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A. .11.: :ufiflulnwb 1.2a.-- , . \tz.’l\,7\.§z .. 1 It... ‘ , . .. . fir... t- .flnfilm2 r... .. 3.... Kfisrxflflt :5 x : a .. . . 3.9L... . i. .3! to... t‘vis‘tv: .. 1.... . . . , . a I I l\ Eli??? C... . g .1. 3 ..|'...coa. _. 1' :7 iii-31".- ..\. .w ban. a... :.............9.......:. .m mu. .vléxn’izoaia. . . 3...: 1.. I3. 19.4.5.5 1 thnnnuti .33.... i 17:0,... ,. . .. 3.1.4 1 “is!!! 14151311}: I iii... 32. { {£43341} [Hill/Ill! llIII/llllllllll/lll/lljlllllllHill/ll(Ill/II!!! 1293 7862 .TliESIE gm fl Y Micmaz . state University This is to certify that the dissertation entitled APOLIPOPROTEIN METABOLISM IN DIABETES presented by Esperanza R. Briones has been accepted towards fulfillment of the requirements for PLD. degree in Nutnjtjon 49/sz 1/1,!" Ci/LL’M‘fiLLk CA} Major professor Date Oct. 26, 1982 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. W 934%! ”8’3 23' APOLIPOPROTEIN METABOLISM IN DIABETES By Esperanza R. Briones A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1982 ./TQY Q/JI‘ ABSTRACT APOLIPOPROTEIN METABOLISM IN DIABETES By Esperanza R. Briones A series of studies were conducted to determine apolipoprotein metabolism in diabetes. The initial study was on methodology. Using an immunoprecipitation technique, apolipoprotein A—II (apo A-II) associated with apolipoprotein A—I (apo A—I), but not with apolipo— proteins C—II (apo C—II) or C-III (apo C-III). The effects of glucose ingestion on plasma levels of glucose, insulin, triglycerides (TC), cholesterol, high density lipoprotein cholesterol (HDL—C), and apo A—I, apo A—II, apo C—II and apo C—III were studied in 6 normal males and 5 non—insulin dependent (NIDDM) male diabetics. After 2 baseline blood samples were drawn, subjects ingested a test dose of glucose (1 g/kg body weight), and repeat blood samples were obtained at 30, 60, 90, 120, 150, 180, 240, and 300 minutes following the glucose load. All the subjects showed an increase of apo A—I, apo A—II, apo C—II, and apo C—III after the oral glucose challenge. There was no significant difference in the post- prandial changes of apo A—I and apo A-II between the normals and diabetics. There was an initial decrease of apo C—II and apo C—III in response to the glucose load in the diabetic subjects. Since apo 0—11 is an activator for lipoprotein lipase, an enzyme responsible for TG clearance, these findings suggest that the initial decrease of apo C-II in the NIDDM patients may be a contributory factor in the persistent hypertriglyceridemia observed in these patients. Measurements were made of fasting plasma levels of cholesterol, TG, HDL—C, and apo A—I, apo A-II, apo C—II, and apo C—III in 170 diabetic men and women: 78 with insulin dependent diabetes mellitus (IDDM) and 92 with non-insulin dependent diabetes mellitus (NIDDM). The same measurements were obtained in 46 age-matched healthy volunteers for comparison. Lipid profiles were similar in the IDDM and NIDDM groups, with significantly elevated plasma TG, decreased HDL—C and normal cholesterol levels in comparison with non—diabetic control subjects. Plasma apo A-I was low in both groups of diabetics and was negatively correlated with TG levels as compared to normal controls. Apo C—II and apo C—III were strongly correlated with plasma TG (r = 0.71, P <0.0001). Forty one percent of the combined group of diabetics had a confirmed diagnosis of coronary artery disease (CAD) and/or peripheral arteriovascular disease (PAD). There was a signifi— cant decrease of apo A—I levels in those with CAD/PAD compared with those with no disease, but only in the IDDM group. These data indicate that apolipoprotein levels are altered in diabetes mellitus and in— directly suggest that insulin difference, whether absolute or relative, contributes to these changes. Diabetes mellitus is associated with low levels of apo A—I which in turn may predispose to macrovascular disease. DEDICATION This thesis is dedicated to my mother ii ACKNOWLEDGEMENTS I wish to express my gratitude to: Dr. Wanda Chenoweth, for her guidance, interest and helpful advice in all my course work, as well as my research work, and providing me with laboratory experience in animal experiments. Dr. Bruce A. Kottke, who gave me the opportunity to do my research work at Mayo Clinic and for being available for consultation at all times. Dr. Pasquale J. Palumbo, for giving me access to his patients and for being available for consultation at all times. Dr. Simon J. T. Mao, for imparting some of his scientific know—how and his patience in teaching me the essence in the field of apolipo— protein metabolism. He made me aware of the shortcomings of analytical methods and the necessity of critical thinking when evaluating data. Dr. Michael O'Fallon, for statistical help. To all members of the laboratory staff at the Atherosclerosis Research Unit of Mayo Clinic, for their friendship, helpfulness and patience while I was learning the laboratory techniques. TABLE OF CONTENTS Page INTRODUCTION AND BACKGROUND INTRODUCTION BACKGROUND Apolipoproteins: Apo A Peptides Apo B Apo C Peptides Apo E Apo D Association of lipoproteins and CHD in diabetes Postprandial apolipoprotein metabolism SPECIFIC AIMS OU)\I\IO\O\J>N t-‘ v—nr—o U1 PUBLICATION AND MANUSCRIPTS Chapter 1 Association between apolipoproteins A-1 and A-II as evidenced by immunochemical approach 17 Chapter 2 Short—term elevation of apolipoprotein A-I in plasma following a glucose load 26 Chapter 3 Acute effects of glucose ingestion on plasma apolipoproteins in normal men and non— insulin dependent diabetics 45 Chapter 4 Plasma lipids and apolipoproteins in insulin dependent and non-insulin dependent diabetics 65 ADDITIONAL RESULTS Chapter 5 A. Effect of minimal weight loss on plasma lipids and apolipoproteins in non—insulin dependent diabetics 95 B. Comparison of oral glucose load and intravenous glucose infusion in a non—insulin dependent diabetic 120 C. Plasma lipids and apolipoprotein levels of impaired glucose tolerant men in comparison with normal men 125 SUMMARY AND CONCLUSIONS 128 LIST OF REFERENCES 133 iv — APPENDIX A. Postprandial levels of plasma glucose, insulin, plasma lipids, HDL cholesterol and apolipoproteins A-I, A—II, C—II and C—III in normal controls and non-insulin dependent diabetics Linear trend in the cumulative profile (Oral glucose load of the normals and diabetics) Non-insulin dependent diabetics: fasting plasma glucose, insulin, lipids, HDL cholesterol, apolipoproteins A—I, A—II, C-II and C—III levels of those on diet, on insulin and oral hypoglycemic agents Radioimmunoassay procedure Working protocols Consent forms Page 143 154 158 160 166 169 LIST OF TABLES INTRODUCTION AND BACKGROUND Table Table CHAPTER 1 Table CHAPTER 2 Table Table CHAPTER 3 Table CHAPTER 4 Table Table Table Table Table 1 2 Composition and properties of human plasma lipoproteins Characteristics of human plasma apoproteins Specificity of the association of 125I-labeled apo A—I, apo C-II and apo C—III with apo A—I as determined by an immunoprecipitation technique Total plasma cholesterol, HDL cholesterol, and apo A—II levels in individual subjects after an oral glucose load Total plasma cholesterol, HDL cholesterol and apo A-II levels in subject 1 after intravenous glucose infusion Clinical characteristics of diabetics and normal controls Characteristics of normals and diabetics Comparison of diabetic characteristics in normal controls and diabetics Plasma lipids and HDL cholesterol in normals and diabetics Plasma apolipoproteins in normals and diabetics Pearson correlation coefficients among plasma glucose, glycosylated hemoglobin, lipids, HDL cholesterol, and apolipoprotein variables in normal and diabetic males and females vi Page \OU) 21 37 38 48 70 71 73 74 83 — u CHAPTER 5 Table Table Table Table Table Table APPENDIX Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table A-I B—l C-2 Weights (kg) of individual NIDDM subjects before and after weight reduction diet Plasma glucose, insulin and triglycerides in a NIDDM subject after an oral glucose load (OGTT) and intravenous glucose infusion (IVGTT) Plasma cholesterol and HDL cholesterol in a NIDDM subject after an oral glucose load (OGTT) and intravenous glucose infusion (IVGTT) Plasma apolipoprotein A—I, A-II, C—II, and C-III in a NIDDM subject after an oral glucose load (OGTT) and intravenous glucose infusion (IVGTT) Plasma glucose, lipids and apolipoproteins in normals and impaired glucose tolerant men (IMPGTT) Correlation coefficients among the variables measured in the impaired glucose tolerant men (IMPGTT) Plasma glucose Plasma insulin Plasma triglycerides Plasma cholesterol Plasma HDL cholesterol Plasma apolipoprotein A-I Plasma apolipoprotein A—II Plasma apolipoprotein C—II Plasma apolipoprotein C-III Plasma HDL apolipoprotein A—I Plasma HDL apolipoprotein A—II Linear trend in the cumulative profile Normal controls Vs non—insulin dependent diabetics (T test of the linear trend) Study 1 Vs study 2 of non—insulin dependent diabetics Study 1 vs study 2 of the non-insulin dependent diabetics (T test of the linear trend) Variables measured in NIDDM men Variables measured in NIDDM females Radioimmunoassay standard curve statistics Density adjustment Page 96 122 123 124 126 127 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 164 165 CHAPTER 1 Figure CHAPTER 2 Figure Figure Figure CHAPTER 3 Figure Figure Figure Figure Figure CHAPTER 4 Figure LIST OF FIGURES Page Association of 125I-labeled apo ArII with apo AeI as demonstrated by immunoprecipi— tation technique using rabbit anti-apo A-I antibodies 20 Postprandial profile of plasma glucose (A), triacylglycerol (A), apo A-I (A) and insulin (B) after an oral glucose load 32 Postprandial profiles of glucose ingestion on subjects 2 and 3 34 Plasma glucose, triacylglycerol, apo A-I, and insulin levels following an intravenous glucose infusion on subject 1 35 Comparison of fasting plasma lipids and apolipoproteins between the NIDDM and normal subjects 52 Postprandial profiles of plasma glucose (A) and insulin (B) after an oral glucose load in NIDDM and normal subjects 53 Postprandial profiles of plasma HDL cholesterol (A) and triglycerides (B) on the two groups studied after an oral glucose load 55 Postprandial profiles of apolipoproteins A-I (A), A-II (B), C-II (C), and C-III (D) on the normals and diabetics studied 56 Percent incremental changes in plasma triglycerides (A), apo C-II (B) and apo C-III (C) on the two groups studied 58 Relationship between total plasma triglycerides and apolipoprotein C-II (apo C-II) determined by radioimmunoassay in the non-insulin dependent diabetes mellitus (NIDDM) subjects 75 viii Figure Figure Figure Figure Figure CHAPTER 5 Figure Figure Figure Figure Figure Figure Figure Figure .5 A—8 Relationship between total plasma triglycerides and apolipoprotein C-II (apo C—II) determined by radioimmunoassay in the insulin dependent diabetes mellitus (IDDM) subjects Relationship between total plasma triglycerides and apolipoprotein C-III (apo C—III) determined by radioimmunoassay in the non—insulin dependent diabetes mellitus (NIDDM) subjects Relationship between total plasma triglycerides and apolipoprotein C-III (apo C-III) determined by radioimmunoassay in the insulin dependent diabetes mellitus (IDDM) subjects Fasting plasma lipid and apolipoprotein profiles of insulin dependent diabetes mellitus (IDDM) and non—insulin dependent diabetes mellitus (NIDDM) men in comparison with normal subjects Fasting plasma lipid and apolipoprotein profiles of insulin dependent diabetes mellitus (IDDM) and non-insulin dependent diabetes mellitus (NIDDM) women in comparison with normal subjects Postprandial profiles of plasma glucose after an oral glucose load in 2 subjects before (study 1) and after (study 2) weight loss Postprandial profiles of plasma insulin after an oral glucose load in 2 subjects before (study 1) and after (study 2) weight loss Postprandial profiles of plasma glucose after an oral glucose load in 2 subjects who maintained weight Postprandial profiles of plasma insulin after an oral glucose load in 2 subjects who maintained weight Postprandial profiles of plasma triglycerides after an oral glucose load in 2 subjects before (study 1) and after (study 2) weight loss Postprandial profiles of plasma triglycerides after an oral glucose load in 2 subjects who maintained weight Postprandial profiles of plasma total cholesterol after an oral glucose load in 2 subjects before (study 1) and after (study 2) weight loss Postprandial profiles of plasma total cholesterol after an oral glucose load in 2 subjects who maintained weight Page 76 78 79 80 81 97 98 100 101 102 103 104 105 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure A-lO A—ll A-12 A—l3 A-14 A—15 A—16 A-17 A-18 Postprandial profiles of plasma HDL cholesterol after an oral glucose load in 2 subjects before (study 1) and after (study 2) weight loss Postprandial profiles of plasma HDL cholesterol after an oral glucose load in 2 subjects who maintained weight Postprandial profiles of apolipoprotein A-I after an oral glucose load in 2 subjects before (study 1) and after (study 2) weight loss Postprandial profiles of apolipoprotein A—I after an oral glucose load in 2 subjects who maintained weight Postprandial profiles of apolipoprotein A-II after an oral glucose load in 2 subjects before (study 1) and after (study 2) weight loss Postprandial profiles of apolipoprotein A—II after an oral glucose load in 2 subjects who maintained weight Postprandial profiles of apolipoprotein C-II after an oral glucose load in 2 subjects before (study 1) and after (study 2) weight loss Postprandial profiles of apolipoprotein C-II after an oral glucose load in 2 subjects who maintained weight Postprandial profiles of apolipoprotein C-III in 2 subjects before (study 1) and after (study 2) weight loss Postprandial profiles of apolipoprotein C-III after an oral glucose load in 2 subjects who maintained weight Page 106 107 108 109 110 111 112 113 114 115 INTRODUCTION