA Reverse Transcription Kit (Applied Biosystems TM , Life Technologies Inc. TM ), diluted to 10 ng/ µ L - 1 , and stored at - 20°C . Measurement of SLC2A4 , SLC38A2 and INSR Relative mRNA Abundance by Quantitat ive Reverse Transcription PCR Primers for target genes SLC2A4 , SLC38A2, and INSR and for the reference genes GAPDH , ACTB , and SDHA (respec tively encoding glyceraldehyde - 3 - phosphate - actin and succinate dehydrogenase c omplex, subunit A, flavoprotein were designed using equine gene sequences from GenBank (National Center for Biotechnology Information, United States National Lib rary of Medicine) (Table 1) . Gene specific assays for SLC2A4 and SLC5A1 , and for all three reference genes were designed using Taqman ® Gene Expression Assays kit (Applied Biosystems TM , Life Sciences Technologies Inc. TM ) . Custom Taqman ® Gene Expression assa ys were designed for SLC38A2 and INSR on human equivalent sequences referenced in GenBank (National Center for Biotechnology Information, United State s National Library of Medicine) blasted against equine sequences (e!Ensembl, EMBL - EBI). Quantification was assessed using reverse transcription quantitative PCR in 96 - well plates (MicroAmp ® Fast Optical Plate, Life Technologies Inc. TM ). Plates were briefly centrifuged at 400 × g for 1 min. at 15°C, loaded into the ABI Prism 7000 Sequence Detection System ( Applied Biosystems TM , Life Technologies Inc. TM ), and analyzed with 7000 RQ Sequence Detection Systems Software (version 2.2.1, Applied Biosystems TM , Life Technologies Inc. TM ). Abundance of each target gene was calculated according to the following equation s (Livak and Schmittgen, 2001; Pfaffl, 2001): C T = C T target gene Average of C T reference genes. C T C T PPID - C T Control 43 Fold Change (FC) = 2 - C T Western Blotting Analysis The western blot analysis was performed acco rding to Annandale et a l. (2004) with the following modifications. The antibody against GLUT4 (rabbit polyclonal GLUT4 antibody) was graciously donated by Dr. Samuel Cushman ( Diabetes Centre, Lund University ) and has been used previously in horse tissues (Annandale et al., 2004 ) . The insulin receptor antibody was purchased from Millipore ( Anti - - Subunit), clone CT - 3 ) and has been previously used i n equine tissues (Kullman et al., 2015 ) . The secondary antibody was a goat anti - rabbit HRP - conjugat e from Pierce (Goat Anti - Rabbit IgG (H+L). C rude membrane fractions were prepare d from gluteal muscle by homogenizing 1 g of muscle tissue in 8 mL buffer [ (25 mM HEPES, 0.5 mM PMSF, 4 mM EDTA, and 5 µL protease inhibitor cocktail ( Protease Inhibitor Cocktail Set III, EMD Chemicals Inc.) at pH 7.4 ] . Samples were centrifuged at 100,000 × g for 1 h at 4°C, the pellet re - suspended in 1 mL of the original buffer + 1% Triton - X - 100, incubated for 1.5 h on ice, and centrifuged for 500 x g for 10 min at 4°C. The supernatant was analyzed for protein content with a Pierce BCA kit ( ThermoFisher S cientific ). SDS - PAGE gels were run with 25 µ g of each sample separated on 8 - 20% polyacrylamide gels (Criterion TGX Stain - Free Precast Gels, Bio Rad) and transferred to PVDF membrane (Trans - blot Turbo transfer pack, Bio - Rad). The membrane was blocked for 1 h with a commercially available blocking reagent (StartingBlock (TBS) Blocking Buffer, Thermo Scientific) and 0.1% non - ionic detergent ( Tween - 20, Sigma Aldrich) and blocked with the primary antibody (1:4000 for GLUT4; 1:1000 for INSR ) overnight at 4°C. The membrane was washed 5 times with 0.1 % (100 mL Tween 20/ Tris - Buffered Saline) TBST and secondary antibody (1:10,000 for GLUT4; 1:5000 for 44 INSR ) and incubated for 1 hr at 4°C. Images were immune - detected with a chemiluminescent kit (West Pico Chemilu minescent Substrate, Thermo Scientific). Statistical Analysis All data was normally distributed based on Gaussian distribution analysis in SAS (version 9.3, SAS Institute Inc., Cary, NC). Analysis of data was performed using the PROC MIXED procedure (SAS version 9.3, SAS Institute Inc.) with treatment as fixed effect and individual horse nested within treatment as random effect . The C T for mRNA and arbitrary units for protein expression. Pairwise d ifferences bet ween treatments were t - test. Treatment effects were represented as fold c hange by back transforming the estimated mean di fference in C T ( C T ) between control and PPID for each transporter of interest using the expression FC= 2 - C T according to Livak and Schmittgen (2001) . Following Steibel et al. (2009) , 95% confidence intervals of mean differences were also back transfor med to provide confidence interval for the estimated f o ld c hange. Fold changes with transformed confidence intervals spanning across the value of 1.0 were considered not significant at = 5%. Results Amino Acid Profile in Plasma The amino acid profile in plasma is presented in Table 1. The majority of amino acids (alanine, arginine, asparagine, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, pro line, serine, taurine, tryptophan, tyrosine, valine, urea, and citrulline) did not differ between PPID and control horses. Two amino acids, aspartic acid and ornithine, were greater ( P = 0.01) and tended to be greater ( P =0.07) in PPID horses when compared to 45 controls. T hreonine ( P = 0.07) tended to be lower, and glutamine ( P = 0.05), methionine ( P = 0.04), 3 - methylhistidine ( P = 0.02) were lower in PPID horses than that of control horses. SLC2A4 , INSR , and SLC38A2 mRNA Abundance in Gluteal Muscle Transcri pt abundance of SLC2A4 , INSR , and SLC38A2 in gluteal muscle of PPID (n=8) relative to control horses (n=4) is presented in Figure 1. The mRNA abundance of SLC2A4 , INSR , and SLC38A2 was no t different in PPID horses relative to control horses. GLUT4 and IN SR C ontent in Gluteal Muscle In equine skeletal muscle lysates, the GLUT4 and INSR antibody detected a single immunoreactive band that migrated with an apparent molecular mass of approximately 45 kDa (Figure 3 A and B) and 95 kDa , respectively (Figure 4 A and B ). Densitometric quantification of Western blot band intensities revealed no differences in protein abundance of GLUT4 or INSR between PPID and control horses (Figure 3A and 4A). 46 Table 3.1. Amino acid profile in the plasma of PPID and non - PPID horses ( µg/mL ). Amino Acid Control PPID P - value Alanine 13.52 ± 2.15 14.77 ± 1.52 0.64 Arginine 14.14 ± 1.41 13.06 ± 1.00 0.55 Asparagine 2.44 ±0.75 1.87 ± 0.53 0.55 Aspartic Acid 0.93 ± 0.20 1.74 ± 0.14 0.01 Cysteine 0 .00 ± 0.00 0 .00 ± 0.00 1 .0 0 Glutamic Acid 4.44 ± 1.75 7.66 ±1.24 0.16 Glutamine 45.24 ± 3.99 34.16 ± 2.82 0.05 Glycine 40.65 ± 3.86 38.17 ± 2.73 0.62 Histidine 11.16 ± 0.65 10.38 ± 0.46 0.35 Isoleucine 6.55 ± 0.68 6.19 ± 0.48 0.68 Leucine 12.65 ± 0.90 11.48 ± 0.64 0.31 Lysin e 15.07 ± 1.74 13.31 ± 1.23 0.43 Methionine 3.70 ± 0.30 2.85 ± 0.21 0.04 Phenylalanine 10.38 ± 0.58 9.62 ± 0.41 0.31 Proline 6.4 ± 0.56 6.73 ± 0.39 0.64 Serine 20.68 ± 2.29 23.52 ± 1.62 0.34 Taurine 4.58 ± 0.46 4.07 ± 0.33 0.38 Threonine 15.68 ± 1.66 11.50 ±1.18 0.07 Tryptophan 12.48 ± 1.54 12.35 ± 1.09 0.95 Tyrosine 10.93 ± 1.05 11.20 ± 0.74 0.83 Valine 20.87 ± 1.37 19.89 ± 0.97 0.57 Urea 298.17 ± 27.58 291.92 ± 19.51 0.86 3 - methylhisti di ne 4.09 ± 0.63 2.04 ± 0.44 0.02 Carnosine 4.63 ± 0.53 3.3 5 ± 0.38 0.08 47 Citrulline 9.99 ± 1.86 11.18 ± 1.31 0.61 Ornithine 6.16 ± 0.73 7.99 ± 0.52 0.07 48 Table 3.2 . Reverse transcription quantitative real - time (RT - qPCR) assay primer information in the gluteal musc le o f PPID and non - PPID horses. Gene Name Symbol Protein Taqman Assay/Accession number 1 Solute carrier family 38, member 2 Solute carrier family 2 (facilitated glucose transporter), member 4 Insulin receptor glyceraldehyde - 3 - phosphate dehydrogenase actin, be ta Succinate dehydrogenase complex subunit A SLC38A2 SLC2A4 INSR GAPDH ACTB SDHA SNAT2 GLUT4 I NS R GAPDH - actin SDHA NC_009149 NM_001081866.1 (10 - 11) XM_001496584.3 NM_001163856 NM_001081838 DQ402987 1 - Applied Biosystems TaqMan® assay product number or GenBank accession number of sequence submitted for Custom TaqMan® Gene Expression Assay design 49 Figure 3.1. Fold change in mRNA abundance of SLC2A4 , INSR , and SLC38A2 in the gluteal muscle for PPID (n=8) relative to control (n=4) horses . SLC2A4 50 Figure 3.2 . Western blot analysis of crude muscle membrane extracts p repared f rom gluteal biopsies for PPID (n=8) , represented as P1 - P9, and control (n=4) , represented as C1 - C4, horses treated with antibodies against GLUT4 depicted by (A) Western blotting showing protein expression level and (B) a graph indicating the relative expre ssion level. Intensities of bands were estimated by using the program "ImageLab" from BioRad. A C1 C 2 C 3 C 4 P1 P2 P3 P4 P5 P6 P7 P8 P9 45 kDa P1 P2 51 Figure 3. 3. Western blot analysis of crude muscle membrane extracts p repared from gluteal biops ies for PPID (n=8) , represented as P1 - P9 , and control (n=4) , represented as C1 - C4, horses treated with antibodies against INSR depicted by (A) Western blotting showing protein expression level and (B) a graph indicating the relative expression level. Intensities of bands were estimated by using the program "ImageLab" from BioRad . C1 A C2 C3 C4 95 kDa P 1 P8 P7 P6 P5 P4 P3 P2 P9 52 Discussion Horses suffering from PPID demonstrate poor physical appearance and muscle wasting while maintaining a high appetite (Donaldson et al., 2004; Aleman et al., 2006; McFarlane, 2011; Klinkhamer et al., 2 011 ; Innerå et al., 2013) . The condition is characterized by chronically elevated concentrations of glucocorticoids, namely ACTH and cortisol (Hillyer et al., 1992; van der Kolk et al., 1993; Love, 1993; Dybdal et al., 1994; van der Kolk et al., 1995; Eile r et al., 1997; Reed, 1998) . We reported earlier (Chapter 1) that horses with PPID in this study had significantly higher serum concentrations of ACTH and cortisol compared to non - PPID horses (Chapter 1). Basal ACTH serum concentrations were 85.6 ± 18. 