AND BACKGROUND INTRODUCTION The two lipids that circulate in the plasma that are important clinically are cholesterol and triglycerides. However, they are not by themselves soluble. When they are synthesized, they combine with specific proteins, called apolipoproteins, to allow them to be trans- ported in plasma in a soluble state. They are known as lipoproteins. Examination of plasma lipids and lipoproteins in diabetes mellitus is of interest because of the increased prevalence of arteriosclerotic cardiovascular disease associated with this disorder. The insulin deficiency of insulin dependent (IDDM) and the obesity and hyperinsu- linemia of non-insulin dependent (NIDDM) diabetics may each affect the factors regulating hepatic and intestinal output of lipoproteins into plasma. Lipoproteins undergo dynamic changes during the postprandial period. Both normal and hypertriglyceridemic individuals experience postprandial increases in plasma triglycerides after a carbohydrate or a fat meal. It is known that changes in very low density lipo- protein (VLDL) metabolism occur following the ingestion of simple carbohydrates; and high carbohydrate diets increase the secretion of VLDL triglycerides by the liver. If human subjects develop a transient accumulation of triglyceride rich lipoprotein remnants during the postprandial period, those that experience prolonged lipemia might 2 have a defect in clearance and reveal differences in postprandial and postabsorptive apolipoproteins. The main purpose of this research was to study the fasting and the postprandial dynamics of apolipoprotein metabolism in normal and in diabetic subjects. BACKGROUND Lipoproteins The function of plasma lipoproteins is to transport lipids in a water soluble form. They are lipid—protein complexes that transport lipids in circulation and regulate lipid synthesis and catabolism. The various types of lipid-protein complexes are classified by two physical chemical methods: 1) ultracentrifugation, where the separation depends on the density of the particles and 2) electro- phoresis in paper, agarose or polyacrylamide gels, where the separation depends on charge and to some extent on particle size. The typical classes of human plasma lipoproteins are shown in Table 1 (1—3). The triglyceride rich particles, hepatic and intestinal VLDL and intestinal chylomicrons are metabolized in similar pathways (4). Chylomicrons carry dietary triglycerides from the intestine to nonhepatic tissues for utilization or storage. VLDL contain tri- glycerides made primarily in the liver. Progressive delipidation of VLDL and chylomicrons leads to the formation of lipoproteins of intermediate density (IDL) and chylomicron remnants, respectively, and finally to the production of low density lipoproteins (LDL). The LDL derived from VLDL catabolism and the high density lipoproteins (HDL) made in the liver contain the bulk of the plasma cholesterol. During the last several years, numerous reviews have summarized the current state of knowledge of the plasma lipoproteins (2—8). The moa< coma HHHIuoa< mon< HHuooa< HHu maononansno cofiuaoamflmmmao mcflwuouaoaflq mEmmHm :manm wo mmfluumaonm mam COHuHmanou H wH£MH 4 following discussion will focus particularly on the specific functions of the major apoproteins in the regulation of the metabolism of lipo- proteins. Apolipoproteins Apo A i The A apoproteins (apo A-I and apo A-II) are the major protein constituents of high density lipoproteins (HDL). The amino acid sequence of both A apoproteins has been determined (9-13). Apo A—I and apo A—II have been shown to have clearly different primary and secondary structures which contribute to their immunologic and bio— chemical characteristics (11, 13). Apo A—I and apo A—II constitute about 90% of total HDL protein with an apo A—I to apo A—II ratio of 3:1 by weight (14). HDL can be divided into two density classes: (HDL2 (d 1.063 — d 1.125 g/ml) and HDL3 (d 1.125 — d 1.21 g/ml). Published reports of apo A—Izapo A—II ratio of HDL compared to HDL 2 are contradictory indicating that it is higher (15-18), lower (19) 3 or the same (20) in HDL The contradiction probably is due to 3. variations in the isolation of these subfractions. The significantly higher levels of HDL cholesterol and apo A-I in women has been attributed entirely to an increase in circulating HDL2 (18). It has been proposed that HDL is the major contributor to the anti—atherogenic 2 role of plasma HDL (18, 21). The sites of apo A synthesis are the liver and intestine (22, 23). Active intestinal synthesis has been demonstrated during fat absorption and chylomicron assembly (22—24). Intestinal synthesis is estimated to contribute up to 59% of the apo A-I pool in the rat (22). Studies of the excretion of apo A-I following fat feeding in two patients with 5 chyluria suggest that intestinal synthesis may contribute 48 to 50% of the Apo A-I pool in the human (25). Apo A-I is an activator of the enzyme lecithin cholesterol acyl- transferase (LCAT) (26), the enzyme responsible for esterification of plasma cholesterol in man. Apo A-II is associated with HDL lipid more tightly than Apo A—I and may play more of a structural role in the HDL particle (27). In normal human plasma, 87% of Apo A—I and 90% of Apo A-II are found in HDL and trace amounts of each apoprotein are found in the other lipoprotein fractions (28). Reported plasma HDL half-life values in normal subjects range from 3.3 to 5.8 days (29, 30). Mean concentrations reported in the literature ranged from 100 to 154 mg/dl for Apo A-I and 34 to 83 mg/dl for Apo A—II (17, 28, 31 - 33). Dif- ferences in values may relate to whether or not the assay techniques incorporated a delipidation step and possibly to variability and purity in the antisera used. Some groups have reported higher total A apoprotein levels in females than in males (33) while others have reported nearly identical levels (17, 31, 32). Plasma levels of Apo A-I and Apo A—II correlate with HDL choles- terol levels (17). Low HDL cholesterol levels have been associated with high prevalence of coronary artery disease in population studies (34 — 37) and it has been suggested that HDL may be important in remo- ving cholesterol from tissues (38). HDL cholesterol levels are elevated in long distance runners (39) and in premenopausal females (40) and can be increased by estrogen (41) or nicotinic acid admi— nistration (29). They are decreased in hypertriglyceridemic patients (35, 42), those with coronary heart disease (35, 43), in diabetics 6 (44, 45), and in subjects consuming high carbohydrate diets (29). Apo B Apo B is the major protein of LDL and is also a major protein constituent of VLDL and chylomicrons. It has been shown to be synthesized in both the liver and intestine (22, 46). Apo B has been identified in human intestine mucosal cells by immunological techniques (47). Apo B is essential for the transport of trigly— cerides out of the liver and the intestine. In patients with abeta- lipoproteinemia, no triglyceride enters the blood stream despite excessive amounts of intracellular triglycerides (48). Apo C The Apo C apoproteins are a group of low molecular weight proteins present as the major apolipoproteins in VLDL and minor constituents of HDL. The amino acid sequence of the three proteins (Apo C-I, Apo C-II, and Apo C—III) are known (49 — 51). Apo C—III is composed of three different polymorphic forms designated as apolipoprotein C—IIIO, apolipoprotein C-III and apolipoprotein C—III depending 1 2’ on their sialic acid content (51). It is now generally accepted that Apo C proteins play an impor— tant role in the catabolism of triglyceride rich lipoproteins (chylomicrons and VLDL) (52). Apo C—II is a specific protein cofactor necessary for triglyceride hydrolysis by lipoprotein lipase of extrahepatic origin (52, 53). Apo C—I also has been reported to activate LCAT (7). Apo C-III may inhibit lipoprotein lipase in vitro (54). However, all three of the C apoproteins may partially inhibit triglyceride hydrolysis when present in surplus amounts. Apo C is synthesized and secreted by the liver (46). Whether 7 Apo C is secreted from the liver with VLDL or HDL or both is not clear. Variable amounts of Apo C can be found with all plasma lipoproteins. Apo C comprises more than 60% of the total protein mass of chylomicrons, 40-80% of the VLDL apoproteins, 2-10% of the HDL apoproteins and is also present in trace amounts in the LDL density range (6). In fasting human plasma, the C apoproteins are mainly found in VLDL and HDL. Apo C-II and Apo C—III concentrations in normal plasma are approximately 5 mg/dl and 11 mg/dl respectively (55, 56). The concentrations of both these apoproteins are increased in hypertriglyceridemic subjects. Apo E Apo E is also known as the arginine rich apolipoprotein. Following synthesis in the liver, Apo E is secreted as part of nascent HDL and is an important constituent of normal human VLDL (57 - 59). A number of studies have suggested that Apo E maybe involved in the plasma transport of cholesteryl esters (60, 61). Recent findings also show that the presence of Apo E in lipoprotein particles confers on them the ability to interact with specific cell membrane receptors, both in the peripheral (62) and hepatic cells (63) and to deliver their cholesterol to these tissues. Apo E concentrations in normal human plasma are approximately between 4 and 10 mg/dl depending on the analytical technique (64 - 66) and are increased two—fold in patients with Type III and Type IV hyperlipoproteinemia (64, 67). Apo D Apo D is also known as "thin line peptide" and has been isolated from HDL density range (68). Together with the enzyme LCAT and Apo A-I, this complex has been reported to synthesize and distribute 8 cholesteryl ester to the acceptor lipoproteins, with Apo D serving as a transfer factor (69). Summary of the characteristics of human plasma apoproteins are shown in Table 2 (5, 70). Association of Lipoproteins and Coronary Heart Disease in Diabetes Atherosclerotic cardiovascular disease is a major cause of mor— tality among the insulin dependent (IDDM) (71) and non—insulin depen- dent (NIDDM) diabetics (72). Abnormalities in blood lipids associated with diabetes mellitus have been reported. Hypertriglyceridemia has been observed frequently (73) as well as hypercholesterolemia (72). Several recent studies have examined the complex interrelationship between diabetes and lipoproteins. These studies are difficult to interpret because there are many factors in diabetes that can influence blood lipid levels, such as insulin levels, obesity, sex, age and the type of diabetes. However, from these studies have emerged several trends. First, insulin deficient diabetics have low HDL cholesterol levels (45, 74, 75) and the HDL cholesterol returns toward normal if insulin is given and when diabetes is controlled. In some (76, 77) but not all studies (78, 79), HDL cholesterol levels have been correlated with such indices of diabetic control as gly- cosylated hemoglobin concentrations or plasma glucose levels. Several investigators have also reported alterations in the HDL composition of diabetic plasma (74, 77, 79) including alterations in subclasss distribution of cholesterol and Apo A proteins. Several mechanisms have been suggested whereby the insulin deficiency of IDDM (80) may induce derangements in lipoprotein metabolism: 1) decreased activity of lipoprotein lipase, thus ocfiumoucfl .Hm>flq scufiQHSCw Amqs s w ooo.mm m a Baum mmum>auonu use mnnnnesHa I as qu.w HHHlo uo>flq GOMum>Huom omega; 0 mm mmw.w HHIU cowum>fiuum ommaflgs na>ag coaum>auom efig uuoamcmuu UH Nm m w m uo>HH .wcfiumouaH m I «ma ooo.mH HHI¢ um>aa .aanumaucH coaum>fluom H o 100 r c 8 “50% so I 1 - - A - .__..q , 100. - #— r E E. I i 60 40 - 50 - 20 1W 0 __L J J 1 1 1 1 j o 1 1 1 1 1 1 1 1 1 O 60 120 180 240 300 O 60 120 180 240 300 Minutes Minutes Figure l Postprandial profiles of plasma glucose (A), triacylglycerol (A), apo A-I (A), and insulin (B) on subject 1 after an oral glucose load. The profile of the same subject not given glucose is shown in panels C and D. Apo A-I concentrations were determined by radioimmunoassay and each point represents the mean of triplicate analyses. 33 to baseline within 90 min. The postprandial time response in the elevation of plasma triacyl- glycerol and glucose is different for each subject showing individual variations. As demonstrated in subjects 2 and 3 (Figure 2), plasma triacylglycerol changes paralleled the rise of plasma glucose. In these subjects, plasma triacylglycerol increased very rapidly (within 30 min). Glucose was infused intravenously to bypass the glucose absorption by the intestine on subject 1. Plasma glucose levels were determined at 10 min intervals. Adjustments in the rate of infusion were made until a rise in plasma glucose (Figure 3) similar to that of the oral glucose profile was achieved (Figure 1). No increases in plasma triacylglycerol were observed after the glucose infusion. Elevation of plasma apo A—I following a glucose load A striking increase of apo A—I following the administration of oral glucose was seen in subject 1 (Figure l). The peak concentration, as determined by radioimmunoassay, increased 64% from the baseline. The increase was apparently not due to the assay technique. Coefficient of variation of the intrassay is normally less than 5% (21). In a control experiment, the subject was fasted for 12 h and given no glucose before the blood samples were drawn. No significant changes in plasma apo A—I and triacylglycerol levels were observed (Figure l). Apo A-I levels of subjects 2 and 3 were also determined (Figure 2). The pattern of apo A—I levels varied among each individual following the glucose load. Nevertheless, in these subjects, two patterns of response were elucidated: l) the maximal elevation of plasma apo A—I was reached later than the glucose elevation; 2) the level of apo A-I increased 34 250 120 A I Apo Al A Glucose 100 . B 200 - O Tnacylglycorol 80 F 150 60 r 100 ‘ 40 r 50 2° , l l o l 1 1 1 1 1 1 J 1 250 lnsulin ( nunits/ ml) 200 Concentrations in plasma (mg/dl) O 150 100 50 o o 60 120 180 240 300 o 60 120 180 240 300 Minutes Minutes Figure 2 Postprandial profiles of glucose ingestion on subjects 2 (A and B) and 3 (C and D). Experimental conditions were identical to that of Figure 1, A and B. 35 250 A e Apo AI 200 A Glucose g g 0 Trlecylglycerol .9 a 1; g 150 - 5 m 5 S 100 8%; i 0 c "' 50 r O J 1 1 i l 1 J_ 0 60 120 180 240 Minutes Figure 3 300 120 100 80 60 401 lnsufin(;Lunns/nfl) 20 1 1 1 1 60 120 180 Minutes Plasma glucose, triacylglycerol, apo ArI (A), and insulin levels (B) following an intravenous glucose infusion on subject 1. 240 300 36 and then fell to almost the baseline level within 30 min regardless of the fluctuations in levels over time. The effect of glucose infusion on subject 1 demonstrated that plasma apo A—I levels continuously increased from 140 mg/dl to 174 mg/dl in a period of 5 h (Figure 3). Concentrations of plasma apo A—II, as determined by radioimmuno— assay, also increased within 90 min of glucose ingestion as observed in all three subjects (Table 1). The increase was also observed within 30 min during the intravenous glucose infusion (Table 2). These findings suggest that an acute ingestion of glucose is associated with increments of apo A-I and to a lesser extent with increments of apo A-II. Tables 6 and 2 show slight fluctuations in total plasma cholesterol and HDL cholesterol after an acute ingestion of glucose. Plasma insulin levels following a glucose load Typical time response curves of insulin levels are shown in Figures 2, 2, and 6, The percent increase in insulin levels ranged from 600-1200 in the three subjects. Elevation of apo A—I did not occur before the insulin peaked. Discussion The purpose in these studies was to determine whether apo A—I and apo A-II levels changed following an oral glucose load since glucose ingestion is known to increase plasma triacylglycerol. The oral glucose load in the normal subjects studied increased plasma triacylglycerol as well as plasma glucose levels within 30—60 min of glucose ingestion. Plasma apo A-I levels also increased, although the timing of the increase differed in the subjects. Whether or not part of the increase in plasma triacylglycerol was directly derived 37 Table 1 Total Plasma Cholesterol, HDL Cholesterol, and ApoA-II Levels in Individual Subjects After an Oral Glucose Load Time Cholesterol HDL Cholesterol ApoA—II Subjects (min) (mg/d1) (mg/d1) (mg/d1) l O 169 37.0 20.2 30 179 33.1 22.5 60 174 33.1 22.5 90 172 36.4 29.4 120 177 33.1 18.2 150 184 34.2 25.3 180 165 38.6 26.2 240 171 34.2 25.9 300 176 36.4 26.5 2 0 233 36.3 21.9 30 210 38.7 20.5 60 222 40.4 25.5 90 215 40.4 26.2 120 221 38.7 22.6 150 203 38.7 24.2 180 225 41.3 23.1 240 210 40.4 19.7 300 209 41.3 20.5 3 0 149 54.3 23.0 30 164 51.4 21.1 60 160 51.4 20.3 90 159 53.9 25.8 120 151 57.3 21.0 150 154 55.6 21.6 180 151 58.1 21.9 240 153 56.4 30.1 300 156 53.9 27.2 38 Table 2 Total Plasma Cholesterol, HDL Cholesterol, and ApoA—II Levels in Subject 1 After Intravenous Glucose Infusion Time Cholesterol HDL Cholesterol ApoA—II (min) (mg/d1) (mg/d1) (mg/d1) 0 208 45.1 22.8 30 185 42.1 28.8 60 208 43.0 27.1 90 200 43.0 27.7 120 203 43.8 21.6 150 197 44.6 24.0 180 194 42.1 28.3 240 186 43.8 29.1 300 201 43.8 27.4 39 from intestine is unclear. Ordinarily, such an increase is due to release of triacylglycerol from the liver (11, 22). Previous studies in this laboratory (9) and others (23, 24) have shown that intestinal triacylglycerol transport is a slow process (includes digestion and absorption) and plasma levels peak between 3—5 h following a fat load. The finding of an increase in apo A—I levels after glucose ingestion has not been reported previously. This observation is in contrast to what has been demonstrated in fat feeding which does not produce an increase in the levels of apo A—I up to 8 h postprandially in normal subjects (9, 10). Studies in rats (8) indicate that the production of apo A—I by the small intestine is not regulated by the rate of intestinal triacylglycerol transport. The intestine of the rat also secretes discoidal HDL lipoproteins (25) which are an addi— tional source of apo A—I for plasma. This phenomenon has not been documented in humans. Apo A—I has been shown to leave the surface of chylomicrons after their secretion into the plasma and then be transferred to the HDL. In humans, it has been estimated that as much as 30% of the total daily synthesis of apo A—I may originate from the intestine and directly influence HDL metabolism (26). Most of the glucose ingested orally is actively absorbed in the upper gastrointestinal tract and is transported to the liver via the portal vein. The insulin response to oral glucose in these subjects was higher than that seen when glucose was administered intravenously (Figures 1, 2, and 6). This is a well-known phenomenon (27). The release of more insulin by glucose by the oral route rather than the intravenous route is hypothesized as being due to the release of various 4O gastrointestinal humeral factors (28). The rise in insulin concentrations after oral glucose ingestion is concurrent with the rapid rise of apo A-I. It is possible that the insulin release following glucose ingestion triggers the hepatic output of apo AeI, contributing to its increased plasma levels. However, this peak seems to only last for about 1 h. The present study shows that a glucose load (oral and intravenous infusion) does not alter total plasma cholesterol and HDL cholesterol. However, this is an acute effect of a glucose load and it seems to alter mainly apo A—I and apo A—II without affecting HDL cholesterol. This finding suggests that a common mechanism may be involved in the regulation of apo A—I and apo A—II concentration in plasma. In longer term studies (4-5 days), high carbohydrate diets have been shown to decrease apo A-I levels in normal men (24). This change has been hypothesized as resulting from the secretion of altered lipoproteins or from diet—induced alterations in apolipoprotein catabolism. The metabolic significance of the short temporary increase of apo A—I concentrations after glucose ingestion is not clear. It can be speculated that the increase of plasma apo A—I levels may facilitate the secretion of triacylglycerol into the circulation. Further studies are being continued in this laboratory including studies of diabetic patients in order to compare their responses to that of normal subjects. Acknowledgement The authors thank Deanna Nash for glucose determinations, Joan King for insulin determinations, the nursing and dietary staff of the Clinical Research Center for their excellent help in the execution of the study, and Susan Woychik for expert secretarial assistance. 41 References 1. 10. Miller GJ, Miller NE. Plasma high density lipoprotein concentration and development of ischemic heart disease. Lancet 1975;1:16-l9. Gordon T, Castelli WP, Hjortland MC, Kannel WB, Dawber TR. High density lipoprotein as a protective factor against coronary heart disease. Am J Med 1977;62:704—714. Mao SJT, Gotto AM, Jackson RL. Immunochemistry of plasma HDL. Radioimmunoassay of apo A—II. Biochemistry 1975;14:4127—4131. Wu AL, Windmuller HG. Relative contributions by liver and intestine to individual plasma apolipoproteins in the rat. J Biol Chem 1979;254:7316—7322. Schonfeld G, Bell E, Alpers DH. Intestinal apoproteins during fat absorption. J Clin Invest 1978;61:1539-1550. Schaefer EJ, Jenkins LL, Brewer HB. Human chylomicron apolipoprotein metabolism. Biochem Biophys Res Commun 1978;80:405—412. Tall AR, Green PHR, Glickman RM, Riley JW. Metabolic fate of chylomicron phospholipids and apoproteins in the rat. J Clin Invest 1979;64:977—989. Windmuller H, Wu AL. Biosynthesis of plasma apolipoproteins by rat small intestine without dietary or biliary fat. J Biol Chem 1981;256:3012-3016. Barr SI, Mao SJT, Kottke BA. Postprandial distribution of apolipo— proteins A—I, A—II, C—II, in normal and hypertriglyceridemic individuals. (submitted 1982). Kay RM, Rao S, Smott C, Miller NE, Lewis B. Acute effect of the pattern of fat ingestion on plasma high density lipoprotein com— ponents in man. Atherosclerosis 1980;36:567-573. 11. 12. 13. 14. 15. 16. 17. 18. 42 Quarfordt SH, Frank A, Shames DM, Berman M, Steinberg D. Very low density lipoprotein triglyceride transport in type IV hyper— lipoproteinemia and the effects of carbohydrate-rich diets. J Clin Invest 1970;49:2281—2297. Schonfeld G. Changes in the composition of very low density lipoprotein during carbohydrate induction in man. J Lab Clin Med 1970;75:206—211. Patton JG, Dinh DM, Mao SJT. Phospholipid enhances triglyceride quantitation using an enzyme kit method. Clin Chim Acta 1982; 118:125-128. Department of Health, Education and Welfare. Lipid research clinical program manual of laboratory operations, 1974. Bethesda, MD: Department of Health, Education and Welfare (NIH 75—628). Chung BH, Wilkinson T, Geer JC, Segrest JP. Preparative and quantitative isolation of plasma lipoproteins: rapid, single discontinuous density gradient ultracentrifugation in a vertical rotor. J Lipid Res 1980;21:284—291. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 1955;34:1345-1353. Mao SJT, Kottke BA: Tween—20 increases the immunoreactivity of apolipoprotein A—I in plasma. Biochim Biophys Acta 1980; 620:447-453. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measure— ment with the folin phenol reagent. J Biol Chem 1951;193:265-275. 20. 21. 22. 23. 24. 25. 26. 27. 43 Mao SJT, Miller JP, Gotto AM, Sparrow JT. Structure of apo A—I in human HDL radioimmunoassay using surface specific antibodies. J Biol Chem 1980;255:3448-3454. Herbert V, Lau KS, Chester W, Gottlieb CW, Bleicher SJ. Coated charcoal immunoassay of insulin. J Clin Endocrinol Metab 1965; 25:1375-1384. Barr SI, Kottke BA, Chang JY, Mao SJT. Immunochemistry of human plasma apolipoprotein C-II as studied by radioimmunoassay. Biochim Biophys Acta 1981;663:491—505. Schonfeld G, Pfleger B. Utilization of exogenous free fatty acids for the production of very low density lipoprotein triglycerides by livers of carbohydrate fed rats. J Lipid Res 1971;12:614—621. Schlierf G, Jessel S, Ohm J et al. Acute dietary effects on plasma lipids, lipoproteins and lipolytic enzymes in healthy normal males. Eur J Clin Invest 1979;9z319—325. Schonfeld G, Weidman SW, Witztum JL, Bowen RM. Alterations in levels and interrelations of plasma apolipoproteins induced by diet. Metabolism 1976;25:261-275. Green PHR, Tall AR, Glickman RM. Rat intestine secretes discoid high density lipoprotein. J Clin Invest 1978;61:528—534. Green PHR, Glickman RM, Saudek CD, Blum CB, Tall AR. Human intestinal lipoproteins. Studies in chyluric subjects. J Clin Invest 1979;64:233—242. Cataland S, Crockett SE, Brown JL, Mazzaferri EL. Gastric inhibitory polypeptide (GIP) stimulation by oral glucose in men. J Clin Endocrinol Metab 1974;39:223—228. 44 28. Perley MJ, Kipnis DM. Plasma insulin responses to oral and intravenous glucose: studies in normal and diabetic subjects. J Clin Invest 1967;46:1954—1962. CHAPTER 3 Acute effects of glucose ingestion on plasma apolipoproteins in normal men and non—insulin dependent diabetics ABSTRACT Apolipoprotein levels were studied in diabetic and control subjects. Five men with non—insulin dependent diabetes mellitus (NIDDM) and six healthy men (control subjects) were admitted to the Clinical Research Center after consuming a high carbohydrate diet for three days. After fasting for 12 h, the subjects ingested l g glucose/kg body weight. Blood samples were drawn at 30, 60, 90, 120, 150, 180, 240, and 300 min following the glucose load. The NIDDM patients were found to have low fasting plasma levels (mg/d1) of apolipoprotein A—I (apo A—I) (147.7 f 19.1) and apolipoprotein A—II (apo A-II) (20.9 i 4.6) and significantly high levels of apolipoprotein C—II (apo C—II) (7.8 t 2.1) and apolipo- protein C-III (apo C—III) (15.2 t 2.0) as compared to normal control subjects. All the subjects showed an increase of apolipoproteins A—I, A—II, C—II, and C-III after the oral glucose challenge. Since apo C-II and apo C—III are not synthesized in human intestine, these findings suggest that the liver is primarily responsible for the increase of the C apolipoproteins following a glucose load. There was no significant difference in the postprandial changes of apo A-I and apo A-II between normal and diabetic subjects. However, an initial decrease of apo C-II and apo C-III was seen in the diabetics in response to the glucose load. Total plasma triglyceride levels were elevated at all time points in the diabetic subjects. Since apo C-II is an activator for lipoprotein lipase, an enzyme responsible for triglyceride clearance, these findings suggest that the initial decrease of apo C—II in the NIDDM subjects may be a contributory factor in the persistent hypertriglyceridemia observed in these subjects. Whether hyperinsulinemia and associated apparent cellular insulin insensitivity account for these findings is not known. 45 46 Introduction Lipoprotein particles are very dynamic. Their apolipoprotein constituents play important roles in the mechanisms involved in the regulation of lipoprotein metabolism. The liver and the intestine are primarily responsible for producing the apolipoprotein components of the plasma lipoproteins (1—4). Liver and intestine provide sig- nificant quantities of apolipoproteins A and B (5, 6). The liver contributes most of the C apolipoproteins (5). Glucose absorption from the gastrointestinal tract will induce secretion of very low density lipoprotein (VLDL) triglyceride in the liver. In short term carbohydrate feeding, the lipids and protein composition of the lipoproteins have been shown to be altered (7, 8). These postprandial changes are associated with transfers of apolipo— proteins and can alter the composition of high density lipoproteins (HDL). Previous studies have suggested that HDL may play roles both in triglyceride clearance and in the removal of cholesterol from tissues. Apolipoprotein A-I (apo A—I) and apolipoprotein A-II (apo A—II) are the major apoproteins of HDL, while apolipoprotein C—II (apo C—II) and apolipoprotein C-III (apo C—III) are the major apoproteins of VLDL and chylomicrons. The apolipoprotein C peptides are distributed in both HDL and triglyceride—rich lipoproteins (9). It is known that apo C—II is the activator for lipoprotein lipase (LPL), the enzyme responsible for the catabolism of triglyceride-rich lipoproteins (10). Measurements of the concentrations of apo C-II and apo C—III in normal and hypertriglyceridemic humans show that their distribution among lipoproteins is influenced by the plasma triglyceride level (11, 12). 