5 a nd 23.0 ± 26.1 pg/mL in PPID and non - PPID horses (Chapter 1). Basal and post - dexamethasone cortisol concentrations were 158.0 ± 36.7 and 1 0 .0 ± 2 1 .9 nmol/L for control horses and were 156.0 ± 25.9 and 99.3 ± 14.5 nmol/L , respectively, for PPID horses . Gluc ocorticoids play a role in accelerating muscle proteolysis in rats with sepsis ( Löfberg et al., 2002) . High doses of prednisolone lead to a sharp increase in net protein catabolism in human patients (Ferrando et al., 1996) . In humans patients diagnosed wit - body protein synthesis and an increase in the rate of protein breakdown ( Burt et al. , 2007) suggested muscle atrophy to be a result of high circulating concentrations of g lucocorticoids. The high levels of glucocorticoids in horses diagnosed with the PPID condition are proposed to be responsible for the decrease in skeletal muscle tissue . In human patients diagnosed with decrease in the rate of whole - body protein synthesis and an increase in the rate of protein breakdown (Aleman et al., 2010) . The loss of epaxial and gluteal muscle s s een in PPID horses (McFarlane, 2011) result s from atrophy in types 1A and 2B muscle fibers , and loss of type 2B myofibers (Coderre et al., 1995) . However, the 53 mechanism responsible for the muscle wasting characteristic to PPID horses remains largely unknown. Co rtisol reduces GLUT4 translocation to the cell surface in rat skeletal muscle (Weinst ein et al., 1995; Dimitriadis et al., 1997; Ekstrand et al., 1996) and increase s lipolysis in human fat tissue (Kennedy et al. 1999, Filipovsky et al., 1996) . Insulin resistance and central fat deposit in men has been well established to be associated with excess blood cortisol concentration and cortisol production rates (Stolk et all., 1996; Phillips et al., 1998) . In this study, horses with PPID had significantly higher serum concentrations of ACTH and cortisol compared to non - PPID horses (Chapter 1). Ba sal ACTH serum concentrations were 85.6 ± 18. 5 and 23.0 ± 26.1 pg/mL in PPID and non - PPID horses (Chapter 1). Basal and post - dexamethasone cortisol concentrations were 158.0 ± 36.7 and 1 0 .0 ± 2 1 .9 nmol/L for control horses and were 156.0 ± 25.9 and 99.3 ± 14.5 nmol/L , respectively, for PPID horses . Similarly, horses with PPID have abnormal fat deposits across the crest of the neck, tail head, and abdominal region and several studies have reported insulin resistance in horses with PPID (Schott, 2002; Garcia and Beech, 1986 ) . It has been suggested that the major mechanism of insulin resistance may be related to a defect in the events post - receptor versus a defect at the INSR level (Glass, 2003) . Protein accretion and breakdown signaling pathways are affected by insulin (Glass et al., 2010; Suagee et al., 2011) therefore a decrease in I NS R protein abundance could explain muscle wasting in horses diagnosed with PPID. Suagee et al. (2011) reported reduction in GLUT4 and INSR in horses with elevated insulin levels . Studies in mice and humans indicate reductions in peripheral tissue responsiveness to insulin may be due to hyperinsulinemia (Nankervis et al., 1895; Marban and Roth, 1996; Barr et al., 1997) . The first determinant of insulin - dependent peripheral glucose uptake is the availability on the cell membr ane of the GLUT4 transporter (Andrew and Walker, 54 1999) . T he effect of glucocorticoids on insulin receptors has been examined with limited consensus among studies. Some studies have shown insulin resistance in PP ID horses when compared to non - PPID horses (Schott, 2002; Garcia and Beech, 1986) . However, horses were not age matched and it is possible that the decrease in insulin sensitivity seen was a function of age rather than the PPID status (Vick et al., 2007) . The INSR and GLUT4 mRNA abundance and protein expression was not different in PPID horses relative to control horses in our study. This is further confirmed in our previous work with no difference being found in either insulin sensitivity or glucose toler ance during either OGTT or FSGITT when comparing horses with PPID to their age - matched non - PPID control ( Chapter 1 ). Although the DI was noticeably greater in PPID than control horses, the large variation in particular in the control group precluded detect ing a statistical difference. Nonetheless, the positive and relatively strong correlation between ACTH concentration and the DI, and the sustained higher insulin concentration following the i.v. insulin administration is indicative that there was compensa tion to some degree of insulin resistance in the PPID group. Hyperinsulinemia is associated with insulin resistance (Carter et al., 2009; Frank et al., 2011) . Whether the indications of insulin sensitivity reported here and elsewhere (Schott, 2002; Garcia and Beech, 1986) are due to the severity of the PPID condition or age alone remains inconclusive. Metab ol ic acidosis in humans with chronic renal failure has been shown to cause wasting of lean tissue (Mitch et al., 1994) , si milar to the muscle wasting seen in horses with PPID. Muscle wasting in these patients has been attributed to low pH causing inhibition of L - Gl n transport via SNAT2 (Evans et al., 2007) . Our study reported no effect of PPID status on the expression of SNAT2 in the gluteal muscle. Fu rther, we sought to evaluate the plasma amino acid profile differences in PPID horses when compared to non - PPID control horses. Studies hav e 55 shown that SNAT2 inhibition indirectly depleted other amino acids whose intracellular concen trations are maintained by the L - Gln gradient across the plasma membrane (Evans et al., 2007) . In this respect, SNAT2 may determine intracellular anabolic amino acid levels thus regulating the signaling that affects protein mass, nucleotide/nucleic acid metabolism, and cell grow th (Evans et al., 2007) . A study by Bergstrom et al. (1990) has shown the depletion of intramuscular free amino acids to be an early step in muscle wasting of human uremia patients. Interestingly, the correction of acidosis reverses intramuscular free ami no acid depletion (Löfberg et al., 1997) . This depletion is sensed through the protein synthesis stimulating mammal ian target of rapamycin (mTOR) (Proud, 2004) . As shown in Mastro et al. (2014) , a numerical reduction in mTOR signaling was indicated in PPID horses when compared to control horses, thus potentially making it difficult for horses afflicted with the PPID condition to detect a depletion in free amino acids. A study in geriatric humans with trauma by Jeevanandam et al. (1990) demonstrated a reduct ion in lean body mass to be related to a reduced protein metabolic response to trauma, namely due to changes in free pl asma amino acid concentrations. T he PPID horses of our study showed a similar decrease in methionine and an increase in ornithine concent ration, suggesting that the change in free amino acid concentrations seen in PPID horses could be related to the stress of the PPID condition. Further, the urinary excretion of 3 - methylhistidine (3 - MH) has been used widely in humans and animals as a biolog ical marker for skeletal protein breakdown (Young et al., 1972; Long et al., 1988) . In our study, horses with PPID had a reduced concentration of 3 - MH in their plasma compared to control horses, indicating that they had a reduced product of protein degrada tion. This is contrary to what would be expected. However, in times of prolonged starvation in human patients in a study by Long et al. (1977) , 3 - MH excretion in urine decreases. This decrease in 3 - MH is an adaptive attenuation 56 of muscle breakdown and a re duction in lean body mass (Long et al., 1977) . Horses with PPID are widely characterized by a poor physical appearance and muscle wasting, mimicking the effect of a body during times of prolonged starvation (Donaldson et al., 2004; Aleman et al., 2006; McF arlane, 2011; Klinkhamer et al., 2011; Innera et al., 2013) . Human patients with severe abdominal sepsis, display a decreased plasma concentration of glutamine, indicating intracellular glutamine formation in muscle tissue may be compromised (Roth et al., 1982) . In addition, reduced plasma glutamine concentration has been reported in patients with untreated diabetes mellitus, diet - induced metabolic acidosis, and in the recovery period following high intensity intermittent exercise (Walsh et al., 1998) . Comm on among the above mentioned states of stress are the rises in plasma cortisol and glucagon concentrations and an increased tissue requirement for glutamine for gluconeogenesis (Walsh et al., 1998) . Together, this suggests that intracellular glutamine form ation in the muscle tissue plays a role in the characteristic muscle wasting of PPID horses. Aleman et al. (2010) reported that muscle wasting in PPID horses was not caused by reduced expression of major proteolytic systems and growth regulators. However , the numerical reduction in mTOR signaling in PPID horses when compared to non - PPID control horses could potentially explain, in part, some of the mechanism behind muscle wasting (Aleman et al., 2010) . Mastro et al. (2015) reported that muscle fractional protein breakdown rate was not affected by PPID status when compare to control horses without PPID. Additionally, in this same study, there was n o impact of PPID status relative to control horses on the abundance or activation of positive and negative regu lators of protein synthesis and positive regulators of protein breakdown . Other factors must be at work to account for the muscle wasting typically seen in the PPID condition. Early reports by Ralston et al. (1989) reported a decrease in apparent crude 57 pro tein digestion in aged horses in relation to younger control animals, but were later followed by showing no decrease (Ralston et al., 2001) . A study in rats found the splanchnic extraction of amino acids to be reduced by 20 - 30% in aged rats when compared t o younger controls (Jourdan et al., 2011) . Human studies have found a similar trend in whole body muscle protein synthesis declining with age (Short et al., 2004) . Future studies in PPID horses should be compared not only to aged non - PPID horses, but youn ger non - PPID and PPID affected horses in an effort to better understand those characteristics which are a function of age and which are related to the PPID condition. In summation, w hether a decrease in utilization of glucose and amino acid at the system ic level contribut es to the pathology of PPID remains largely unknown. Additionally, whether the indications of insulin sensitivity reported here and elsewhere (Schott, 2002; Garcia and Beech, 1986) are due to the severity of the PPID condition or age alon e remains inconclusive. Although the gene expression of SLC2A4 , INSR , and SLC38A2 was not different in PPID horses compared to controls, additional factors related to muscle atrophy such as posttranscriptional and posttranslational mechanism that affect pr otein production, activation or inactivation, and function may be responsible and should receive further attention. The changes in free amino acid concentrations in the PPID horses of our study could be related to the stress of the PPID condition . Specific ally, the role intracellular glutamine formation plays in the muscle tissue plays is indicative of muscle wasting of PPID horses. We have investigated the role of the SI in reduced nutrient absorption and utilization of glucose and glutamine in regards to the structural and functional integrity of the promixal and mid small intestinal mucosa and found lower morphological changes in villi and cryptal layers, and decreased glucose absorption across the jejunal brush border membrane in PPID horses compared to controls ( Chapter 3 ). Future studies 58 should focus on distinguishing the effects of age versus the PPID condition alone. The results of this study show that the muscle wasting associated with the pathology of the PPID condition is not due to a reduced utili zation of glucose and amino acid at the systemic level in regards to SLC2A4 , INSR , and SLC38A2 abundance and content. Many factors affect nutrient absorption and muscle wasting and thus diligent research should be continued in PPID horses to determine the mechanisms behind the condition. In conclusion, our results suggest that geriatric horses and those in particular with PPID should be carefully managed with feeding diets containing low concentration of soluble carbohydrates in order to reduce the risk of laminitis . 