47 To understand the relationship between triglycerides and lipo- proteins in a common disease associated with abnormal glucose metabolism as well as abnormal triglyceride levels, patients with non-insulin dependent diabetes mellitus (NIDDM) were studied. The present studies were undertaken to determine the response of plasma levels of apo A—I, apo A—II, apo C-II, apo C-III, total triglycerides, total cholesterol, HDL cholesterol, glucose, and insulin to stimulation of VLDL secretion by a glucose load and to compare these responses in normal and NIDDM men. The study subjects were limited to one sex (men) because of potential effect of sex difference on lipid metabolism, and because of availability of these subjects. Materials and Methods Subjects Six healthy male control subjects and five male NIDDM patients were recruited. None of the patients was taking insulin or using oral hypoglycemic agents at the time of the study. The diabetic subjects were being managed with diet alone. The control subjects and the diabetics were of comparable age, with the diabetics having a higher mean relative weight (Table 1). Ideal weights were based on desirable weights in relation to heights for adults (13). All were instructed to consume a high carbohydrate diet (approximately 300 g/day) for three days and to abstain from alcohol for at least one week prior to the study. After giving fully informed written consent, each subject was studied for approximately 6 h after an overnight fast. Plasma samples Each subject was given 1 g glucose/kg body weight orally as a 48 unwaea HaeeH ustwB HmnuoHuMHom uLmHoz mw¢ mammHm mammHm wcHummm wGHummm Anm H cmmev mHouucoo Hmauoz new mUHumpwHQ mo mUHumHHmuomumno HMUHGHHU H wanna 49 34% solution. Blood samples were drawn from an indwelling venous catheter into tubes containing EDTA (0.1%) for lipoprotein and lipid analyses and into separate tubes with NaFl for glucose and insulin analyses. Blood samples were centrifuged immediately to obtain the plasma. Baseline blood samples were drawn at -15 and 0 min and thereafter at 30, 60, 90, 120, 150, 180, 240, and 300 min after glucose ingestion. Lipid and apolipoprotein determinations Plasma triglycerides were determined in duplicate on the day of the study using a modification of an enzymatic method (14). Plasma total cholesterol was also determined enzymatically using the choles- terol modification of the Beckman enzyme kit method (Beckman Instru— ments, Inc., Fullerton, CA). Both methods are comparable to that of established methods used in the Lipid Research Clinics (15). HDL cholesterol was analyzed using VLDL and low density lipoprotein (LDL) depleted samples. The VLDL and LDL were removed by a modification of a single spin ultracentrifugation technique (16). Plasma apolipoproteins A—I, A—II, C—II, and C—III were determined by radioimmunoassays as previously described (17-19). All dilutions of plasma, antibodies, and apolipoproteins were made with a standard radioimmunoassay buffer containing 0.1% bovine serum albumin, 0.1 M sodium borate, 1.0 mM EDTA, and 0.1% NaN3, pH 8.5. Diluted plasma samples were stored at —200C and all samples were determined in the same assay run. Tween—20 (100 pl), at concentrations 1.5, 0.2, and 0.1% were added for apo A—I, apo C—II and apo C-III assays, respectively. Specificity of the assays have been established (17-19). The apoli— poprotein values reported are the means of triplicate samples at each 50 time point of plasma measurements for each subject. Intraassay coefficients of variation for the assays were less than 5% for apo A-I, apo A—II, apo C-II, and apo C-III. Other methods Plasma glucose was measured immediately using the glucose oxidase method with a YSI glucose analyzer (Yellow Springs Instruments, Yellow Springs, OH). Insulin levels were determined by radioimmunoassay of the frozen stored plasma samples (20). Statistical methods Summary statistics include the mean f SD of the mean in a table and the mean f SEM in figures for the diabetic and normal samples. The statistical analyses were performed using two-tailed, non—paired .6 tests (comparison of plasma values between normal and diabetic subjects), and paired 6 tests (% change from baseline after the glucose load for either the normal or diabetic subjects). Results Clinical evaluation of subjects As shown in Table 1, the mean age for the diabetic group (N=5) was 49.0 yr (range 39—58), while for the normal subjects (N=6) it was 48.0 yr (range 33-60). Mean fasting plasma glucose level was higher in the diabetics as compared with the normal subjects. The difference was not statistically significant due to the high degree of variability among the individual subjects. The mean fasting plasma insulin concentration was elevated to 18.4 pU/ml in the diabetics in comparison with 13.1 pU/ml in the controls. The diabetics were mildly overweight as shown from their relative weight. 51 Fasting plasma lipids and apolipoprotein levels The comparisons of the fasting plasma lipids and apolipoprotein values between the diabetic and normal groups are shown in Figure 1. Using "0" as the mean level of all the variables measured in the normal subjects (N = 6), the diabetics (N = 5) tended to have sig— nificantly elevated plasma triglycerides (P< 0.02), apo C—II (P <0.02), and apo C-III (P< 0.002) and significantly decreased HDL cholesterol (P <0.005) and low levels of apo A—I and apo A-II. There was no difference in total plasma cholesterol between the two groups. Postprandial changes in plasma glucose and insulin Figure 2 shows the average responses of the five diabetic and six control subjects. In the control subjects, mean plasma glucose level following a glucose load reached a maximum value after 30 min and gradually declined to baseline values at 150 min. The maximum plasma insulin value was observed 90 min after administration of glucose; the levels returned to baseline values at 300 min. The corresponding responses of the diabetics to the glucose load showed a maximum glucose value at 90 min, which remained elevated for 150 min, and then declined to baseline values at 300 min. The insulin peak reached a maximum at 120 min and remained elevated for 180 min, then gradually declined, but did not return to initial levels within 300 min of observation. The plasma glucose rise was sig— nificantly greater in the diabetics in comparison with the controls. Postprandial changes in plasma cholesterol, HDL cholesterol, and triglycerides following the glucose load Comparisons of changes in HDL cholesterol and triglyceride in 52 Non-insulin dependent diabetics (N: 5) RELATIVE SCORE '4 ' is HDL ApoA-ll Apoc-III CHOL Total A-I A c-II CHOL A” °° Figure 1 Comparison of fasting plasma lipids and apolipoproteins between the NIDDM and normal subjects. Relative score = (value of each diabetic subject) - (means of normals)/ (pooled SD of normals) i SEM. 53 350 A 300 l- , . A 5 Datum \ : a) 250 - m 5) E g 200 - 0 ‘_§, 150 - a, ‘5 100 '- E w 2 50 _ Normals CL (n=6) 0 l 1 l l l l l l 1 1 I 150 0 B E \ .2 5 10° ' Dmbeuc 1 (n=5) é Normals .3 (n=6) .9 5° ' m E m .S (L o I I l l 1 l l l 1 1 l O 60 120 180 240 300 Time (min) Figure 2 Postprandial profiles of plasma glucose (A) and insulin (B) after an oral glucose load in NIDDM and normal subjects. Values are mean t SEM. 54 control and diabetic subjects are shown in Figure 3. There were no significant changes in plasma total cholesterol (not shown) or HDL cholesterol during the 300 min of observation in either groups throughout the study. The diabetics exhibited significantly lower levels of HDL cholesterol (P <0.001) in comparison to the control subjects. In normal controls, maximum triglyceride concentration was reached at 30 min, while in the diabetic subjects, the maximum triglyceride concentration was observed at 60 min. Comparisons of absolute values at each time point showed statistical differences between the two groups; the diabetics exhibiting significant elevations (P <0.02) at all time points. HDL cholesterol showed negative correlation with triglyceride levels (r = -0.44). Postprandial changes in apolipoproteins A—I, A—II, C—II, and C—III following glucose load Changes in apolipoprotein levels are shown in Figure 4. Post— prandial fluctuations were observed in apo A—I and apo A-II con— centrations. There were no statistical differences in plasma apo A—I and apo A—II postprandial changes between the two groups of study subjects. Several peaks were observed in plasma apo C-II of normal controls. The first peak of apo C—II paralleled the triglyceride changes with its major peak at 240 min, representing a significant increase from the baseline (P <0.02). No significant changes in apo C-II concentrations were observed in the diabetic group. Plasma apo C-III in normal controls started to increase at 60 min reaching its maximum peak at 90 min. These increases were significant 1 L.1‘.‘1.‘ I 55 50 4O - (n=6) 30 - s - WW \ DIabetIc g1 20 '- (n=5) 2 P tn 10 l' A .E Q I 1 l 1 1 1 4 u 1 J .E m 350 8 z 300 - I: Diabetic “ _ m=m s 250 8 o 200 - C) 150 - Normals 100 - W 50 - B o I 1 1 l 1 J J J 1 l 1 0 60 120 180 240 300 Time (min) Figure 3 Postprandial profiles of plasma HDL cholesterol (A) and triglycerides (B) on the two groups studied after an oral glucose load. Values are mean t SEM. 56 Concentrations in plasma (mg/6|) C I I I I o oo I20 TIme (mm) Figure 4 Postprandial profiles of apolipoproteins A—I (A), A—II (B), C-II (C) and C-III (D) on the normals (A) and diabetics (5) studied. The apolipoprotein concentrations were determined by radioimmunoassay and each point represents the mean of triplicate analyses. Intraassay coefficient of variation was less than 5%. 57 from the baseline values (P <0.002). No significant changes in apo C-III concentrations were observed in the diabetic group. The increased ratio of triglyceride to apo C-II levels were ob— served during the 300 min of observation in the diabetic subjects. Incremental changes in triglycerides, and apolipoproteins C-II and 9:111 Figure 5 summarizes the percent incremental changes in plasma triglycerides, apo C—II, and apo C-III concentrations of the normal and diabetic subjects. The 0 value represents the basal level of the two groups. Although the absolute levels of the three variables were elevated at all time points in the diabetics, the degree of the response to glucose ingestion differed quantitatively. The trigly- ceride maximum peak of the normal controls was reached at 30 min and the rise paralleled the plasma glucose profile. Plasma trigly— ceride of the diabetics had a smaller rise, reaching its major peak at 60 min and leveling off at 150 min. Plasma apo C—II and apo C—III in normal controls started to rise at 30 min. Apo C—II peaked at 240 min which differed signifi— cantly from the diabetics (P <0.05). Plasma apo C—III levels in normal controls reached maximum peak at 90 min and differed signifi— cantly from the diabetic group (PI<0.05). There was an initial postprandial decrease in plasma apo C—II and apo C—III concentrations observed in the diabetic group. Discussion Apolipoprotein levels are different in NIDDM vs normal controls both in the fasting state and after a glucose challenge. The NIDDM subjects chosen were not on treatment with exogenous insulin which 58 5O 30 - 20 - Normals 10 _ (n=6) 0 . W I TD:;1;e;;cT A n 1 1 1 l 1 I l 1 ~20 Normals (n=6) 20 - Dmbel-c (n=5l % change lrom basal OP<005 Normals (n=6) Dnabenc ln=5I Time (min) Figure 5 Percent incremental changes in plasma triglycerides (A), apo C—II (B), and apo C-III (C) on the two groups studied. Values are mean t SEM above the basal value (0) for each group. The asterisk indicates significant differences between the response of the normal and NIDDM subjects to a glucose load. I “fill .uu‘t ’ 59 may affect lipoprotein synthesis. They have endogenous insulin levels which were, in fact, higher than the control subjects. The glucose tolerance test results and immunoassayable insulin levels are consistent with NIDDM. The glucose and insulin responses to the oral glucose challenge were greater in the diabetics in terms of the magnitude of the increase throughout the 5 h of measurements. However, the initial increases (at 30 min), both of plasma glucose (60% increase in normals vs 30% increase in NIDDM) and plasma insulin (210% increase in normals vs 95% increase in NIDDM), were greater in the normal subjects. The relative weights of the diabetic subjects were greater than the normal controls. Obesity is a frequent contri— butory factor in hyperglycemia and hyperinsulinemia of diabetics and this may be the case in this group. Plasma total cholesterol and HDL cholesterol levels remained stable following the acute glucose load in both groups. The same effect was observed when fat was given to normal and hypertriglyceridemic subjects (19). Fluctuations seen in apolipoprotein values, especially apo A-I and apo A—II, cannot be attributed to under or over estimation in the radioimmunoassay technique, but rather to the individual variations evident in all the subjects. To determine the accuracy of the radio- immunoassay method, the plasma of one of the subjects was divided into several aliquots with dilutions of 1:5000. Apo A-I was measured in triplicates. The values obtained were consistent and reproducible. The reason for the observed decreases in apo A—I and apo A—II levels in the NIDDM subjects from 90-150 min are not clear. These decreases paralleled the increases in plasma glucose and insulin levels. 60 Differences were seen in the postprandial changes of apo C-II and apo C-III between the two study groups. Fasting plasma apo C-II levels were higher in diabetics vs normal control subjects; plasma apo C-II concentrations increased proportionately with plasma triglyceride in the control subjects. The initial decrease in plasma apo C-II and apo C-III may be related to unresponsiveness to endogenous insulin levels and may have contributed to the hypertriglyceridemia in the diabetic group. It is also possible that the delayed decrease in insulin levels in diabetics associated with a rise in apo C-II and apo C-III may have contributed to the decreased triglyceride responses in this group. It is evident from other studies that only a small amount of apo C—II is required for the activation of lipoprotein lipase (11). The increase in apo C—II induced in the diabetic group at 150 min may have provided enough apo C-II to enhance lipoprotein lipase activity causing triglyceride concentrations to return to the initial fasting level of 240 min. The mechanism by which the glucose load caused initial decreases of plasma apo C—II and apo C—III in the diabetic subjects is not known. The observed increase in the ratio of triglyceride to apo C-II levels from the fasting levels during the 300 min of observation in the diabetic subjects vs the normal control subjects suggests a possible reduction of apo C—II to activate lipoprotein lipase and thereby may account for the persistent hypertriglyceridemia in the diabetic patients. Absence of apo C-II in human plasma has been described to produce severe hypertriglyceridemia (21). The plasma of patients with an absence of apo C—II failed to activate lipoprotein lipase in vitro. Thus, the C apolipoproteins play a very important 61 role in triglyceride metabolism. The apparent insulin resistance observed in the NIDDM patients in conjunction with a defect in apo C—II synthesis, secretion and/or degradation may be contributory factors in the persistent hypertriglyceridemia in the NIDDM patients. Due to the difficulty of monitoring the daily biosynthesis of apolipoproteins in humans, results of this study suggest the possibility that measuring apo C following their acute induction by glucose may serve as a future test to detect abnormalities in lipoprotein metabolism in the diabetic patients. Acknowledgment The authors thank Deanna Nash for glucose determinations; Joan King for insulin assays; Dr. Alan Zinsmeister for statistical help; the nursing staff, especially Suzanne LeBlanc; the dietary staff, especially Barbara Moenke of the Clinical Research Unit, for their excellent help in the execution of the study; and Susan Woychik for expert secretarial assistance. This research was funded in part by National Institutes of Health Grants HL-O7329 and HL—27114. "" " ‘Ww «mfimm 62 References 1. 10. Schaefer EJ, Eisenberg S, Levy RL. Lipoprotein apoprotein metabolism. J Lipid Res 1978;19:667—687. Smith LC, Pownall HJ, Gotto AM. The plasma lipoproteins: structure and metabolism. Ann Rev Biochem 1978;47:751-777. Jackson RL, Morrisett JD, Gotto AM. Lipoprotein structure and metabolism. Physiol Rev 1976;56:259—316. Osborne JC, Brewer HB. The plasma lipoproteins. Adv Protein Chem 1977;31:253—337. Windmuller HG, Herbert PN, Levy RI. Biosynthesis of lymph and plasma lipoprotein apoprotein by isolated perfused rat liver and intestine. J Lipid Res 1973;14:215—223. Wu AL, Windmuller HG. Relative contributions by liver and intestine to individual plasma apolipoproteins in the rat. J Biol Chem 1979;254:7316—7322. Schonfeld G, Weidman SW, Witztum JL, Bowen RM. Alterations in levels and interrelations of plasma apolipoproteins induced by diet. Metabolism 1976;25:261—275. Falko JM, Schonfeld G, Witztum JL, Kolar JB, Salmon P. Effects of short—term high carbohydrate, fat—free diet on plasma levels of apo C-II and apo C—III and on the apo C subspecies in human plasma lipoproteins. Metabolism 1980;29:654—660. Eisenberg S, Levy RI. Lipoprotein metabolism. Adv Lipid Res 1975;13:1-89. LaRosa JC, Levy RI, Herbert PN, Lux SE, Frederickson DS. A specific apoprotein activator for LPL. Biochem Biophys Res Commun 1970;41:57—62. ll. 12. 13. 14. 63 j Kashyap ML, Srivastava LS, Chen CY, Perisutti G, Campbell M, Lutmer RF, Glueck CF. Radioimmunoassay of human apolipoprotein C—II. A study in normal and hypertriglyceridemic subjects. J Clin Invest 1977;60:171—180. Schonfeld G, George PK, Miller J, Reilly P, Witztum J. Apoli- poprotein C-II and C-III levels in hyperlipoproteinemia. Metabolism 1979;28:1001—1010. Bray GA. The obese patient, Vol IX. Major problems in internal medicine. WB Saunders Co, Philadelphia, 1976, p. 8. Patton JG, Dinh DM, Mao SJT. Phospholipid enhances triglyceride ‘ quantitation using an enzyme kit method. Clin Chem Acta 1982; 118:125—128. Department of Health, Education and Welfare. Lipid Research Clinical Program Manual of Laboratory Operation, 1974. Bethesda, MD: Department of Health, Education and Welfare (NIH 75—628). Chung BH, Wilkinson T, Geer JC, Segrest JP. Preparation and quantitative isolation of plasma lipoproteins: rapid, single discontinuous density gradient ultracentrifugation in a vertical rotor. J Lipid Res 1980;21:284—291. Mao SJT, Kottke BA. Tween—20 increases the immunoreactivity of apolipoprotein A-I in plasma. Biochim Biophys Acta 1980; 620:447—453. Barr SI, Kottke BA, Chang JY, Mao SJT. Immunochemistry of human plasma apolipoprotein C—II as studied by radioimmunoassay. Biochim Biophys Acta 1981;663:491—505. 19. 20. 21. 64 Barr SI, Mao SJT, Kottke BA. Postprandial distribution of apolipo— proteins A-I, A-II, C—II, C—III in normal and hypertriglyceridemic individuals. (Submitted) 1982. Herbert V, Lau KS, Chester W, Gottlieb CW, Bleicher SJ. Coated charcoal immunoassay of insulin. J Clin Endocrinol Metab 1965; 25:1375—1384. Miller NE, Rao SN, Alaupovic P, Noble N, Slack J, Brunzell JD, Lewis B. Familial apolipoprotein C—II deficiency; plasma lipoproteins and apolipoproteins in heterozygous and homozygous subjects and the effects of plasma infusion. Eur J Clin Invest 1981;11:69-76. . m “Mancunian CHAPTER 4 Plasma lipids and apolipoproteins in insulin dependent and non—insulin dependent diabetics 1 11le u‘ ABSTRACT Fasting plasma levels of cholesterol, triglycerides, high density lipoprotein (HDL) cholesterol, apolipoproteins (apo) A—I, A—II, C-II, and C—III were studied in 170 diabetic patients and 46 age-matched healthy control subjects. The diabetics were separated into two major groups: insulin dependent diabetes mellitus (IDDM, 78) and non-insulin dependent diabetes mellitus (NIDDM, 92). The plasma lipid profiles were similar in the IDDM and NIDDM groups with signi- ficantly elevated plasma triglycerides, decreased HDL cholesterol, and normal cholesterol levels compared to controls. Plasma apo A—I levels were low in both groups of diabetics but more so in the IDDM { subjects, and negatively correlated with plasma triglyceride levels. Apo A—II was also decreased in the diabetics. Apo C-II and apo C—III levels were strongly correlated with plasma triglyceride levels (apo C—II, r = 0.71, P< 0.0001, apo C—III, r = 0.72, P< 0.0001) in the com- bined group of diabetics. Forty-one percent of the diabetics had a clinical diagnosis of coronary artery disease (CAD) and/or peripheral arteriovascular disease (PAD). Univariate analysis showed a significant decrease of apo A—I levels in those with CAD/PAD compared to those without the disease, but only in the IDDM group. Diabetes is therefore associated with low levels of apo A—I which may predispose to macro— vascular disease. The HDL cholesterol levels were higher in the IDDM vs NIDDM indicating that the apo A—I of HDL varies, suggesting a possible different mechanism for macrovascular disease in IDDM vs NIDDM. The significant decrease in the ratio of plasma apo C—II to triglyceride levels in the diabetics suggests a relative reduction of apo C-II availability for activation of lipoprotein lipase which may contribute to the hypertriglyceridemia of the diabetics. 65 66 ? These data indicate that apolipoprotein levels are altered in diabetes mellitus quantitatively and perhaps qualitatively, and indirectly suggest that insulin deficiency, whether absolute or relative, accounts for these_changes. Insulin may modulate apolipoprotein metabolism either through control of blood glucose or through a mechanism separate from glucose regulatory activity. Introduction Lipoprotein metabolism is altered in diabetes with associated differences in apolipoprotein levels in diabetics vs control subjects. These differences may account for the increased frequency of arterios— l clerotic vascular disease in diabetes (1—4). The role of insulin, endogenous and exogenous, in lipoprotein metabolism appears to be separate from its effect on glucose levels and hyperglycemia. Insulin modulates the activity of lipoprotein lipase (LPL) and may influence lecithin cholesterol acyltransferase (LCAT) (5). Insulin deficiency, whether absolute or relative, results in hypertriglyceridemia (6, 7) and decreased high density lipoprotein (HDL) cholesterol levels (8—10). Availability of radioimmunoassay measurements for apolipoproteins has heightened interest in these protein moieties as possible cardio— vascular risk factors separate from circulating lipids. It has been suggested that apolipoproteins may be better predictive factors than lipids for macrovascular disease (11, 12). Low levels of apolipoprotein (apo) A—I have been shown to be a risk factor for the development of early coronary heart disease (13). Apo A—I and apo A-II levels were decreased in patients with abnormal carbohydrate tolerance (14). Among elderly diabetics, there were significantly decreased levels of apo A—I and apo A—II in well controlled cases. Apo C-II and apo C—III are 67 increased in hypertriglyceridemic subjects (15, 16). These observations provide suggestive evidence that macrovascular disease in the diabetic is related to abnormalities of apolipoprotein metabolism. The purpose of our study is to provide the apolipoprotein charac— teristics of defined diabetic cohorts, categorized as to type of diabetes (insulin dependent diabetes mellitus, IDDM; and non-insulin dependent diabetes mellitus, NIDDM), sex, and presence of arteriosclerotic vas- cular disease (coronary artery disease, CAD, and peripheral arteriovas- cular disease, PAD). The relationship of the levels of apo A-I, apo A—II, apo C-II, and apo C—III to each other, to total plasma lipids in diabe- tic and control subjects, and to macrovascular disease in diabetic subjects was evaluated. Materials and Methods Subjects Patients were recruited from consecutive patients seen at the Lipid and Diabetes Clinic at the Mayo Clinic. A total of 170 diabetics were classified into NIDDM (92) and IDDM (78) based on the criteria proposed by the National Diabetes Data Group (17). There were 95 men and 75 women. By definition, all IDDM patients require insulin therapy and were therefore on such therapy at the time of the study. The NIDDM patients were treated by diet (N = 41), oral hypoglycemic agents (N = 14), or insulin (N = 37). The control subjects (25 males, 21 females) were comparable by age and sex to the diabetic subjects. Plasma samples Subjects continued to consume their usual diets during the study. After the subjects had fasted overnight (minimum 12 h postabsorptive), blood samples were drawn into vacutainer tubes containing EDTA (0.1%). 68 Blood samples were centrifuged immediately to obtain the plasma for immediate analysis of plasma glucose, glycosylated hemoglobin, HDL cholesterol, cholesterol, and triglycerides. Aliquots of plasma were stored at —200C for later measurements of apolipoproteins A-I, A-II, C-II and C-III. Lipid and apolipoprotein determinations Plasma triglycerides were determined in duplicate using a modifi- cation of an enzymatic method (18). Plasma total cholesterol was also determined enzymatically using the cholesterol modification of the Dow enzyme kit method (Dow Chemical Company, Indianapolis, Indiana). Both methods were comparable to that of established methods used in the Lipid Research Clinic (r = 0.98, P< 0.001) (19). HDL cholesterol was determined by measurement of the lipid in the supernatant after precipitation of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) in 1 ml of plasma with 50 U1 of 2.5 MnCl and 50 pl of sodium heparin (4000 units/ml). Cholesterol 2 concentration of the supernatant was determined enzymatically using the cholesterol modification of the Dow enzyme kit method (Dow Chemical Company, Indianapolis, Indiana). Plasma apolipoproteins A-I, A—II, C—II, and C-III were determined by radioimmunoassay previously described (20-22). Plasma samples for radioimmunoassay were frozen and all samples were determined in the same assay run. Tween-20 (100 p1), at concentrations 1.5, 0.2, and 0.1% were added for apo A—I, apo C—II, and apo C-III assays, respectively. Intraassay coefficients of variation were 4% for apo A-I, apo A-II, apo C-II, and apo C—III. 69 Other methods Plasma glucose was measured by a modification of the method of Trinder with a normal fasting range of 70 to 100 mg/dl (23). Glyco— sylated hemoglobin was measured by the Isolab Fast Hemoglobin Test System (Isolab Inc., Akron, Ohio). Statistical methods Pearson correlation coefficients were used to evaluate the degree of linear association between two variables. Coefficients of skewness and kurtosis were evaluated to test deviations from normal distribution. Transformations of measurements of plasma triglycerides and apo A—II to logarithms were used to achieve a Gaussian distribution before statistical analyses. Non—paired E tests were performed to compare significant differences of the variables measured between the diabetics and the normal controls. Results Characteristics of normals and diabetics The mean age of the diabetic groups, IDDM and NIDDM men and women was similar (Table 1). Mean age for men and women and for diabetic and control subjects were comparable, ranging between 60 to 64 yr. There was very little difference between the mean relative weight of the IDDM group and the normal control subjects. However, the NIDDM subjects tended to be overweight with diabetic women exhibiting the highest mean relative weight. Comparison of diabetic characteristics in normal controls and diabetics As expected, fasting plasma glucose concentrations of the IDDM and NIDDM subjects were significantly elevated (P <0.001) in comparison with the control subjects (Table 2). Similarly, fasting plasma levels Characteristics of Normals and Diabetics* 70 Table l Diabetics Normal Controls IDDM NIDDM Males No. of Subjects 25 45 50 Age (yr) 62.2 t 5.0 61.5 t 9. 