59 CHAPTER FOUR : Morphology of the s mall intestin al mucosa in horses with pituitary pars intermedia dysfunction J.L. De Vries a , H.C. Schott III b , J. Lubitz a, 2 , and N.L. Trottier a,* a Department of Animal Scieonce, Michiga n State University, 474 S. Shaw Lane, East Lansing MI 48824 USA b Large Animal Clinical Sciences, Department of Veterinary Medicine, A118 Veterinary Medical Center, East Lansing MI 48824 USA * Corresponding author. Tel.: +1 517 4325140. E - mail addre ss: trottier@msu.edu (N.L. Trottier). 2 Present address: Veterinary Medical Center, 736 Wilson Rd, East Lansing MI 48824 USA 60 Abstract The objective was to evaluate the morphology of the small intestinal mucosa of horses with PPID and without PPID. Eight PPID and 4 non PPID (control) of m ultiple breeds and genders, and over 19 years of age were donated for this study . Horses were maintained on second cutting grass timothy hay and senior feed at 1.8 and 0.5% of their BW, respectively for a period of 5 d. H orses were euthanized , the entire gastrointestinal tract removed and samples collected from the duodenum at 25 and 50 cm distal to the pyloric sphincter, the mid - jejunum and the mid - ileum. Compared to control horses, villus height proximal average of PPID horses was lower ( P < 0.05) and di d not differ in duodenum at 25 cm or 50 cm, mid - jejunum or ileum . Villus width did not differ between PPID and control horses across any segment or proximal average . Compared to control horses, v illus crypt depth of PPID horses tended to be greater in mid - jejunum ( P < 0.10 ) , and did not differ in duodenum at 25 or 50 cm, mid - jejunum, or the ileum. Compared to control horses, VH:CD of PPID horses was lower in mid - jejunum ( P < 0.0 5), and did not differ between PPID and control horses in duodenum at 25 or 50cm , ileum, or proximal average. In conclusion, PPID in horses is associated with small intestinal villi atrophy and greater VH:CD in proximal to mid regions of the small intestine. Keywords: histology; small intestine; pituitary pars in termedia dysfunction, horse 61 Introduction Pituitary pars intermedia dysfunction (PPID) is an endocrine disorder primarily affecting the geriatric horse (Schott, 2006) . It is a progressive condition characterized by hypertrophy, hyperplasia, adenoma and loss of dopaminergic inhibition of the pars intermedia of the pituitary gland resulting in increased circulating concentration of pro - opiomelanocortin peptides (POMC), including ACTH ( Hillyer et al., 1992 ; S Love, 1993; van der Kolk et al., 1993 ; Dybdal et al., 1994 ; van der Kolk et al., 1995 ; Eiler et al., 1997 ; SM Reed, 1998 ) . Horses diagnosed with PPID exhibit a variety of clinical signs, most commonly including poor body condition, appearance of muscle wasting, abnormal fat deposits, hirsutism, and heig htened risk for secondary infections ( Donaldson et al., 2004; Aleman et al., 2006; McFarlane et al., 2010; Klinkhamer et al., 2011; Innerá et al., 2013 ). Pituitary pars intermedia dysfunction has also been associated with an increased risk for laminitis (D onaldson et al., 2004; Carter et al., 2009; Geor, 2009 ). Despite a characteristically high appetite (K.G., Mountjoy, 2010) , physical appearance of horses with PPID indicate that poor nutrient absorption may play a role in the patho physiology of the conditi on . A variety of ailments targeting the small intestinal mucosa have been shown to cause malabsorption , including lesions (Platt, 1986), chronic inflammatory and lymphoproliferative disorders of the small intestine (Pass and Bolton, 1982), alimentary l ymphomas (Platt, 1986) and mycobacterial infections (Merritt et al., 1975). Changes in the histomorphology of the small intestinal mucosa, including villi atrophy and increased cryptal layer, are related to poor nutrient absorption and malnutrition in anim als (Stevenson et al., 2013) and humans (Dewar and Ciclitira, 2005; and Ventura et al., 2013). The clinical and gross pathological features of these conditions are not readily distinguishable and thus histopathological examination of intestinal biopsy or 62 p ost - mortem specimens for diagnosis are necessary (Platt, 1986). Histomorphometric analysis of the small intestinal mucosa in horses afflicted with PPID has not been previously documented. We hypothesized that mucosal villus height and crypt depth of the small intestine is lower and greater, respectively in horses with PPID . The objective was to evaluate the morphology of the mucosa in the proximal, mid and distal regions of the small intestine in horses clinically confirmed with PPID and no PPID . Mater ials and Methods All methods were approved by the Institutional Animal Care and Use Committee at Michigan State University (03 - 12 - 064 - 001). Animals and H ousing Eight horses confirmed with PPID and 4 non PPID (control) were donated for this study following all within the fall of 2012 as des cribed in Chapter 2 . Horses were of multiple breeds and genders (mare and gelding) across groups ranging from 19 to 39 years with an average age of 25.3 ± 2.11 years for control and 28.6 ± 1.49 years for PPID horse. Following admission for the study, a ll horses were 3.05m × 3.05m or 3.66m × 3.66m ) at the Michigan State University College of Veterinary Medicine Research an d Instructional Building. All horses were fed second cutting mixed o rchard grass ( Dactylis glomerata L. ) and b rome - grass hay ( Bromus mollis L.) , and a commercial grain concentrate ( Equine Senior, Purina, St. Louis, MO) containing 14% crude protein , 1.8% fa t and 18% crude fiber at 1.8 and 0.5% of their body weight, respectively. 63 Clinical E valuation Clinical evaluation has been described in detailed elsewhere ( Chapter 2 ). In brief, horses were subjected to ACTH testing as described in Beech et al. (2009) , an overnight dexamethasone suppression test (ODST) as described by Innera et al. (2013), an oral glucose tolerance test (OGTT) per Pratt et al. (2006), and a frequently sampled intravenous glucose tolerance test (FSGITT) was performed as described in Tót h et al. (2009). Collection of T issue Horses were euthanized on the day following FSGITT (i.e., day 6) with pentobarbital (120 mL Fatal Plus at 392 mg/mL) administered intravenously using the jugular catheter already in place. Following confirmation of death, the skull was opened on the dorsal aspect to excise the brain and access the pituitary. The pituitary gland was gently dissected out, weighed, and measured for length and diameter. The gland was cut in half with one half flash - frozen in liquid N fo r tissue archive and the other half transferred into 10% formalin solution (10mL Formalin/100mL H20) and submitted to the Histopathology lab for histo pathological examination. Slides were stained with hematoxylin and eosin (HE) and graded as described in Miller et al. (2008). Immediately following confirmation of death, horses were positioned in either the right or left lateral recumbent position and an incision was made along the ventral midline of the abdominal cavity to expose and remove the entire gas trointestinal tract. S amples (25 - cm long segment) were collected from the duodenum at 25 cm (Duo25) and 50 cm (Duo50) distal to the pyloric sphincter, the mid - jejunum (Jej) , and the ileum (Ile) at 15 cm proximal to the cecum. 64 Approximately 5 - cm long tran sversal cut of each collected segments were kept intact and gently rinsed in ice - cold NaCl solution (0.9 g NaCl/100 mL H 2 O ) , and transferred in formalin - distilled water solution (10% vol/vol) for fixation over a 24hour period. Following fixation, segments were transferred to an ethanol - distilled water solution (30% vol/vol) until histological processing. Histological Examination of the Small I ntestine For histological visualization and morphometric an alysis of the intestinal mucosa, each segment was cut longitudinally to expose the mucosa, with two transverse sections (approximately 0.4 - cm thick) at the proximal and dorsal end and placed in histological cassettes. Cassettes were sent to the Michigan State University Histopathology Laboratory for processin g. Tissue samples were vacuum infiltrated with paraffin (Excelsior tissue processor, ThermoFisher Scientific Inc., Waltham, MA) and embedded (HistoCentre III, ThermoFisher Scientific Inc., Waltham, MA). The resulting blocks were cut using a microtome (Reic hert Jung 2030 rotary microtome, Rankin Biomedical Corporation, Holly, MI) in fine sections at 4 - 5 microns and mounted via incubation at 56 °C for 2 - 24 hrs. Slides were stained with Hematoxylin and Eosin ( Pureview® Hematoxylin and Eosin Stain System , Cance r Diagnostics Inc. Durham, NC ) according to Porter and Joseph (2015). Histological visualization of the intestinal mucosa was performed at 200 × magnification (Nikon Eclipse Ti Inverted Microscope) and measurements performed using a microscope imaging sof tware (NIS Elements Microscope Imaging Software) at 100 × magnification. For each segment, a minimum of 10 well - defined villi and associated crypts were measured for villus 65 height (VH), villus width (VW), and crypt depth (CD) . T he ratio of height to crypt depth (VH:CD) and average or proximal segments (Duo25, Duo50 and Jejunum) was calculated. Statistical Analysis Normal distribution of data was assessed using the Gaussian distribution analysis in SAS (version 9.3, SAS Institute Inc., Cary, NC). Analysis of data was performed using PROC MIXED procedure of SAS (SAS version 9.3, SAS Institute Inc., Cary, NC) . The model includ ed segment and treatment as fixed effects and horse (treatment) as the random term . S egment was included as a repeated measure. D iffer ence in VH, VW, CD and V:C between treatments were determined t - test with s ignificant differences evaluated at P 0.05 and tendency at P 0.1. Results Compared to control horses, villus height of PPID horses was lower in the proximal average of segments and did not differ in the duodenum at 25 cm or 50 cm, mid - jejunum or ileum . Villus width did not differ between control and PPID horses across any segment or the average of proximal segments . Compared to control horses, v illus crypt depth of PPID horses tended to be greater in the mid - jejunum ( P < 0.10 ) , and did not differ in the duodenum at 25 or 50 cm, ileum , or the average of proximal segments . Compared to control, V:C of PPID horses was lower in the mid - jejunum ( P < 0.0 5) and did not differ in the duodenum at 25 cm or 50 cm, Ileum, or the average of proximal segments. The proximal average did not differ between control and PPID horses in VH ( P = 0.181), VW ( P = 0.803), CD ( P = 0.334), or V:C ( P = 0.244). The proximal average of villus height in control horses was 518 ± 56.7 and 418 ± 40.1 in PPID 66 horses. Villus width proximal average in control horses was 183 ± 9.07 and 185 ± 6.42 in PPID horses. The proximal average of crypt depth in control horses was determined to be 189 ± 13.1 in non - PPID horses and 173 ± 9.23 in PPID horses. The V:C proximal average in control horses was 2.80 ± 0.26 and 2.41 ± 0.18 in PPID h orses. 67 Figure 4.1. Villus height of the small intestine in PPID (n=8) and control (n=4) horses. Duo25, duodenum at 25 cm; Duo50, 50 cm distal to the pyloric sphincter; Jejunum, mid - jejunum; Ileum, mid - ileum 15 cm pr oximal to the cecum. 68 Figure 4.2. Villus width of the small intestine in PPID (n=8) and control (n=4) horses. Duo25, duodenum at 25 cm; Duo50, 50 cm distal to the pyloric sphincter; Jejunum, mid - jejunum; Ileum, mid - ileum 15 c m proximal to the cecum. 69 Figure 4.3. Villus crypt depth of the small intestine in PPID (n=8) and control (n=4) horses. Trending difference from control horses ( P Duo25, duodenum at 25 cm; Duo50, 50 cm distal to the pyloric sphincter; Jejunum, mid - jejunum; Ileum, mid - ileum 15 cm proximal to the cecum. 70 Figure 4.4. Villus height:Crypt Depth (V:C) of the small intestine in PPID (n=8) and control (n=4) horses. *Significantly different from con trol horses ( P Duo25, duodenum at 25 cm; Duo50, 50 cm distal to the pyloric sphincter; Jejunum, mid - jejunum; Ileum, mid - ileum 15 cm proximal to the cecum. 71 Discussion Clinical manifestations of PPID in horses include poor body condition, abnormal fat deposits, muscle atrophy , lethargy and hirsutism (Donaldson et al., 2004; Aleman et al., 2006; McFarlane et al., 2010, Klinkhamer et al., 2011; Innerá et al., 2013). The PPID condition is characterized by excess ACTH production, resulting in high concentra tion of cortisol. Sustained high levels of cortisol are associated with suppression of the immune and inflammatory response systems in humans (Reiche et al., 2004). Humans with PPID have chronic elevated cortisol concentration and suffer from d egeneration of muscle, bone, and neuronal tissue (Melzack and Katz, 2004), impaired digestion (Jones and Quinn, 2005) and a weakened immune system ( Cohen and Williamson, 1991 ; Andersen, et al., 1994 ). High doses of corticosteroids have also been shown to cause mucosa l lesions on the duodenum in humans (Kiziltas et al., 1998), which decreases the protective barrier function of the mucosa (Travis and Menzies, 1992). Insulin resistance and central fat deposit in men has been well established to be associated with excess blood cortisol concentration and cortisol production rates (Filipovsky et al., 1996; Stolk et al., 1996; Phillips et al., 1998; Purnell et al., 2009) however the mechanisms of action remains largely misunderstood (Rosmond, 2005) and poorly documented in h orses. While the etiology of PPID is multifaceted, horses suffering from this condition demonstrate poor physical appearance suggesting that optimum nutrient utilization and (or) absorption may be compromised. I ntestinal morphology is of vital importance in dictating nutrient absorption (Dewar and Ciclitira, 2005; Stevenson et al., 2013; Ventura et al., 2013) and gut health (Xu et al., 2003). Gut health is compromised in the face of toxins, resulting in rapid changes of the intestinal mucosa morphology as seen by a reduction in villus height and increase in crypt depth of broiler chickens (Yason et al, 1987). In humans affected by Celiac disease, 72 inflammation of the small intestinal mucosa induces a malabsorption syndrome and intestinal lesions including re duced villus height, increased villus width, and increased cryptal layer (Murray, 1999). There is very little information on the intestinal mucosa histomorphometry of horses and to date, the impact of PPID on intestinal mucosa morphology has not been repo rted. In this study, horses suffering from PPID displayed alterations in their mucosal architecture including reduced villi height, greater crypt depth, and lower villus height to crypt depth ratio in the proximal and mid regions of the small intestine. The VH values along the small intestinal mucosa ranged from 491 ± 24.8 to 559 ± 73.1 µm in control and 372 ± 47.6 to 490 ± 17.5 µm in PPID horses. Values for the control horses nearly fall within range to some degree with those reported by Lindberg and K arlson (1985), i.e., 600 - 860 µm. This discrepancy may be attributed to the age difference between the horses in this study,i.e., > 19 years and those of Lindberg and Karlson (1985), i.e., > 4 years). Villus height decreased with age in rabbit jejunal muco sa when comparing young and weanling rabbits (Keelan et al., 1985). Studies in male rats have shown a similar trend of decreased villus height when moving from the proximal to distal region of the small intestine seen in the horses of our study (Hohn et al ., 1978). Villus width in our study ranged from 167 ± 8.22 to 193 ± 11.6 µm in control and 173 ± 5.93 to 196 ± 8.18 µm in PPID horses, which fall within previous range of 150 to 250 µm reported by Lindberg and Karlson (1985). The ratio of villus height: c rypt depth (V:C) is considered to be a better indicator of mucosal integrity and nutrient absorption than villus height alone (Montagne et al., 2003). The V:C increased from Duo25 to Duo50 and decreased from Duo50 to the jejunum in both PPID and control ho rses, and increased in control and decreased in PPID from jejunum to ileum. In pigs, V:C also decreased from the proximal to distal regions moving from the duodenum to jejunum (Guay et al., 2006). The V:C ranged from 2.54 ± 0.23 to 3.18 ± 0.25 in control a nd 2.22 73 ± 0.15 to 2.55 ± 0.25 in PPID horses and are comparable to those in pigs ranging from 1.5 ± 0.11 to 3.29 ± 0.25 (Spreeuwenberg et al., 2001; Guay et al., 2006), and those of humans ranging from 1.59 ± 0.12 to 2.50 ± 0.28 (Pironi et al., 1994). Grea ter villus height and shallower crypt depth are related to increased nutrient absorption (Xu et al., 2003). Increased villus extrusion in the duodenum and ileum is a common response to infection (Domeneghini et al., 20 06). In an effort to maintain function al integrity, the intestine compensates for infection by greater activity in the crypts causing them to become deeper (Domeneghini et al., 20 06). The mechanisms by which PPID may potentially compromises in testinal function are unknown, however, m orphologic al changes in intestinal mucosa of horses with PPID are similar to those seen in horses and other animal species exposed to immunological challenges. For instance, horses with equine granulomatous enteritis (EGE) display villous atrophy and pronounced len gthening of the crypts (Lindberg, 1984). Equine granulomatous enteritis is associated with carbohydrate malabsorption and excessive protein loss into the gut (Meuten et al., 1978). Other species exhibit similar morphological changes as seen in this study. In humans , c eliac disease is an inflammatory bowel disease characterized by inflamed and flattened villi, resulting in impaired absorption function (Murray et al., 1999; Dewar et al., 2005). Severe villi atrophy in all segments of the small intestine is ch aracteristic of porcine epidemic diarrhea virus resulting in malabsorption (Stevenson, 2013). The absorptive capacity of humans with Giardiasis is decreased due to villi shortening (Ventura et al., 2013). Intestinal mucosa development and maintenance is a chieved through cell renewal and extrusion (Yamauchi et al., 1996). A balance between these events ensures constant turnover of old to new cells and thus maintenance of villus height (Yamauchi et al., 1996). For instance, long periods of fasting are assoc iated with large lysosomal autophagic vacuoles causing cell death 74 which leads to an increased rate of extrusion and reduction in villus size (Yamauchi et al., 1996). Crypt depth in our study ranged from 174 ± 12.9 to 198 ± 24.7 µ m in control horses and 164 ± 9.13 to 222 ± 8.64 µ m in PPID horses, which compare to previous reported values in pigs ranging from 102 ± 16.4 to 169 ± 14.4 µ m (Pluske et al., 1996). The crypts are a source of replacement cells for the entire villus; a greater crypt depth, as seen in the jejunum of PPID horses when compared to controls in our study, indicates a high demand for new tissue and thus a greater rate of tissue turnover (Yason et al., 1987). This increased turnover requires a greater energy and protein demand from diet in or der to sustain gut morphology (Luk et al., 1980; Alican and Kubes, 1996; Burrin and Reeds, 1997; Reeds et al., 1997). Overall, if the increased energy and protein demand from diet is not adequately met, a change in intestinal morphology can result reducing amino acid availability for maintenance of muscle mass . The inflammatory response in the small intestine has been extensively studied for many disorders. Our study did not address this area, but additional studies should seek to address this aspect based on the following available literature. Celiac disease is characterized by increased numbers of lymphocytes, plasma cells, and macrophages in the lamina propria and increased lymphocytes numbers in the surface layer of the epithelium (Murray, 2014). Entero cytes of Celiac - affected individuals are decreased in height, increased in width, and deeper in crypt depth, and more poorly nucleated in comparison to enterocytes of healthy individuals (Murray, 2014). Additionally, the intestinal damage as seen in Celiac disease is mediated largely by cellular immunity. Studies by MacDonald (1990) have shown in Celiac disease that the inflammatory response likely affects both the structural support and microcirculation of the villus resulting in villus collapse. It is unk nown whether immunological factors play any roles in the pathology of PPID and the associated mucosal morphological change. Human studies in patients with 75 dopami nergic neurons by activated macrophage glial cells (Dauer et al., 2003) which secrete inflammatory cytokines and free radicals (McGeer et al., 1988). Glucocorticoid levels higher in horses affected by the PPID condition (Wilson et al., 1982; Horowitz et al ., 2003), and as such they may be immunologically compromised. However, McFarlane and Holbrook (2008), reported that horses with PPID exhibit decreased pro - inflammatory cytokine expression when compared to healthy aged horses, which may be related to their high plasma concentration of anti - inflammatory hormones. These same authors reported increased expression of