63.0 f 5. Height (cm) 173.9 f 4.9 173.5 f 5. 173.5 f 6. Weight (kg) 82.6 i 8.2 81.8 t 12. 87.3 i 14. Relative Weight 1 26 t 0 1 1.26 t 0. 1.33 i 0. Females No. of Subjects 21 33 42 Age (yr) 1 63.5 r 6.0 60.0 i 10. 63.7 t 6. height (cm) 160.0 f 6.4 161.6 t 5. 159.4 r 5. Weight (kg) 64.1 t 5.0 67.5 t 13. 75.7 i 15. Relative Weight 1.24 i 0.1 1.28 t 0. 1.46 i 0. *Mean I SD. Relative Weight = Actual Weight Ideal Weight. 71 Table 2 Comparison of Diabetic Characteristics in Normal Controls and Diabeticsk Diabetics Normal Controls IDDM NIDDM Males No. of Subjects 25 45 50 Plasma Glucose 96.7 i 11.5 193 o t 86.4** 180.4 f 59.3** (mg/dl) Glycosylated + + ** + ** Hemoglobin (%) 7.6 _ 0.5 12 l _ 2 4 10.9 _ 2.2 Females No. of Subjects 21 33 42 Plasma Glucose 91.5 i 7.8 182 4 i 84.1** 190.8 E 69.2** (mg/dl) Glycosylated + ** + ** Hemoglobin (%) 7.6 _ 0.9 11 3 t 1.8 11.5 _ 2.6 *Mean I SD. **P< 0.001 significantly different from normal controls. 72 of glycosylated hemoglobin were also significantly elevated (P< 0.001) in both groups of diabetics vs control subjects. A significant corre- lation was found between the fasting plasma glucose levels and glyco— sylated hemoglobin (r = 0.68, P< 0.0001) in the two groups combined. Plasma lipids and HDL cholesterol in normals and diabetics In the diabetics, highly significant elevations (P< 0.005) of fasting plasma total triglycerides were observed in the IDDM men and NIDDM men and women vs control subjects (Table 3). Although not statistically significant, the plasma triglyceride levels of IDDM women were also elevated in comparison to normal controls. Total plasma cholesterol levels were comparable in all the groups (diabetics vs controls). HDL cholesterol was lower in both groups of diabetics compared to controls; in NIDDM the decrease was statistically signi— ficant (P <0.005). HDL cholesterol was negatively correlated with plasma triglycerides in both diabetic groups (r = -.32). Plasma apolipoproteins in normals and diabetics Apo A—I levels were lower in both groups of diabetics compared to controls, but significant lower values were found only in the IDDM groups (P< 0.005) (Table 4). No significant differences were observed in apo A-II levels in the 3 groups studied. Although plasma apo C-II and apo C-III concentrations were slightly elevated in both groups of diabetics, only in the IDDM women was apo C—III significantly increased in comparison with the age—matched controls. Total plasma triglycerides strongly correlated with apo C—II in the NIDDM (r = 0.69, P< 0.0001) and IDDM subjects (r = 0.73, P< 0.0001) (Figures 1 and 2). The same trend was found with plasma triglycerides and apo C-III concentrations in the NIDDM (r = 0.73, 73 Table 3 Plasma Lipids and HDL-Cholesterol in Normals and Diabetics* (mg/d1) Diabetics Normal Controls IDDM NIDDM Males No. of Subjects 25 45 50 Total Triglyceride 93.7 137.2** 146.4** (Geometric Mean) Total Cholesterol 213.1 f 34.7 209.0 f 38.8 208.6 1 51.7 HDL-Cholesterol 52.1 i 13.4 43.9 i 14.3 42.3 t l4.4** Females No. of Subjects 21 33 42 TOtal Triglycerlde 103.7 140.4 l74.4** (Geometric Mean) Total Cholesterol 234.0 f 40.0 232.2 I 51.5 235.0 I 51.1 HDL—Cholesterol 56.6 t 11.8 50.8 + 17.8 44.0 t 12.7** *Mean I SD. **P< 0.005 significantly different from normal controls. 74 Table 4 * Plasma Apolipoproteins in Normals and Diabetics (mg/d1) Diabetics Normal Controls IDDM NIDDM Males No. of Subjects 25 45 50 ApoA—I 179.2 I 33.5 151.8 I 28.8** 170.8 I 36.7 ApoA—II (Geometric Mean) 46'2 41'0 45'1 ApoC—II 9.5 t 2.5 10.9 t 4.0 10.5 i 3.5 ApoC—III 14.1 i 3.0 16.2 i 4.6 16.5 i 4.3 Females No. of Subjects 21 33 42 ApoA—I 190.0 f 30.1 165.2 f 28.7** 177.4 f 29.9 ApoA—II (Geometric Mean) 53'3 50'3 49'4 ApoC-II 9.8 i 1.8 11.4 t 4.0 11.1 f 4.2 ApoC—III 16.1 t 1.5 18.8 f 3.1** 18.3 t 3.6 *Mean I SD. **P< 0.005 significantly different from normal controls. 75 20 . g 15 — \ c» _ E :F 10 - o I— g NIDDM subjects <1: 5 — 0 °. r = 0.69 _ . ' . p <0.0001 n = 92 0 1 l I I I 4 5 6 Total plasma triglycerides (log) Figure 1 Relationship between total plasma triglycerides and apolipoprotein C—II (apo C—II) determined by radioimmunoassay in the non—insulin dependent diabetes mellitus (NIDDM) subjects. l 25 20 15 1O Apo C-ll (mg/dl) 76 l— o " . IDDM subjects "' .. . o r = 0.73 __ ’ . . ' ‘ p <0.0001 o . n = 78 ' ' J I l 1 1 4 5 6 Total plasma triglycerides (log) Figure 2 Relationship between total plasma triglycerides and apolipoprotein C-II (apo C-II) determined by radioimmunoassay in the insulin dependent diabetes mellitus (IDDM) subjects. 77 P< 0.0001) and IDDM subjects (r = 0.70, P< 0.0001) (Figures 3 and 4). On the other hand, only in the IDDM group was apo A-I negatively correlated with plasma triglycerides (r = -0.28, P< 0.01). A significant decrease in the ratio of apo C-II to plasma trigly— ceride levels were observed in both diabetic groups in comparison with normal controls (P< 0.005). Effect of treatment on the NIDDM subjects When the NIDDM group was separated into those that were being treated with diet alone (48% men, 40% women), with insulin (34% men, 48% women), and oral hypoglycemic agents (18% men, 12% women), no £' significant differences were observed in the variables measured among the different groups. However, the diabetics on oral hypoglycemic agents (men and women) had lower HDL cholesterol and higher plasma triglycerides in comparison with those diabetics that were on insulin and on diet alone. Summary of plasma lipids and apolipoprotein profiles of the diabetics in comparison with normal controls Using 0 as the mean level of each variable measured in the normal controls, both groups of diabetics, men and women, exhibited similar profiles (Figures 5 and g). The profile in the diabetics showed sig- nificant elevations of plasma triglycerides with the NIDDM group showing highest elevations. No significant difference in total plasma cholesterol concentrations was observed between the diabetics and normal controls. HDL cholesterol was significantly decreased in the NIDDM group. Apo A-I and apo A—II levels were low in the diabetics with the IDDM subjects showing significant decreases in apo A-I. Apo C-III was significantly elevated in the IDDM women. 30 c 25 __ O O D O \ '_ 0 oz E 20 — C) 15 — 8 _ NlDDM Subjects < 10 —- r = 0.73 . . g. p <0.0001 _- o n = 92 5 i I l l 4 5 6 Total plasma triglycerides (log) Figure 3 Relationship between total plasma triglycerides and apolipoprotein C—III (apo C—III) determined by radioimmunoassay in the non-insulin dependent diabetes mellitus (NIDDM) subjects. 79 € \ O) E (3 10 L :. . IDDM subjects 8 . r = 0.70 < _ . p <0.0001 5 —. n = 78 0 I l J l l 4 5 6 Total plasma triglycerides (log) Figure 4 Relationship between total plasma triglycerides and apolipoprotein C-III (apo C-III) determined by radioimmunoassay in the insulin dependent diabetes mellitus (IDDM) subjects. 80 3 ,_ D insulin dopondont (n=45) - Non-Insulin dependent (n=50) Normals (n=25) Standardized Differences -4 l- "‘ Trigly- Total HDL Apo A-I Apo C-ll cerides CHOL CHOL Apo A-II Apo C-lll Figure 5 Fasting plasma lipid and apolipoprotein profiles of insulin dependent diabetes mellitus (IDDM) and non—insulin dependent diabetes mellitus (NIDDM) men in comparison with normal subjects. Zero is the mean value of all the variables measured in the normal subjects. Standardized difference is the difference between the two means (diabetics and normals)/pooled SD. 81 3 ._ D Insulin dependent (N=33) . Non-Insulin dependent (n=42 Standardized Differences O Normals (n=21) -1 -2 -3 .4 i— ‘ Trigly- Total HDL Apo A-I Apo C-II cerides CHOL CHOL Apo A-II Apo C-lll Figure 6 Fasting plasma lipids and apolipoprotein profiles of insulin dependent diabetes mellitus (IDDM) and non-insulin dependent dependent diabetes mellitus (NIDDM) women in comparison with normal subjects. 82 Correlations between plasma glucose, glycosylated hemoglobin, lipids, and apolipoprotein variables in normals and diabetics Pearson correlation coefficients are given in Table 5. As expected, glycosylated hemoglobin was strongly correlated with fasting plasma glucose in both groups of diabetics, but not in the normal subjects. In all the groups (diabetic and control groups), apo A-1 and apo A—II were strongly correlated with each other; negative correlations were observed between total plasma triglycerides and HDL cholesterol; apo C-11 and apo C—III were strongly correlated with plasma triglyceride levels; apo C—II was strongly correlated with total cholesterol. Apo C-11 and apo C—III were strongly correlated with each other only in the diabetic groups. As evident from Table 5, weaker correlations were seen between the other variables of the study. Relationship of CAD/PAD to apo A—I levels The relationship between the presence of CAD/PAD and apo A-I levels was evaluated by univariate analysis. Forty—one percent of the diabetics had a confirmed diagnosis of CAD and/or PAD. Apo A—I was decreased significantly in those that have CAD/PAD. However, this was evident only in the IDDM subjects. We did not find any significant difference in the apo A—I levels of those without and those with the disease in the NIDDM group of patients, although their apo A—I levels were still low in comparison with normal subjects. Discussion These data show an influence of diabetes on apolipoprotein metabo— lism. The differences noted in apolipoprotein levels in the diabetics may account for hypertriglyceridemia and decreased HDL cholesterol levels through possible effects on LPL and LCAT either by hyperglycemia, 83 HHHIUOL< MN.O HHIUOL< «aaanq.o ~o.o HHI izfiwuhu iOLo 10:0 1050; adobe An: HNuOh .00xao mmHnEwL vcm mama: ufiuanofin new HmELoz cw monLcfipc> amoucuacnchg< no: ._616nu6~c:o-ae: .mnsasa .c«ao~mo&u: vouwnzmouz—U .omaoagu ozmamm uncr< ussamuwgacou Eamon—:nu:u Compton m uaanh HHHIUOQ< ««aHU «0n.0 n~.0 HH.0| «H.0i m~.0 no.0I m00.0i ««am0.0 01m 3: u E scab -~100a< Hmluon< HHI<00< ~i- Plasma HDL cholesterol displayed several peaks, with the subjects who lost weight showing lower plasma concentrations in study 2 (Figure A-9). Slightly higher HDL cholesterol concentrations were shown by the subjects who maintained weight in study 2 (Figure A—lO). Plasma apo A—I concentrations fluctuated in both groups, showing lower concentrations in study 2 (Figures A-ll, A:12). Plasma apo A-II concentrations fluctuated throughout the 300 min of observation in both groups (Figures_A:13, A:14). Plasma apo C—lI concentrations of the 2 subjects who lost weight increased slightly in study 2 (Fégure A-15). This was not evident in the 2 subjects who maintained weight (Figure A-l6). Plasma apo C-III concentrations were lower in study 2 for the 2 groups (Figures A-l7, A_-1§>. Obesity is associated with reduced levels of HDL cholesterol (1—3). In some studies, there was a significant correlation between HDL cholesterol and several indices of overweight (4). However, other piano—'0 omooc~m 158v 188 100 1 J l J. J I MIN Figure A-3 J. l 4 V 8 38 68 98 128 158 188 218 248 278 388 Postprandial profiles of plasma glucose (mg/d1) after an oral glucose load (OGTT) in 2 subjects who maintained weight. There was a 3—month interval between the first (study 1) and the second OGTT (study 2). of the 2 subjects. Values are means macaw—=0 3 »~—-c m 3 w 15%» 18}- 101 Figure A-4 Postprandial profiles of plasma insulin ( uUnits/ml) after an oral glucose load (OGTT) in 2 subjects who maintained weight. There was a 3-month interval between the first (study 1) and the second OGTT (study 2). Values are means of the 2 subjects. carom—d) OH 2881- 102 JA .4. ~. A ........ ,3 ................... a .‘A 9 study 1 o ."A studg 2 "X A ............ g 1 4 ~41 .L t J. i 4 t 4 1 8 38 68 98 128 158 188 218 248 278 388 MIN Figure A—5 Postprandial profiles of plasma triglycerides (TG) (mg/d1) after an oral glucose load in 2 subjects before (study 1) and after (study 2) weight loss. OGTT was given at 3 months interval. Values are means of the 2 subjects. DBmflH'U O—I 103 3K}- Efii~ 35}- ......... studg 1 .- “A A ........ A“. 0 ”A" .... A“. fi ................... .A _.' ' "fl ................... StUdg 2 A' ............ 158 4 4. 4 fit 4. 4 i i : : a -38 8 ' 38 68 98 128 158 188 218 248 278 388 MIN Figure A—6 Postprandial profiles of plasma triglycerides (TG) (mg/d1) after an oral glucose load (OGTT) in 2 subjects who maintained weight. There was a 3-month interval between the first (study 1) and the second OGTT (study 2). Values are means of the 2 subjects. ”Swab-‘0 ~01nr+mo~oro 1a}- 175.. 178" 168 -38 104 J J u 1 8 38 68 98 128 158 188 218 248 278 388 MIN Figure A-7 Postprandial profiles of plasma total cholesterol (mg/d1) after an oral glucose load (OGTT) in 2 subjects before (study 1) and after (study 2) weight loss. OGTT was given at 3 months interval. Values are means of the 2 subjects. “39900—13 o—Oflflflwflp—oojn 218" 198" 1901- 178» 168" 158 105 8 3060 98128 158188 21824827818 mm Al- Figure A-8 Postprandial profiles of plasma total cholesterol (mg/d1) after an oral glucose load (OGTT) in 2 subjects who maintained weight. There was a 3-month interval between the first (study 1) and the second OGTT (study 2). Values are means of the 2 subjects. ~01or+wn~oro FUI 106 24" -38 8 38 68 98 128 158 188 218 248 278 388 MIN Figure A-9 Postprandial profiles of plasma HDL cholesterol (mg/d1) after an oral glucose load in 2 subjects before (study 1) and after (study 2) weight loss. OGTT was given at 3 months interval. Values are means of the 2 subjects. F’UI ~010dmo~orn 240 107 study 1 O study 2 8 38 68 98 128 158 188 218 248 278 388 MIN Figure A—lO Postprandial profiles of plasma HDL cholesterol (mg/d1) after an oral glucose load in 2 subjects who maintained weight. There was a 3—month interval between the first (study 1) and the second OGTT (study 2). Values are means of the 2 subjects. 