IL - 8 and suggested that horses with PPID have a decreased ability to respond to pathogen challenge. Specific nutritional guidelines for PPID hor ses have yet to be established. However, digestion is improved and incidence of GI diseases reduced when smaller, more frequent meals are fed, access to water increased, and starch (corn) intake reduced in geriatric horses (Ralston, 1990; Ralston, 1999; Pu gh, 2000). Based on our previous results and the ones presented herein, we suggest that geriatric horses and those in particular with PPID should be carefully managed with feeding diets containing low concentration of soluble carbohydrates. Currently, ther e is no cure for the PPID condition. Serotonin antagonists (cyproheptadine) and dopamine agonists (pergolide mesylate or Prascend® ) are drugs commonly used to treat the PPID condition (van der Kolk, 1997; Schott, 2002). The use of cyproheptadine has been d iscouraged (cited in Schott, 2006) based on an improvement in the condition with management alone. While pergolide is considered the drug of choice, no studies have been performed in horses to verify its safety nor bioavailability and metabolism. Additiona lly, there is no evidence that early medical intervention has an effect on long - term outcome. Therapies targeted at improving GI function of the PPID affected horse are needed. P robiotics and prebiotics in mice reduce intestinal turnover and 76 modulate the i mmune system (Bi et al., 2004). Santos Rocha et al. (2012) reported that supplemental feeding of threonine or mannan oligosaccharides in Salmonella birds increased villus height, decrease the crypt depth, and subsequently reduces V:C when compared to healt hy birds. The results of this study show that the structural integrity of the proximal to mid small intestinal mucosa is compromised in horses suffering from PPID. Based on the notion that structural mucosa integrity is associated with nutrient absorption , we propose that the poor nutritional status of PPID horses is related, in part, to the functional morphology of their small intestinal mucosa. 77 CHAPTER FIVE Jejunal brush border membrane uptak e of D - glucose and L - glutamine and s mall intestinal mucosa SLC5A1 and SLC38A2 mRNA abundance in horses with pituitary pars intermedia dysfunction J.L. De Vries, * H.C. Schott II, C.W. Enst, * J.P. Steibel, * C. Diard, * A.D. Woodward, and N.L. Trottier *, 2 * Department of Animal Science, Col lege of Agriculture and Natural Resources, Michigan State University, 474 S. Shaw Lane, Anthony Hall, East Lansing MI 48824 USA Department of Large Animal Clinical Sciences, College of Veterinary Medicine, 736 Wilson Rd., Veterinary Medical Center, Ea st Lansing MI 48824 USA 1 We acknowledge Sue Wismer, Jenna Lubitz, Lee - Anne Huber, and Dave Chamberlin for their help in sample collection. We would also like to acknowledge Janet Ireland for assistance with insulin analysis and Nancy Raney for assistanc e with quantification of mRNA abundance. 2 Corresponding author: trottier@msu.edu 78 A bstract The goal of this study was to test the hypothesis that D - glucose and L - Gln transport capacity across the apical membrane of the jejunal mucosa, and mRNA abundance of SLC5A1 and SLC38A2 throughout the small intestinal mucosa are lower in horses with pituitary pars intermedia dysfunction (PPID) compared to horses without PPID. The objectives were 1) to measure transport capac ity (V max ) of D - glucose and L - Gln across brush border membrane (BBM) of the jejunal mucosa and 2) to determine mRNA abundance of SLC5A1 and SLC38A2 in the duodenal, jejunal and ileal mucosa of PPID and non - PPID horses (control). Twelve horses of multiple b reeds and genders, and of similar age ( 28.6 ± 1.5 and 25.3 ± 2.1 yr for PPID and control, respectively) were donated for this study. Horses were subjected to overnight dexamethasone suppression test and histopathological evaluation of the pituitary glan d, and clearly classified as PPID (n=8) or non - PPID (control; n=4). Following euthanasia, mucosal scrapings were collected from the duodenum at 25 and 50 cm distal to the pyloric sphincter, the mid - jejunum and the mid - ileum. Mucosal scrapings were flash fr ozen in liquid N and stored at - 80°C. Maximal rate ( V max ) and affinity ( K m ) of D - glucose and L - Gln transport across the jejunal apical membrane was determined in vitro using brush border membrane vesicles. Messenger RNA abundance was assessed by quantitati ve Reverse Transcription PCR and results expressed as fold change relative to control. Compared to control horses, D - glucose uptake across BBM of PPID horses was lower at 5 m M ( P = 0.045) and 7.5 m M ( P = 0.016). L - Glutamine uptake did not differ between PP ID and control horses at any L - Gln concentrations. Compared to control, PPID Glucose V max was lower ( P = 0.021) and K m did not differ. L - Glutamine V max and K m did not differ between PPID and control horses. The mRNA abundance of SLC5A1 and SLC38A2 was not different in PPID horses relative to control horses in any segment. In conclusion, the 79 poor physical appearance, muscle wasting, and laminitis associated with PPID horses is not due to lack of nutrient transporter abundance in the small intestinal mucosa c ompared to healthy horses of similar ages, but may in part be related to a function of age or glucose intolerance. 80 Introduction Pituitary pars intermedia dysfunction (PPID) is a progressive endocrine disorder that primarily affects the geriatric horse (Schott, 2006). The condition is characterized by hypertrophy, hyperplasia, adenoma and loss of dopaminergic inhibition of the pituitary gland pars intermedia. This loss of inhibitory control results in increased circulating concentration of pro - opiomelanocortin peptides (POMC), including ACTH (Eiler et al., 1997; van der Kolk et al., 1995). Horses with PPID exhibit a variety of clinical signs, generally including poor body condition, appearance of muscle wasting, abnormal fat deposits, hi rsutism, and heightened risk for secondary infections (Innerá et al., 2013; Klinkhamer et al., 2011; McFarlane et al., 2010). Similarly, humans with PPID have chronic elevated cortisol concentration and suffer from degeneration of muscle, bone, and neuron al tissue (Melzack and Katz, 2004), and a weakened immune system (Cohen and Williamson, 199 1 ; Andersen, et al., 1994 ). Despite characteristically high appetite, clinical signs associated with PPID in horses are typical of poor nutritional status. I mpaired digestion is reported in human subjects with PPID (Jones and Quinn, 2005). Peripheral dexamethasone administration to broiler chickens causes dose response jejunal morphological changes including increased crypt depth, and decreased in villus height, abs orptive surface area and villus height/crypt depth ratio (Li et al., 2009). We have shown hitherto (Chapter 3) a degree of histomorphological changes in the small intestinal mucosa of PPID horses that would suggest some impairment in nutrient absorption. T he impact of PPID on intestinal nutrient transport in horses has not previously been investigated. Brush border membrane vesicles (BBMV) preparation from small intestinal mucosa is a useful ex vivo tool to precisely characterize transport kinetics of nutr ients in response to changes in physiological or dietary processes. Dietary AA are a major fuel for the small intestinal mucosa and are necessary for maintaining the 81 intestinal mucosal morphology and function in neonatal piglets (Wu, 1998). Glutamine is a preferred nutrient of intestinal enterocytes for mucosal growth and function (Ganapathy et al, 1994; Salloum et al., 1994; Windmueller and Spaeth, 1980) . Glutamine is transported by the s odium - dependent neutral amino acid transporter 2 (SNAT2), which is en coded by SLC38A2 (solute carrier family 38, member 2). In horses, glucose is transported across the apical membrane of small intestinal epithelial cells (Dyer et al., 2002) by the high - affinity, Na + - glucose cotransporter SGLT1, encoded by SLC5A1 (solute c arrier family 5, member 1). We hypothesized that the transport capacity (V max ) of D - glucose and L - Gln across the apical membrane is reduced in PPID compared to non - PPID horses, and that reduced V max is related to lower mRNA abundance of SLC38A2 and SLC5A1 . The objectives of this study were to 1) measure transport capacity of D - glucose and L - Gln across jejunal BBMV of PPID and non - PPID horses; and 2) quantify the mRNA abundance of SLC5A1 and SLC38A2 in duodenal, jejunal and ileal mucosa of PPID and non - PPID horses . Materials and Methods All methods were approved by the Institutional Animal Care and Use Committee at Michigan State University (03 - 12 - 064 - 001). Animals and Housing Twelve horses, 8 confirmed with PPID and 4 confirmed free of PPID (control) w ere donated for this study between the months of September 2012 and December 2012 following 19 to 39 yr of age (25.3 ± 2.11 yr for control and 28.6 ± 1.49 yr for PP ID horses) (Table 1). No ponies were admittedfor this study. All horses were housed indoor in individual stalls (3.05 m × 3.05 m or 3.66 m × 3.66 m) with an ambient temperature maintained at 21 °C. All horses were 82 fed second cutting mixed orchardgrass ( D actylis glomerata L. ) and bromegrass hay ( Bromus mollis L.) , and a commercial grain concentrate ( Equine Senior, Purina, St. Louis, MO) containing 14% CP, 1.8% fat and 18% crude fiber. Hay and concentrate were fed at 1.8 and 0.5% of body weight, respective ly. Horses were monitored for medical stability for a period of 5 d prior to being tested for presence and severity grade of PPID. Briefly, horses were subjected to ACTH testing as described in Beech et al. (2009) (Chapter 1) and an overnight dexamethason e suppression test (ODST) as described by Innera et al. (2013) (Chapter 1). Body condition scoring (BCS) was performed based on the Henneke scoring method (Henneke et al., 1983) and body weight (BW) recorded on the day of admission. Collection of Tissue H orses were euthanized and intestinal segments (approximately 24 - cm long samples) including the proximal and distal duodenum, jejunum and ileum were removed. Segments were rinsed in ice - cold NaCl solution (0.9 g NaCl/100mL H 2 0), cut and opened longitudinal ly to expose the mucosal lining, and rinsed again in fresh ice - cold NaCl solution. The mucosal layer was scraped from the serosal layer using a glass microscope slide. Scrapings were transferred to 15 - mL plastic conical tubes (Corning, Sigma - Aldrich Corp. , St. Louis, MO), immediately flash frozen in liquid nitrogen and stored at 80°C . Each tube contained approximately 3 g for a total of approximately 12 g of mucosal scraping collected from each segment. Preparation of Brush Border Membrane Vesicles Preparation of brush border membrane vesicles (BBMV) was based on methods descr ibed by Fan et al. (1998, 2004) using jejunal mucosal scrapings (Woodward et al. 2012). Approximately 2 g of tissue was weighed into homogenization buffer kept on ice (52 mL; 50 m M D - Mannitol, 10 m M HEPES, 0.2 m M phenylmethanesulfonylfluoride; pH = 7.4) an d 83 homogenized (Ultra - Turrax T25, Janke & Kunkel, IKA® - Labortechnik, Staufen, DE). A portion of this crude homogenate was set aside and kept on ice for measurement of protein concentration and alkaline phosphatase activity. The remaining crude homogenate wa s further processed for purification of the BBM. It was first centrifuged at 2000 × g for 15 min at 4°C, the supernatant recovered and the pellet discarded. To the recovered supernatant, a final volume of 10 m M MgCl 2 was achieved by adding 200 m M MgCl 2 , g ently shaken on ice for 15 min, and centrifuging at 2,400 × g for 15 min at 4°C. The supernatant was collected and the pellet discarded. The supernatant was transferred to ultracentrifuge tubes for centrifugation at 19,000 × g for 30 min at 4°C. The super natant was discarded and the pellet (representing the crude BBM) was suspended using a Pasteur pipette in vesicle pre - loading buffer kept on ice (150 m M potassium thiocyanate, 10 m M D - Mannitol, 5 m M HEPES; pH = 7.4). Final centrifugation was completed at 3 9,000 × g for 30 min at 4°C, the supernatant discarded and the pellet re - suspended with vesicle pre - loading buffer. A sample of purified BBMV homogenate was saved and kept on ice for measurement of protein concentration and alkaline phosphatase activity. The remaining BBMV homogenate was kept on ice until use for the measurement of D - glucose and L - Gln uptake as described later. Measurement of Protein Concentration and Alkaline Phosphatase Enzyme Activity Protein concentration of the crude homogenate sam ple and the BBMV suspension was determined with a Lowry DC TM Protein Assay Kit (Bio - Rad Laboratories, Inc., Hercules, CA) of BSA at 1.5 m M , 1 m M , 0.5 m M , 0.25 m M and 0.125 m M . The purity of the BBMV was verified by measuring the specific activity of alkaline phosphatase (EC 3.1.3.1) (AP) in both the crude mucosal homogenate and the BBVM suspension. The AP was measured according to 84 EngstÖm (1964) with modificatio ns as described by Fan et al (1998) using P - nitrophenyl phosphate as the substrate. Measurement of D - Glucose and L - Gln Uptake into Jejunal Brush Border Membrane Vesicles Fresh BBMV - 1 with vesicle preloading buffer. Uptake of D - Glucose and L - Gln was determined using the rapid filtration M D - glucose or 10 m M l - Gln, 5 m M HEPES, 150 m M sodium thiocyanate; pH = 7.4). Nitrocellulose membrane filters (0.22 µm) (Millipore, Billerica, MA) were soaked in the uptake buffer prior to the initiation of each uptake experiment. Uptake buffer kept at room temperature and containing either 3 H - D - glucose or 3 H - L - Gln and varying concentrations of D - glucose or L - Gln, respectively (0, 0.1, 0.25, 0.5, 1, 2.5, 5, and 7.5 m M ), was dispensed (50 µL) directly into the bottom of polystyrene tubes (brand, company). N ext, two 5 µL BBMV droplets were added to the side of the tube and allowed to warm to room temperature (22 - 24°C) for approximately 5 s. Uptake was initiated with a foot - switch activated vibromixer connected to an electronic timer (Timer model 545, GraLab C orporation, Centerville, OH) for 3 s for D - glucose (Woodward et al, 2012) and 15 s for L - Gln (Salloum, 1993). Uptake was terminated with addition of ice - cold stop and wash solution (1.2 mL; 150 m M potassium thiocyanate, 10 m M mannitol, 10 mM HEPES, 0.1 m M HgCl 2 ; pH = 7.4). The uptake solution was transferred onto filters mounted on a vacuum connected Manifold Filtration Unit (Millipore, Billerica, MA), washed with the ice - cold stop and wash buffer, and the filters were transferred to scintillation vials ( Research Products International Corps., Mount Prospect, IL) containing 10 mL scintillation fluid. Filters were allowed to equilibrate for a minimum of 30 min and radioactivity determined by liquid 85 scintillation counting (LS6500, Beckman, Brea, CA). The rem aining uptake solutions at each At the beginning and end of each uptake experiment, non - specific binding was determined by adding the stop and wash solution to uptake buffer (without BBMV sample). All uptake experiments were performed in triplicate. Jejunal samples from two horses were processed per experiment, from opposing treatment groups when possible. Estimation of D - Glucose and L - Gln Kinetics Uptake at different concentrations of D - glucose or L - Gln was determined using the following equation: J = [(( R F R B ) × S ) / R I ]/ ( W × T ), [1] Where J is the initial rate of D - glucose or L - Gln uptake into BBMV ( J, - 1 s - 1 ); Rf is the radioactivity in disintegration per minutes on filters (DPM/ Filter); RB is the radioactivity for non - specific binding to filters (DPM/ filter); S is the extravesicular concentration of D - glucose of L - Gln (m M ), R I is the radioactivity in the uptake media (DPM/ µL); W is the amount of membrane protein provided for the incubation (mg of protein) and T is the time of incubation for initial uptake (s). The kinetic parameters V max and K m for D - glucose and L - Gln transport across the BBMV were determined using a multi - parameter curve fitting program (Curve Fitter, MathSol - Mathematical Solutions Development, Institute of Mathematics and Statistics, Helsinki, FI) based on the following equation: J = V max [ S ] + D [ S ] K m [ S ] [2] 86 Where J is the initial rate of uptake into BBMV (J, - 1 s - 1 ) ; V max is the maximal transport rate ( - 1 - 1 ); S is the extravesicular D - glucose or L - Gln concentrations (m M ); K m is the Michaelis constant (50 % saturation; m M ); and D is the diffusion component ( - 1 s - 1 ) . Real - time quantitative PCR (qRT - PCR) analysis Messenger RNA was isolated from the intestinal mucosa scrapings (proximal and distal duodenum, jejunum and ileum) according to the ma mir Vana TM miRNA Isolation Kit (Ambion ® , LifeTechnologies Inc. TM , Carlsbad, CA). Approximately 0.1 mg of tissue was weighed and homogenized (Kinematica TM Polytron TM , Luzern, CH) on ice for 3 - s intervals in 10 volumes of l ysis/binding buffer per tissue mass. The purity of the isolated mRNA concentration was quantified by spectrophotometry (Nanodrop 1000, Thermo Scientific, Waltham, MA; Agilent Bioanyalyzer 2100, Palo Alto, CA). Complementary DNA was prepared using a High - Ca pacity cDNA Reverse Transcription Kit (Applied Biosystems TM , Life Technologies Inc. TM , Foster City, CA), diluted to 10 ng/µL and stored at - 20°C. Microarray results were confirmed through q uantitative real - time reverse transcript ion PCR (qRT - PCR) in 96 - well plates (MicroAmp ® Fast Optical Plate, Life Technologies Inc. TM , Carlsbard, CA) using TaqMan® technology (Applied Biosystems) . Each assay consisted of two unlabeled PCR primers and a FAM TM dye - labelled TaqMan® MGB (minor groove binder) probe. Three o f the primer - probe sets ( SLC5A1 , GAPDH , and ACTB ) were commercially available from Applied Biosystems and the fourth primer - probe set ( SLC38A2 ) was custom designed by Applied Biosystems for this project (Custom TaqMan® Gene Expression Assay). TaqMan® prime r - probe information is provided in Table 1. Plates (MicroAmp ® Fast Optical 87 Plate, Life Technologies Inc. TM , Carlsbard, CA) were briefly centrifuged at 400 × g for 1 min and 15°C, loaded into a real time PCR System (Step One Plus TM Real - Time PCR Systems, T hermo Fisher Scientific Inc., Waltham, MA) and analyzed with the associated software (Step One TM Software, Thermo Fisher Scientific Inc., Waltham, MA). Cycling conditions were 95 °C for 10 min, followed by 40 cycles of 95°C for 15 s and 60 °C for 1 min. Tr anscription levels for SLC38A2 and SLC5A1 were normalized using both genes GAPDH and ACTB . Abundance of each target gene was calculated according to the following equations (Pfaffl, 2001 and Livak and Schmittgen, 2001): C T = C T target gene Average of C T reference genes [3] C T C T ( PPID ) - C T ( Control ) [4] Fold Change (FC) = 2 - T [5] Statistical Analysis All data was found to be normally distributed based on the Gaussian distribution analysis in SAS (ve rsion 9.3, SAS Institute Inc., Cary, NC). Analysis of data ( uptake kinetic parameters and mRNA quantification ) was performed using the PROC MIXED procedure (SAS version 9.3, SAS Institute Inc.) with treatment as fixed effects and the random effect of each individual horse nested within treatment . The response variable was cycles to threshold ( C T ) for mRNA expression. Pairwise differences between t - test. Fold change were calculated by back transforming the estima ted mean difference in C T ( C T ) between control and PPID for each gene of interest using the expression FC= 2 - C T (Livak and Schmittgen, 2001). Following Steibel et al. (2009), 95% confidence intervals of mean differences were also back transformed to p rovide the confidence interval for the estimated fold change. 88 Fold change means with transformed confidence interval spanning across the value 1.0 were considered not significant at = 5%. R esults Membrane Purity Alkaline phosphatase activity in the mucosal scrapping crude homogenate of PPID and control hors es did not differ and was 58.08 and 58.75 ± 14.52 U for control and PPID horses, respectively. In the purified BBVM of PPID an d control horses, AP did not differ and was 152.2 and 171.4 ± 30.26 U, respectively. The average enrichment factor, i.e., AP BBMV/AP crude homogenate, was 2.83 ± 0.28 fold, indicating that the BBMV preparations were enriched in brush border membrane. Init ial Time Course of D - Glucose and L - Gln Uptake into Brush Border Membrane Vesicles Extravesicular D - glucose and L - Gln concentrations of 0.00, 0.10, 0.25, 0.50, 1.00, 2.50, 5.00, and 7.50 m M were based on Woodward et al. (2013) for D - glucose and L - Lys uptak e in equine BBMV preparations. Incubation time of 3 s for D - glucose was based on studies by Dyer et al. (2002), Wolfram et al. (1986) and Woodward et al. (2010), and of 15 s for L - Gln was based on one study by Salloum et al. (1993) in horses and Fan et al. (1998) in pigs. D - Glucose and L - Gln Uptake into Brush Border Membrane Vesicles Initial uptake rates of D - glucose and L - Gln are presented in Figures 1 and 2, respectively. In all figures, the diffusion and saturable uptake are represented. Compared to c ontrol horses (Figure 1A), D - glucose uptake across BBVM of PPID horses was lower at 5 m M ( P = 0.045) and 7.5 m M D - glucose ( P = 0.016) (Figure 1B). L - Glutamine uptake did not differ between PPID (Figure 2B) and control horses (Figure 2A) at any L - Gln concen tration. 