0‘!) 1681 158“ 143» 108 128 -38 studg 1 O x studg 2 'A A ............ 8 38 68 98 128 158 188 218 248 278 388 MIN Figure A—ll Postprandial profiles of apolipoprotein A—I (apo A—I) (mg/d1) after an oral glucose load (OGTT) in 2 subjects before (study 1) and after (study 2) weight loss. OGTT was given at 3 months interval. Values are means of the 2 subjects. Apolipoprotein concentrations were determined by radioimmunoassay and each point represents the mean of triplicate analysis. 1mib 13}- 128 109 1 \\ x,“ ‘5 '9 studg 1 O study 2 128 158 188 218 248 278 388 MIN 8 8 §}l Figure A—12 Postprandial profiles of apolipoprotein A-I (apo A-I) (mg/d1) after an oral glucose load (OGTT) in 2 subjects who maintained weight. There was a 3 month interval between the first (study 1) and the second OGTT (study 2). Values are means of the 2 subjects. OUD ND 24.- 16.. 14-- 12 -38 110 Figure A-13 Postprandial profiles of apolipoprotein A—II (apo A-II) (mg/d1) after an oral glucose load (OGTT) in 2 subjects before (study 1) and after (study 2) weight loss. OGTT was given at 3 months interval. Values are means of the 2 subjects. 01) ND 24-- 18 38 68 98 128 111 158 188 218 248 278 388 Figure A-l4 Postprandial profiles of apolipoprotein A-II (apo A—II) (mg/d1) after an oral glucose load in 2 subjects who maintained weight. There was a 3 month interval between the first (study 1) and the second OGTT (study 2). Values are means of the 2 subjects. 0"!) NO 112 7.5“ 7+ 6.5" 6 4. 4 : i i . 4 4 i i 4. ~38 8 38 68 98 128 158 188 218 248 278 388 MIN 1 1 Figure A-15 Postprandial profiles of apolipoprotein C—II (apo C—II) (mg/d1) after an oral glucose load (OGTT) in 2 subjects before (study 1) and after (study 2) weight loss. OGTT was given at 3 months interval. Values are means of the 2 subjects. 113 9.. 8.5“ R p 6.. O C 7.5 2 7.. Studg 1 O 6.5" study 2 6 -38 Figure A—16 Postprandial profiles of apolipoprotein C-II (apo C—II) (mg/d1) after an oral glucose load (OGTT) in 2 subjects who maintained weight. There was a 3 month interval between the first (study 1) and the second OGTT (study 2). Values are means of the 2 subjects. 0‘0 (1)0 114 ,- 8 38 68 98 128 158 188 218 248 278 388 Figure A—l7 Postprandial profiles of apolipoprotein C—III (apo C-III) (mg/d1) in 2 subjects before (study 1) and after (study 2) weight loss. OGTT was given at 3 months interval. Values are means of the 2 subjects. 013D (.00 18v 164r 15‘ Y 141' 13.. 121F- 115 ’I a .U .- oooo o .- ;I .. 68 98 128 158 188 218 248 278 388 MIN (9 &§.L Figure A—18 Postprandial profiles of apolipoprotein C-III (apo C-III) (mg/d1) after an oral glucose load (OGTT) in 2 subjects who maintained weight. There was a 3 month interval between the first (study 1) and the second OGTT (study 2). Values are means of the 2 subjects. 116 studies did not reveal any close relationship between HDL cholesterol and relative body weight (1, 5). Very few studies have evaluated the influence of weight loss on the levels of HDL cholesterol and other lipoproteins. In one study in which obese subjects and controls were compared, obese subjects were found to have lower values of HDL cholesterol and apo A—I (6). Effects of weight loss on HDL cholesterol are conflicting. It has been reported that weight loss produce an increase in HDL choles— terol (3, 6), no change (7, 8) or a decrease (9). A recent study demonstrated that lipid patterns of men and women differ in response to weight loss. Men showed an increase in HDL cholesterol, while women showed a decrease (10). The decreases in mean fasting plasma total cholesterol and tri— glycerides in the 2 subjects who lost weight are consistent with the studies showing decreases in levels of cholesterol and triglycerides after weight reduction (11, 12). It was estimated by Kannel et a1 (11) that each percentage increase or decrease in body weight is accompanied by a corresponding 1.1 mg/dl change in the cholesterol level. The mean weight loss of the 2 subjects was 5.2%, with an 11.0 mg/dl decrease in mean fasting plasma total cholesterol. It is difficult to evaluate the present results statistically due to the limited number of patients. Improvements in mean plasma triglyceride concentrations as well as the decreases in absolute concentrations of apo C—III, after the second oral glucose load were the favorable changes observed in the 2 subjects who lost weight. However, this was also evident in the 2 subjects who maintained weight. Why there were slight decreases in HDL cholesterol, and 117 apo A-1; and increases in postprandial plasma total cholesterol are not clear. More patient studies should be performed until statistically quantifiable data can be presented. The mechanisms by which caloric restrictions influence plasma lipids and apolipoprotein levels are not well understood. It is believed that low carbohydrate intake diminished the production of endogenous plasma triglycerides and VLDL. However, this has not been proven directly. Plasma triglyceride turnover had been studied during prolonged starvation (13). An appreciable decrease in trigly— ceride production rate was found, with evidence of concomitant impair- ment of the triglyceride removal efficiency. Lipoprotein lipase activity has been found to be reduced in human adipose tissue (14, 15) and postheparin plasma (16) during low caloric intake. All the above studies were observed in obese patients without the presence of disease. With the presence of diabetes, other hormonal factors may influence apolipoprotein synthesis and secretion which may alter apolipoprotein levels. 118 References 1. Gordon T, Castelli WP, Hjortland MC, Kannel WB, Damber TR. High density lipoprotein and a protective factor against coronary heart disease. Am J Med 1977;62:707-714. Miller CJ, Miller NE. Plasma high density lipoprotein concentration and development of ischaemic heart disease. Lancet 1975;I:16—l9. Wilson DE, Lees RS. Metabolic relationship among the plasma lipo— proteins. Reciprocal changes in the very-low and low density lipOproteins in man. J Clin Invest 1972;51:1051-1057. Rabkin SW, Bayko E, Streja DA. Relationship of weight loss and cigarette smoking to changes in high density lipoprotein cholesterol. Am J Clin Nutr 1981;34:1764-1768. Miller NE, Forde OH, Thelle DS, Mjos OD. The Tromso heart study. High density lipOprotein and coronary heart disease: a prospective case-control study. Lancet l977;I:965-968. Avogaro P, Bittolo Ben C, Cazzolato G, Quinci GB. Lipoproteins and apolipoproteins A—1 and B in obese subjects in basal state and following a hypocaloric diet. Diet and drugs in atherosclerosis. Ed. Noseda G, Lewis B, Paoletti R. 1980 pp. 67-72. Nestel PJ, Miller NE. Mobilization of adipose tissue cholesterol in high density lipoprotein during weight reduction in man. .13 High density lipoproteins and atherosclerosis. Eds. Gotto AM, Miller NE, Oliver MF. New York, Elsevier North Holland Inc. 1978; pp. 78-95. Hulley SB, Cohen R, Widdowson G. Plasma high density lipoprotein cholesterol level: influence of risk factor intervention. J Am Med Assoc 1977;238:2269-2271. 10. 11. 12. 13. 14. 15. 16. 119 Thompson PD, Jeffery RW, Wing RR, Wood PD. Unexpected decrease in plasma high density lipoprotein cholesterol with weight loss. Am J Clin Nutr 1979;32:2016-2021. Brownell KD, Stunkard AJ. Differential changes in plasma high density lipoprotein cholesterol levels in obese men and women during weight reduction. Arch Intern Med 1981;141:1142-1146. Kannel WB, Gordon T, Castelli WP. Obesity, lipids, and glucose intolerance: The Framingham study. Am J Clin Nutr 1979;32:1238- 1245. Olefsky J, Reaven GM, Farquhar JW. Effects of weight reduction on obesity: studies of lipid and carbohydrate metabolism in nor- mal and hyperlipoproteinemic subjects. J Clin Invest 1974;53: 64—76. Streja DA, Marliss EB, Steiner G. Effects of prolonged fasting on plasma triglyceride kinetics in man. Metabolism 1977;26:505. Persson B, Hood B, Angervall G. Effects of prolonged fast on lipOprotein lipase eluted from human adipose tissue. Acta Med Scand 1970;188:225. Guy-Grand B, Bigorie B. Effect of fat cell size, restrictive diet and diabetes on lipoprotein lipase release by human adipose tissue. Horm Metab Res 1975;7:47l. Huttunen JK, Ehnholm C, Nikkila EA, Ohta M. Effect of fasting on two postheparin plasma triglyceride lipases and triglyceride removal in obese subjects. Europ J Clin Invest 1975;5z435. B. Comparison of oral glucose load and intravenous glucose infusion in a non-insulin dependent diabetic COMPARISON OF ORAL GLUCOSE VS INTRAVENOUS GLUCOSE INFUSION IN A NON-INSULIN DEPENDENT DIABETIC A 40 year old male patient with NIDDM who participated in the first oral glucose load was tested after 3 months, using an intra— venous glucose infusion in order to determine if the response would be different from the oral load and if differences exist between the intravenous response of the diabetic and the normal subject. Procedures used in the intravenous glucose infusion were similar to those described previously (Chapter 2). This subject had the same response as the normal subject with regards to the following variables (Tables B—l to B—3): 1) 2) 3) 4) 2) 3) A rise in plasma triglycerides occurred after the glucose load but not during the glucose infusion. The insulin response to oral glucose was higher than that seen when glucose was administered orally. No significant changes were observed in plasma cholesterol. HDL cholesterol absolute values were lower during the intravenous glucose infusion. The differences observed between the normal and the diabetic subject: Although absolute levels of apo C—II were similar in the normal and diabetic subject, the diabetic displayed more fluctuations during the 300 min of observation. The diabetic subject had higher levels of apo C—III during oral glucose in contrast with the normal subject, who had higher levels during the intravenous infusion. There were greater increases in apo A—I levels in the normal subject during the oral glucose load. 120 121 These results are difficult to interpret because only 2 subjects had been studied. Variable responses were shown both by the normal and diabetic subjects following an oral glucose load (Chapters 2 and 3). A larger number of subjects would have added statistically quanti— fiable data. However, the two intravenous infusion studies were performed to give preliminary answers not provided by the oral glucose load. 122 Table B—1 Plasma Glucose, Insulin and Triglycerides in a NIDDM Subject After an Oral Glucose Load (OGTT) and Intravenous Glucose Infusion (IVGTT) Glucose Insulin Triglycerides (mg/d1) (pUnits/ml) (mg/d1) Time OGTT IVGTT OGTT IVGTT OGTT IVGTT 0 107 115 9.5 12.5 105 113 30 194 214 19.0 15.0 118 106 60 234 255 32.0 33.6 129 110 90 217 248 44.0 38.4 128 106 120 189 203 40.0 32.8 117 100 150 167 161 27.2 37.6 112 100 180 142 133 24.8 28.8 114 98 240 98 95 23.2 24.0 108 92 300 73 74 22.4 20.0 105 100 123 Table B-2 Plasma Cholesterol and HDL cholesterol in a NIDDM Subject After an Oral Glucose Load (OGTT) and Intravenous Glucose Infusion (IVGTT) Total Cholesterol HDL Cholesterol (mg/d1) (mg/d1) Time OGTT IVGTT OGTT IVGTT 0 196 193 24.5 22.4 30 198 191 23.7 16.1 60 193 188 22.0 16.1 90 193 188 23.7 16.1 120 187 189 23.7 16.1 150 192 195 23.7 17.0 180 ‘ 188 192 22.8 16.1 240 197 193 23.7 16.1 300 199 202 23.7 17.0 124 0.00 0.00 0.0 0.0 0.00 0.00 0.000 0.000 000 0.00 0.00 0.0 0.0 0.00 0.00 0.000 0.000 000 0.00 0.00 0.0 0.0 0.00 0.00 0.000 0.000 000 0.0 0.00 0.0 0.0 0.00 0.00 0.000 0.000 000 0.0 0.00 0.0 0.0 0.00 0.00 0.000 0.000 000 0.00 0.00 0.0 0.0 0.00 0.00 0.000 0.000 00 0.0 0.00 0.0 0.0 0.00 0.00 0.000 0.000 00 0.0 0.00 0.0 0.0 0.00 0.00 0.000 0.000 00 0.0 0.00 0.0 0.0 0.00 0.00 0.000 0.000 0 000>0 0000 000>0 0000 00000 0000 000>0 0000 wEH H. 0000080 000\000 0000080 0000080 000:0 600 00-0 600 00-0 600 0-0 600 A090>Hv cowmswcH 0000500 msocm>muuaH 0cm AHHUOV 0600 omOUSHU ammo 66 06600 0660000 20002 6 00 000-0 066 .00.0 .0010 .0i0 60606006000600 606600 0-0 60060 C. Plasma Lipids and Apolipoprotein Levels of Impaired Glucose Tolerant Men in Comparison with Normal Men PLASMA LIPIDS AND APOLIPOPROTEIN LEVELS OF IMPAIRED GLUCOSE TOLERANT MEN (IMPGTT) IN COMPARISON WITH NORMAL MEN Twenty men with IMPGTT were also included in the survey study (Chapter 4). Similar measurements and methods were used in the evaluation of the variables. Age was comparable with the normal groups. The IMPGTT group was overweight in comparison with the normal subjects. Fasting plasma glucose and triglycerides were sig— nificantly elevated; HDL cholesterol was significantly decreased; apo A—I and apo A—II were decreased; apo C—II was elevated and apo C-III was significantly increased. These results were similar to the findings found in the NIDDM subjects, thus supporting the hypothesis that abnormal carbohydrate metabolism alters apolipoprotein levels (Table C-l). Apo A—I and apo A-II were strongly correlated with each other (Table C—2). HDL cholesterol negatively correlated with plasma triglycerides. A negative correlation was observed between HDL cholesterol and glucose which was significant at the 0.001 level. Weaker correlations were shown between the other variables in the study. 