89 Kinetic parameters (V max and K m ) are presented in Table 2. Compared to control horses, D - glucose V max of PPID horses was lower ( P = 0.021), and K m and diffusion did not differ. L - Glutamine V max and K m did not differ between PPID and control hor ses. SGLT1 and SNAT2 mRNA Abundance in Intestinal Mucosa Transcript abundance of SLC5A1 and SLC38A2 in the duodenal, jejunal, and ileal mucosa of PPID (n=8) relative to control horses (n=4) is presented in Figure 3 and 4, respectively. The mRNA abundanc e of SLC5A1 and SLC38A2 was not different in PPID horses relative to control horses in any segment. 90 Table 5.1. Reverse transcription quantitative real - time (RT - qPCR) assay primer information in the intestinal mucosa of PPID and non - PPID hors e. Gene Name Gene Symbol Protein Taqman Assay/ Accession Number 1 Solute carrier family 5 (Sodium/Glucose Cotransporter, member 1 SLC5A1 SGLT1 NM_001081872 Solute carrier family 38, member 2 SLC38A2 S N AT2 NC_009149 actin, beta ACTB - actin NM_001081838 G lyceraldehyde - 3 - phosphate dehydrogenase GA P DH GAPDH NM_001163856 1 - Applied Biosystems Taqman® assay product number or GenBank accession number of sequence submitted for Custom Taqman® Gene Expression Assay design 91 Table 5.2. Kinetic paramet ers of D - glucose and L - Gln uptake in equine small intestinal (jejunum) brush border membrane vesicles of control and PPID horses of similar ages. Each parameter measured is representat ive of the LSMEAN ± SEM from four (control) and eight ( PPID ) horses . Kinetic Parameter Control Control PPID P - value V max 1 D - Glucose 262.4 33.43 146.2 25.14 0.021 L - Gln 44.77 27.62 60.28 ± 19.53 0.656 K m 2 D - Glucose 0.21 0.16 0.27 0.12 0.762 L - Gln 0.75 0.47 0.67 ± 0.33 0.883 Diffusion 3 D - Glucose 42.43 11.76 31.74 7.70 0.469 L - Gln 11.87 4.88 9.84 ± 3.45 0.741 1 - ( - 1 s - 1 ) ; 2 - ( m M ) ; 3 - ( - 1 s - 1 ) 92 Table 5.3. Kinetic parameters of D - glucose and L - Gln uptake in several mammalian and chicken jejunal brush border membrane vesicles. Animal Segment Substrate V max 1 K m 2 Sou rce Human Jejunum D - Glucose 931 0.86 Harig et al., 1989 Human Jejunum D - Glucose 805 0.75 Malo and Berteloot, 1991 Rabbit Jejunum D - Glucose 470 0.57 Ikeda et al., 1989 Rat Jejunum D - Glucose 233 0.43 Thiesen et al., 2003 Rat Jejunum D - Glucose 270 0.5 3 Boudry et al., 2007 Chicken Jejunum D - Glucose 392 2.40 Garriga et al., 2006 Pig Jejunum D - Glucose 2595 0.09 Woodward et al, 2013 Horse Jejunum D - Glucose 918 0.49 Dyer et al., 2002 Pony Jejunum D - Glucose 655 0.27 Woodward et al., 2013 Horse Jejunum D - Glucose 262.4 0.21 This study Human Jejunum L - Gln 637 1.36 Said et al., 1989 Rabbit Jejunum L - Gln 4.7 0.34 Talukder et al., 2008 Rat Jejunum L - Gln 222 2.23 Salloum et al., 1993 Dog Jejunum L - Gln 393 0.97 Bulus et al., 1989 Pig Jejunum L - Gln 71 0.77 Fan et al., 1998 Horse Jejunum L - Gln 308 0.52 Salloum et al., 1993 Horse Jejunum L - Gln 45 0.75 This Study 1 - ( - 1 s - 1 ) ; 2 - ( m M ) ; 3 - ( - 1 s - 1 ) 93 A. B. Figure 5.1 . Kinetics of D - g lucose uptake into equine jejunal Control (A) and PPID (B) brush border membrane vesicles. Brush border membrane vesi cles we re loaded with a buffer containing 150 m M KSCN, 10 m M Mannitol, 5 m M HEPES ( pH 7.4) and incubated with another buffer containing 150 m M NaSCN and 5 m M HEPES ( pH 7.4) , 0.8 µM D [ 3 H] - g lucose at concentrations of 0, 0.1 0 , 0.25, 0.5 0 , 1 .00 , 2.5 0 , 5 .00 , and 7. 5 0 m M D - g lucose. D - Mannitol was added to maintain osmolarity. Each point is representative of the LSMEAN ± SEM from four (control, A) and eight ( PPID , B) horses in triplicate uptake experiments per horse . 94 A. B. Figure 5.2 . Kinetics of L - Gl n uptake into equine jejunal Control (A) and PPID (B) brush border membrane vesicles. Brush border membrane vesicles were loaded with a buffer (150 mM KSCN, 10 m M Mannitol, 5 m M HEPES ( pH 7.4) and incu bated with another buffer containing 150 m M NaSCN and 5 m M HEPES ( pH 7.4) , 0.8 µ M L [ 3 H] - Glutamine at concentrations of 0, 0.1, 0.25, 0.5, 1, 2.5, 5, and 7.5 m M L - Gl n. D - Mannitol was added to maintain osmolarity. Each point is representat ive of the LSMEAN ± SEM from four (control, A) and eight ( PPID , B) horses in triplicate uptake experiments per horse . 95 Figure 5.3 . Fold change in mRNA a bun dance of SLC5A1 in PPID horses relative to Contr ol. Duo25, duodenum at 25 cm; Duo50, 50 c m distal to the pyloric sphincter; Jejunum, mid - jejunum; Ileum, mid - ileum 15 cm proximal to the cecum. 96 Figure 5.4 . Fold change in mRNA abundance of SLC38A2 in PPID horses relative to Contr ol. Duo25, duodenum at 25 cm; Duo50, 50 cm distal to the pyloric sphincter; Jejunum, mid - jejunum; Ileum, mid - ileum 15 cm proximal to the cecum. 97 D iscussion This is the first study reported to characterize transport of D - glucose and L - Gln at the molecular and phenotypic level along the small intestinal mucosa of horses with PPID. While the etiology of PPID is multifaceted, horses suffering from this condition commonly demonstrate poor physical appearance while maintaining normal appetite, suggesting that optimum nutrient utilization and (or) absorption may be compromised. Chronic elevated concentration of cortisol have been shown to impact peripheral tissue metabolism by reduction in GLUT4 translocation to the cell surface in muscle tissue (Dimitriadis et al., 1997; Coderre et al., 1996; Oda et al., 1995; Weinstein et al., 1995; Owen et al., 1973) and increased lipolysis (Kennedy et al., 1993; Ekstrand et al., 1992; Ong et al., 1992). Insulin resistance and central fat deposit in men has been well established to be associated with excess blood cortisol concentration and cortisol production rates (Purnell et al., 2009; Phillips et al., 1998; Filipovsky et al., 1996; Stolk et al., 1996). The impact of chronic elevated cortisol concentration on nutrient absorption is less well documented and not available f or horses. In this study, horses with PPID had significantly higher serum concentrations of ACTH and cortisol compared to non - PPID horses (Chapter 1). Basal ACTH serum concentrations were 85.6 ± 18.5 and 23.0 ± 26.1 pg/mL in PPID and non - PPI D horses (Chapter 1). Basal and post - dexamethasone cortisol concentrations were 158.0 ± 36.7 and 10.0 ± 21.9 nmol/L for control horses and were 156.0 ± 25.9 and 99.3 ± 14.5 nmol/L, respectively, for PPID horses . These horses also presented histomorphologic al changes in the small intestinal mucosa of PPID horses that would suggest some impairment in nutrient absorption (Chapter 3). Gastrointestinal and hepatic conditions like inflammatory bowel diseases and chronic active hepatitis are commonly treated with high doses of oral glucocorticoids (references?). 98 Oral administration of glucocorticosteroids increased the rate of fructose transport across the jejunal BLM weaning and adult rats, but not of glucose (Theisen et al, 2003). (Thiesen et al., 2003). In t his study, the maximal capacity for glucose uptake in PPID horses was near half that of control horses. Across both treatment s , glucose V max was nonetheless considerably lower than w e previously reported for ponies (Woodward et al. 2013). Transport of D - g lucose via the Na+ - glucose co - transporter activity in ageing human subjects was reported to remarkably decrease as age increased with (Vincenzini et al., 1989). Those results were co mparable to that observed in intestinal membrane vesicles of young patients suffering from Crohn's disease. The relationship between age and nutrient digestion in horses is unclear. Ralston et al. (1989) reported lower apparent digestibility of crude prote in when comparing geriatric horses to young horses, but failed to show similar results in later studies (Ralston et al., 1996; 1989). Variation in dentition structure and function across aging horses may render it difficult to clearly demonstrate a direct impact of aging on digestive functions per se. The affinity for D - glucose transport in horses has been shown to be 0.49 mmol/L suggesting a high affinity for the isoform SGLT1 (Dyer et al., 2002). The SGLT1 protein is a high - affinity, Na + - dependent tran sporter located in the apical membrane of small intestinal epithelial cells in many species including horse and specific for glucose and galactose (Dyer et al., 2002). The K m for D - glucose in this study was even lower, supporting high affinity transport f or glucose, and was comparable to values previously reported in ponies by Woodward et al. (2013). Dyer et al. (2009) reported an increased transport rate to be related to an enhancement in V max , and not K m . Thus, the reduced D - glucose V max rate of PPID a fflicted horses when compared to control horses is a strong indication that D - glucose transport capacity across the 99 jejunal epithelium was limited in PPID horses. We expected mRNA abundance of SLC5A1 to be lower in horses with PPID, in particular given th e Na + - dependent D - glucose V max uptake in those horses was near half that of control horses. In horses fed increasing levels of carbohydrates, D - glucose uptake across intestinal BBMV paralleled transcriptional abundance of SLC5A1 (Dyer et al., 1997). In contrast, o ral prednisone in rats decreased SLC5A1 mRNA abundance without reduction in glucose Vmax transport across BBBV (Theisen et al, 2003), suggesting possible disassociation between expression of SLC5A1 protein abundance and transporter SGLT1 activit y. Glutamine is a functional amino acid with diverse physiological roles in protecting the gut from atrophy and injury under various stress conditions (Wang et al., 2014), making it a preferred nutrient of intestinal enterocytes for mucosal growth and func tion (Ganapathy et al, 1994; Salloum et al., 1994; Windmueller and Spaeth, 1980). Glutamine transport is facilitated by both Na + - dependent transport mechanisms in horses (Souba et al., 1992), human (Stevens et al., 1992, 1982), dogs(Bulus et al., 1989) and rat (Said et al., 1989), and Na + - independent transport mechanisms in horses (Salloum et al., 1993). Transporter SNAT2 is a sodium - coupled neutral amino acid co - transporter (Sugawara et al., 2000; Yao et al., 2000). Increased mRNA expression of SLC38A2 is indicative of an increased ability to transport L - Gln across the intestine in vitro (Reimann et al., 2004). We expected horses with PPID to have lower L - Gln Vmax and transcript abundance of SLC38A2 . Our L - Gln V max values in this study compare well with th ose reported in pig ( Fan et al., 1998) and rat jejunal BBMV (Salloum et al., 1993) but lower than reported by Salloum et al. (1993) in horse and dog jejunal BBMV (Table 3). Our K m results are consistent with the values previously reported for L - Gln transpo rt in rats (Salloum et al., 1993) and dogs (Bulus et al., 1989). The only other study of L - Gln transport in horses by Salloum et al. (1993) 100 has K m values which differ marginally from those seen in our study (Table 3). The study by Salloum et al. (1993) how ever had a very limited number of horses (n = 3). In conclusion PPID horses have a remarkably lower maximal uptake capacity of glucose by the jejunal brush border membrane compared to horses with no PPID. This reduced capacity may explain in part the bio logical me chanism behind increased risk of dietary induced laminitis in horses with PPID. Thus, dietary management consisting in diets containing minimal concentration of non - structural carbohydrates should be considered. Conversely, there appears to be n o difference in the maximal uptake capacity of glutamine nor SNAT2 transporter abundance by the jejunal brush border membrane in PPID horses compared to horses without PPID. These findings suggest AA nutrient utilization is not limiting in PPID horses. Ano ther mechanism is still responsible and unknown for the muscle wasting characteristically associated with the PPID condition. In conclusion, the poor physical appearance, muscle wasting, and laminitis associated with PPID horses is not due to lack of nutri ent transporter abundance in the small intestinal mucosa compared to healthy horses of similar ages, but may in part be related to a function of age or glucose intolerance. 101 CHAPTER SIX : SUMMARY AND CONCLUSIONS The overarching goal of this t hesis was to determine the impact of PPID on nutrient utilization in the small intestine and the peripheral muscle tissue. The objectives were to (1) measure glucose tolerance and insulin sensitivity, and assess abundance of insulin - dependent molecular ent ities of glucose and neutral amino acid transport in skeletal muscle, and (2) evaluate the impact of PPID on small intestinal mucosa morphology and transport of glucose and glutamine. The data presented in this thesis is novel, as the mechanisms of nutri ent utilization in PPID horses has never been studied before. In the first chapter, horses were clearly classified with PPID condition based not only on clinical symptoms and endocrine testing, but also post - mortem histological examination as well. This st udy was originally designed to include three treatment groups: control, insulin resistant PPID, and non - insulin resistant PPID horses. The OGTT and FSIGTT results clearly demonstrated that PPID horses did not differ from non - PPID horses in parameters defin ing insulin or glucose kinetics. As such, PPID horses could not be classified as either insulin resistant or non - insulin resistant and comparisons were based on two treatment groups including PPID and non PPID (i.e., control). The absence of insulin resi stance was further ascertained by characterizing t he molecular entities of glucose and glutamine utilization. Abundance of mRNA of SLC2A4 , INSR and SLC38A2 and protein GLUT4 and INSR did not differ between PPID and non PPID horses. Our study was carefully designed to include clear, age - matched, non PPID horses, which is in contrast with most of the previously reported studies. Our data corroborate with the only study so far using age - matched control horses (Mastro et al, 2014) indicating that the incidence of insulin resistance in horses with PPID may be overestimated in the current horse population. 102 Mucosal intestinal morphology and glucose uptake differed between PPID and control horses. Horses with PPID had a reduced proximal average of villus height, tendency for greater crypt depth in the mid - jejunum, and a lower villus height to crypt depth ratio in the mid - jejunum. Maximal glucose transport capacity across the apical membrane of the small intestinal mucosa of PPID horses was 146.2 25.1 4 otein - 1 s - 1 compared to 262.4 33.43 protein - 1 s - 1 in control horses, which represents a 44% reduction in maximal glucose uptake. The reduction in glucose transport was not mirrored at the transcriptional level as mRNA abundance of SLC5A1 did not differ between PPID and non - PPID horses. Neither the maximal glutamine transport capacity nor the mRNA abundance of SLC38A2 in any of the small intestinal segments differed between PPID and non PPID horses. It is unclear whether the reduction in glucose transport was associated with the reduction in average proximal villus height and villus height:crypt depth since glutamine transport capacity was unaffected. Transport however was only characterized at the mid - jejnum for both glucose and glutamine and it is possible that glutamine transport was affected in regions either proximal or distal to that examined. It was expected that abundance of SLC5A1 would be lower in PPID horses given the remarkable reduction in glucose transport across BBMV. Albeit not m easured herein, SGLT1 abundance may have paralleled that of glucose transport capacity seen in this work. Other glucose Freeman et al., 1993) however SGLT1 is r eported to be the dominant glucose transporter in the apical membrane of the small intestine of the horse (Dyer et al., 2002). Nonetheless, the fact that neither SLC5A1 nor SLC38A2 differed between PPID and non PPID horses is further indicative that insuli n regulation unlikely played a role in the pathology of PPID in this group of horses. 103 Geriatric horses play a key role in the equestrian industry where they serve as companions and experienced schoolmasters for less experienced riders. A large proportio n of the aging horse population is affected by PPID hence continued research to uncover tools to improve the welfare and reduce costs to owners is essential. The work presented in this thesis showed that horses with PPID may benefit from dietary intervent ions tailored to the maintenance of intestinal integrity and health. Decreased glucose transport rate in the small intestine may lead to increased flow to the large intestinal compartment. Given these notions geriatric horses and those in particular with PPID shou ld be carefully managed with nutritional strategies aiming at increased digestion and nutrient absorption, including feeding sma l l, frequent meals , high in fiber and low in starch. 104 APPENDICES 105 APPENDIX A: CHAPTER TWO MATERIAL 106 Figure A.1. Mean ± s.e acute response of insulin to glucose values derived for 8 PPID and 4 control horses following i.v. administration of 300 mg/kg bwt 50% dextrose solution followed later by the administration of 20 mu/kg bwt insu lin in the FSGITT testing using the minimal model for analysis. 0100 200 300400500 600 7000123AIRgControlPPID107 Figure A.2. Mean ± s.e insulin sensitivity values derived for 8 PPID and 4 control horses following i.v. administration of 300 mg/kg bwt 50% dextrose solution followed later by the administration of 20 mu/kg bwt insulin in th e FSGITT testing using the minimal model for analysis. 01 234 5 6 7 8 9100123SIControlPPID108 Figure A.3. Mean ± s.e glucose sensitivity values derived for 8 PPID and 4 control horses following i.v. administration of 300 mg/kg bwt 50% dextrose solution followed later by the administration of 20 mu/kg bwt insulin in th e FSGITT testing using the minimal model for analysis. 00.511.522.530123 SgControlPPID109 Figure A.4. Mean ± s.e disposition index values derived for 8 PPID and 4 control horses following i.v. administration of 300 mg/kg bwt 50% dextrose solution followed later by the administration of 20 mu/kg bwt insulin in th e FSGITT testing using the minimal model for analysis. 020040060080010001200140016001800 20000123 DIControlPPID110 Figure A.5. Correlation of the Peak Insulin of OGT vs Acute Insulin Response to Glucose (AIRg) Correlation when comparing PPID hor ses to control horses of similar ages. 111 Figure A.6. Correlation of the change in insulin OGT vs Insulin Sensitivity when comparing PPID horses to control horses of similar ages. 112 Figure A.7. Correlation of Insulin Sensitivity vs Acute Response of when comparing PPID horses to control horses of similar ages. 113 Figure A.8. Correlation of the change in insulin OGT vs Insulin Sensitivity when comparing PPID horses to control horses of similar ages. 114 APPENDIX B CHAPTER THREE MATERIAL 115 Figure B.1. Western blot analysis of crude muscle membrane extracts prepared from gluteal biopsies of control horses and PPID horses and treated with antibodies against INSR showing full blot. 116 Figure B.2. Western blot analysis of crude muscle membrane extracts prepared from gluteal biopsies of control horses and PPID horses and treated with antibodies against INSR depicted by a graph indicating the relative expression level. Intensities of bands were estimated by using the program "ImageLab" from BioRad. Values were normalized to the INSR control and expressed relative to the value of control horses. The data are expressed as means ± s.e. using Student™s t- test. 00.050.10.150.20.250.3INSRRelative IntensityControlPPID 117 Figure B.3. Western blot analysis of crude muscle membrane extracts prepared from gluteal biopsies of control horses and PPID horses and treated with antibodies against GLUT4 showing full blot. 118 Figure B.4. Western blot analysis of crude muscle membrane extracts prepared from gluteal biopsies of control horses and PPID horses and treated with antibodies against GLUT4 depicted by a graph indicating the relative expression level. Intensities of bands were estimated by using the program "ImageLab" from BioRad. Values were normalized to the GLUT4 control and expressed relative to the value of control horses. The data are expressed as means ± s.e. using Student™s t-test. 00.050.10.150.20.250.30.350.40.45GLUT4Relative IntensityControlPPID 119 APPENDIX C CHAPTER FIVE MATERIAL 120 Table C.1. Kinetics of glucose uptake rate into equine jejunal control and PPID brush border membrane vesicles. Each point is representative of the mean ± SEM from four control horse's and eight PPID horse™s uptake experiments. Control PPID Control SEM PPID SEM p-value 0.0000 0 0 0 0 0 0.1000 76.7316 67.3569 17.5665 13.2791 0.6803 0.2500 164.64 131.9 39.0554 29.5231 0.5204 0.5000 244.4 194.28 45.6777 34.5291 0.4042 1.0000 227.3 158.22 38.7969 29.3277 0.1892 2.5000 328.63 244.1 55.7127 42.1148 0.257 5.0000 252.041385 139.485019 38.7969 29.3277 0.0449 7.5000 255.393487 141.444856 76.2794 57.6618 0.0449 121 Table C.2. Kinetics of Glutamine uptake rate into e quine jejunal control and PPID brush border membrane vesicles. Each point is representative of the mean ± SEM from four control horse's and eight PPID horse™s uptake experiments. Control PPID Control SEM PPID SEM p-value 0.0000 0 0 0 0 0 0.1000 8.19147907 11.0014254 2.3166 1.5166 0.1316 0.2500 14.7117332 18.203359 6.9997 4.5824 0.3421 0.5000 20.8291951 25.0321454 11.0153 7.2112 0.2637 1.0000 27.3002978 32.8576905 10.3215 6.757 0.1696 2.5000 34.8880588 44.3085268 12.3563 8.0891 0.4786 5.0000 38.993408 48.6574797 21.6667 14.1842 0.1006 7.500 40.6833948 57.9075866 36.4307 21.0333 0.904 122 LITERATURE CITED 123 LITERATURE CITED Aleman M, Nieto JE. Gene expression of proteoly tic systems and growth regulators of skeletal muscle in horses with myopathy associated with pituitary pars intermedia dysfunction. American journal of vete rinary research. 2010;71:664-70. Aleman M, Watson J, Williams D, LeCouteur R, Nieto J, Shelton G. 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