125 126 Table C-l Plasma Glucose, Lipids and Apolipoproteins in Normals and Impaired Glucose Tolerant Men (IMPGTT) IMPGTT NORMAL (n=20) (n=25) Age (yr) 62.7 T 6.1 62.2 T 5.0 , + + Height (cm) 174.9 - 6.9 173.9 - 4.9 Weight (kg) 88.5 T 14.4 82.6 T 8.2 Plasma Glucose (mg/d1) 110.5 T 13.0* 96.7 T 11.5 Glycosylated Hb (x) 7.8 T 0.8 7.6 T 0.5 HDL cholesterol (mg/d1) 43.9 T ll.9** 52.1 T 13.4 Total cholesterol (mg/d1) 225.5 T 43.6 213.1 T 34.7 Total triglycerides 157.8 T 80.7*** 98.7 T 34.3 (mg/dl) + + Apo A-I (mg/d1) 162.5 — 33.3 179.1 - 33.5 Apo A—II (mg/d1) 46.7 T 8.4 49.5 T 18.6 + + Apo C-II (mg/d1) 11.0 - 3.1 9.5 - 2.5 Apo C-III (mg/d1) 17.0 T 2.4* 14.1 T 3.0 + Values are means — S.D. * P = <0.001 ** P = <0.02 significantly ***P = <0.005 different from normal 127 Table C—2 Correlation Coefficients Among the Variables Measured in the Impaired Glucose Tolerant Men (IMPGTT) N r P Fasting plasma glucose Vs Triglycerides 20 0.29 ns Glyco. Hb 20 0.30 ns Body weight index Vs Triglycerides 20 0.42 ns Apo A—I Vs HDL cholesterol 20 0.28 ns Apo A—II 20 0.60 0.005 Triglycerides 20 -0.21 ns HDL Cholesterol Vs Triglycerides 20 -O.50 0.025 Glyco. Hb 20 —O.25 ns Cholesterol 20 —0.09 ns Glucose 20 —0.68 0.001 SUMMARY AND CONCLUSIONS SUMMARY AND CONCLUSIONS These studies have investigated the fasting and the postprandial dynamics of apolipoprotein metabolism in normal and in diabetic state. The initial study, "Association between apo A-I and apo A-II as evidenced by immunochemical approach" is a methodology paper. Both goat and rabbit anti-apolipoprotein A—I antibodies were able to ”co-precipitate" 125I—labeled apolipoprotein A—II, the other major apolipoprotein moeity of HDL, in the presence of apo A—I. The anti- apo ArI antibodies were specific to apo A-I as judged by radioimmuno- assay and an immunodiffusion technique. The physiological significance of the association of apo A-I and apo A—II can be seen in studies that show the parallel degradation rates of apo A-I and apo A—II in normo— lipemic subjects. These studies show the strong association between apo A—I and apo A—II. The finding that apo C-II and apo C-III did not directly associate with apo A-I suggests that apo A-I in HDL may not play a direct role for the net exchange of apo C between HDL and triglyceride rich lipoproteins. The second study discusses the increase and the fluctuations of apo A-I levels in 3 normal subjects following a glucose load. An acute effect of glucose ingestion was associated with increments of apo A-I and to a lesser extent, apo A—II, with no significant changes seen in HDL concentrations. Intravenous infusion of glucose in one of the subjects, thereby bypassing the gastrointestinal tract, 128 129 also was associated with an increase in apo A-I level. These results suggest that the liver may be responsible in part for the increase of apo A-I following an oral glucose load. *It can only be speculated that the increase of plasma apo A-I levels may facilitate the secretion of triacylglycerol into the circulation. The third chapter describes the changes observed in normal and NIDDM subjects following a glucose load. There was no significant difference in the response of apo A—I and apo A—II between the 2 groups. However, there was an initial decrease of apo C—II and apo C—III in response to the glucose load in the diabetic subjects. Since apo C—II is an activator for LPL, an enzyme responsible for trigly- ceride clearance, these findings suggest that the initial decrease of apo C-II in the NIDDM subjects may be a contributory factor in the hypertriglyceridemia observed in these patients. Whether hyper- insulinemia and associated apparent cellular insulin insensitivity contribute to these findings is not known. Following the initial study, the diabetic subjects were instructed on a calorie restricted diet and asked to return for a repeat study after 3 months. The difficulty in motivating these patients to lose weight has been demonstrated in this study. The data presented in additional results is difficult to evaluate statistically due to a limited number of patients. Improvements in mean plasma triglyceride concentrations as well as the decreases in absolute concentrations of apo C-III after the second oral glucose load were favorable changes observed in the 2 subjects who lost weight. However, this was also evident in the 2 subjects who maintained weight. More patient studies should be performed until statistically quantifiable data can be 130 presented. The additional results also include the comparison of oral glucose vs intravenous glucose infusion in a diabetic subject, the plasma lipids and apolipoprotein levels of IMPGTT subjects in comparison with normal subjects. Chapter 4 reports the fasting plasma lipids and apolipoprotein levels of NIDDM and IDDM subjects in comparison with their age matched controls. It is of interest that the NIDDM and IDDM subjects had similar plasma lipid profiles with significantly elevated plasma tri— glycerides, decreased HDL cholesterol and normal cholesterol levels. Plasma apo A—I levels were significantly decreased in the IDDM subjects. Plasma apo C-II and apo C—III were elevated in the diabetics and showed a strong correlation with triglyceride levels. The observed significant decrease in the ratio of apo C-II to triglyceride levels suggests a relative reduction of apo C—II availability for activation of LPL which may contribute to the hypertriglyceridemia in the diabetics. With 41% of the diabetics having confirmed diagnosis of CAD and/or PAD, it can be concluded that diabetes is associated with low levels of apo A—I which in turn may predispose to macrovascular disease. These data indicate that apolipoprotein levels are altered in diabetes mellitus and indirectly suggest that insulin deficiency, whether absolute or relative accounts for these changes in apolipoproteins, affecting lipoprotein levels and total serum lipids. Although findings reported in this dissertation may not have immediate clinical applications, they provide data which are helpful in understanding the dynamics of lipoprotein metabolism. The NIDDM and IDDM subjects presented similar fasting plasma lipids and apo- 131 lipoprotein profiles, with the IDDM exhibiting significant decreases of apo A-I. It is possible to separate disease (CAD/PAD) and non- disease states in the IDDM subjects by their apo ArI levels. This suggests the possibility of a future use of the apo A-I assay as a diagnostic test in screening patients to detect abnormalities in plasma apo A—I concentrations. Available evidence suggests that apo A—I may be the critical element needed for the uptake of free cholesterol from cell membranes by HDL particles. Results of postprandial studies in NIDDM subjects suggest the importance of looking at apolipoprotein level changes during the fed state. The correlation between triglycerides and apo C—II, apo C-III in the NIDDM subjects suggests that the C apolipoproteins are good markers for detecting the abnormal states of triglyceride metabolism. Whether hyperinsulinemia and associated apparent cellular insulin insensitivity account for the abnormal apo C response is not known. The availability of purified apolipoproteins provides many possibilities for detailed studies of apolipoproteins. It is possible to discern some definitive apolipoprotein patterns in the diabetics and some trends are visible that merit further investigation. Apo- lipOprotein profiles may be of considerable clinical importance as a new means for classifying disorders of lipid transport and a device for monitoring progress of therapeutic interventions designed to normalize underlying metabolic disorders. Further research would be fruitful, particularly in the newly diagnosed IDDM individuals where plasma apolipoproteins could be investigated before initiation of insulin. Insulin has marked effects on lipid metabolism. High insulin levels have been shown to enhance the atherosclerotic process 132 in experimental animals, and may play a role in humans. Non-insulin dependent diabetics who are obese, as well as hyperinsulinemic; and insulin dependent diabetics who receive one or more subcutaneous insulin injections per day have arteries exposed to high insulin levels. It appears that exposure to high insulin levels may result in alterations in lipoproteins which might enhance the development of atherosclerosis. 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Postprandial Levels of Plasma Glucose, Insulin, Plasma Lipids, HDL cholesterol and ApolipOproteins A-I, A—II, C-II and C—III in Normal Controls and Non-insulin Dependent Diabetics (NIDDM) Table A—1 Plasma Glucose (mg/d1) Time Normals NIDDM NIDDM (min) (n=6) (n=5) (n=4) Study 1 Study 2 0 104.8 f 2.6 171.6 f 75.4 158.0 f 22.9 30 170.7 f 17.7+ 223.4 f 56.3 220.0 f 26.4++ 60 162.3 f 33.4 287.6 T 54.6*+ 280.3 f 28.8+++ 90 144.3 f 35.9 312.0 f 81.2*+ 300.8 f 41.4+++ 120 120.8 f 34.9 302.2 f 97.2**+ 293.8 f 52.0+++ 150 97.2 i 28.4 294.2 f 112.3**+ 272.5 f 76.8 180 88.3 f 21.0 259.6 f 112.0++ 235.8 f 75.4 240 84.7 i 16.4 197.0 f 99.9 174.8 f 76.8 300 98.0 i 6.9 152.2 f 93.1 136.8 f 63.0 + Values are means - SD *P =< 0.001 significantly different from normal ++ *8 P =< 0.01 + P =< 0.001 significant rise from baseline (paired t-test) +++ P =< 0.002 143 144 Table A-2 Plasma Insulin (LUnits/ml) Time Normals NIDDM NIDDM (min) (n=6) (n=5) (n=4) Study 1 Study 2 0 13.2 i 2.8 18.4 t 6.1 17.1 i 2.6 30 40.5 i 23.5 35.8 f 20.1 34.3 i 15.9 60 56.8 i 38.4 58.2 i 39.8 30.6 i 19.7 90 81.7 f 34.5+ 93.2 i 77.7 57.8 f 35.8 120 63.5 i 31.0 102.4 f 91.2 89.6 i 55.6 150 47.4 f 28.2 92.6 f 74.0 79.0 f 51.6 180 42.0 i 32.6 89.4 i 66.5 75.0 i 33.5 240 20.2 i 12.5 50.4 i 28.9 38.0 t 9.7 300 13.2 i 2.6 36.9 f 14.3* 47.8 i 24.8 Values are means f SD *P =< 0.02 +P =< 0.002 significantly different from normal significant rise from baseline (paired t—test) 145 Table A-3 Plasma Triglycerides (mg/d1) Time Normals NIDDM NIDDM (min) (n=6) (n=5) (n=4) Study 1 Study 2 0 85.2 i 32.8 221.4 f 101.6 182.3 f 45. 30 103.8 f 26.5+ 224.8 4 105.1* 193.8 f 49. 60 101.0 f 21.0 244.4 f 100.3 203.0 f 36. 90 92.7 i 19.0 241.0 f 93.8 206.3 f 36. 120 78.0 f 20.8 233.4 f 97.4 199.8 f 32. 150 80.3 f 20.5 222.2 f 96.3 200.8 f 37. 180 82.5 i 19.7 225.2 4 104.3 196.5 f 40. 240 83.5 f 17.8 216.6 f 95.0 198.0 f 42. 300 88.0 f 21.1 217.8 f 97.5 204.5 f 44. + Values are means — SD *P = < 0.05 +P = < 0.02 significantly different from normal significant rise from baseline (paired t—test) 146 Table A—4 Plasma Cholesterol (mg/d1) Time Normals NIDDM NIDDM (min) (n=6) (n=5) (n=4) Study 1 Study 1 o 183 0 — 33.3 180 o i 31.4 185.3 - 40. 30 187.7 f 24.5 181.2 T 28.6 187.8 f 39. 60 182.3 f 27.1 175.2 f 31.1 183.8 f 38. 90 184.7 f 19 2 174 8 f 28.8 185.5 f 40. 120 178.3 f 25.6 172.6 f 29.8 185.8 T 39. 150 178.5 f 23.2 173.2 f 36.1 187.5 f 42. 180 177.2 T 29.1 174.6 f 35.6 188.5 f 48. 240 178.3 f 20.4 179.6 f 34.1 192.5 f 47. 300 177.7 f 19 4 183 8 i 29.5 199.5 f 49. + Values are means — SD 147 Table Ar5 Plasma HDL Cholesterol (mg/d1) Time Normals NIDDM NIDDM (min) (n=6) (n=5) (n=4) Study 1 Study 2 0 42.5 T 6 5 27.4 T 6 4* 25.4 T 7.1 30 42.4 T 6 3 25.7 T 5.3** 24.9 T 6.6 60 43.1 T 6.2 24.5 T 4.8** 23.8 T 7.4 90 44.4 T 5.9 25.5 T 4.9** 25.3 T 7.1 120 44.3 T 8.1 26.7 T 6.8* 25.3 T 7.3 150 44.3 T 7.4 24.8 T 6.3** 25.3 T 7.3 180 45.9 T 6.8 26.3 T 6.3** 25.1 T 6.4 240 45.0 T 7 6 25.7 T 5.9** 25.1 T 7.4 300 45.1 T 6 0 26.9 T 5.6** 26.4 T 7.2 Values are means f SD *P =<=0.004 significantly different from normal **P =< 0.001 148 Table A—6 Plasma Apolipoprotein A-I (mg/d1) Time Normals NIDDM NIDDM (min) (n=6) (n=5) (n=4) Study 1 Study 2 0 161. T 30. 147. T 19. 144.2 T 17. 30 159. T 28. 154. T 28. 149.3 T 32. 60 177. T 24. 155. T 18. 136.3 T 17. 90 167. T 38. 144. T 14. 131.8 T 16. 120 152. T 21. 153. T 13. 137.5 T 15. 150 165. T 47. 143. T 33. 134.4 T 25. 180 161. T 27. 162. T 22. 142.1 T 22. 240 177. T 36. 154. T 15. 138.9 T 12. 300 166. T 22. 154. T 15. 129.2 T 24. + Values are means — SD 149 Table A-7 Plasma Apolipoprotein A—II (mg/d1) Time Normals NIDDM NIDDM (min) (n=6) (n=5) (n=4) Study 1 Study 0 22.1 T 3 0 20.9 T 4.6 19.4 T 30 23.2 T 2 7 19.5 T 2 8 25.6 T 60 22.3 T 2.4 20.6 T 5 9 21.1 T 90 25.2 T 5.1 18.1 T 5.3 20.6 T 120 23.5 T 4.2 18.5 T 6.6 20.9 T 150 23.1 T 2.2 15.9 T 3.6 22.6 T 180 21.6 T 4.0 21.1 T 7.0 20.5 T 240 23.7 T 4 4 18.6 T 6 2 22.0 T 300 24.8 T 3 2 20.4 T 5 8 21.3 T Values are means f SD 150 Table A—8 Plasma Apolipoprotein C—II (mg/d1) Time Normals NIDDM NIDDM (min) (n=6) (n=5) (n=4) Study 1 Study 0 .7T 1.8 7.8T2.1 8.1T 30 .0T 1.6 7.7T 1.7 7.4T 60 .6T0.9 7.2T 2.6 7.8T 90 .6T 1.6 7.0T 2.1 8.2T 120 .1 T 1.3 7.4T2.6 7.9T 150 .6T 1.3 8.2T 1.7* 8.4T 180 .2T1.9 8.0T1.3 8.2T 240 .8T1.6 8.2T 1.4 8.2T 300 .6T1.3 8.8T 1.7* 9.2T + Values are means - SD *P = < 0.004 significantly different from normal 151 Table A—9 Plasma Apolipoprotein C—III (mg/d1) Time Normals NIDDM NIDDM (min) (n=6) (n=5) (n=4) Study 1 Study 2 0 10.7 T 1.4 15.2 T 2.0* 12.5 T 2.6 30 10.8 T 1.2 15.4 T 2.5* 12.3 T 2.6 60 11.5 T 1.1 15.2 T 2.8 12.3 T 2.5 90 12.2 T 1.0 15.2 T 2.5 11.9 T 2.2 120 11.9 T 1.6 15.7 T 3.0** 12.3 T 1.3 150 12.1 T 1.4 16.0 T 2.7*** 12.7 T 2.1 180 12.1 T 1.4 15.9 T 2.3* 13.1 T 1.6 240 11.8 T 1.5 16.6 T 3.1* 13.3 T 2.2 300 10.9 T 1.0 15.4 T 2.1**** 13.3 T 1.2 Values are means T SD *P =< 0.002 significantly different from normal **P =< 0.02 ***P =< 0.01 ****P =< 0 001 152 Table A-10 Plasma HDL Apolipoprotein A-I (mg/d1) Time Normals NIDDM NIDDM (min) (n=6) (n=5) (n=4) Study 1 Study 2 0 138.2 T 14. 128.8 - 23. 139.2 T 24. 30 131.2 T 21 121.0 T 19. 116.6 T 31. 60 132.8 T 10. 126.5 T 24. 118.0 T 33. 90 132.2 T 13. 120.2 T 17. 114.2 T 28. 120 134.0 T 14. 118.0 T 12. 122.2 T 27. 150 122.2 T 13. 114.2 T 16. 118.2 T 18. 180 137.8 T 22. 125.6 T 13. 117.1 T 28. 240 137.3 T 18. 132.9 T 21. 116.4 T 29. 300 143.9 T 9. 118.5 T 18. 131.7 T 37. Values are means SD Table A—ll Plasma HDL Apolipoprotein A—II (mg/d1) Time Normals NIDDM NIDDM (min) (n=6) (n=5) (n=4) Study 1 Study 2 0 18.3 T 2.3 19.1 T 6.5 19.0 T 4.5 30 17.5 T 2.8 17.9 T 6.0 19.0 T 5.4 60 19.1 T 3.3 17.4 T 7.3 19.4 T 4.4 90 18.2 T 3.5 18.1 T 7.2 19.0 T 4.3 120 18.2 T 3.7 19.2 T 4.0 21.2 T 5.6 150 19.0 T 3.1 18.2 T 3.2 20.7 T 6.6 180 19.9 T 3.5 18.1 T 3.9 20.3 T 6.3 240 20.0 T 4.2 17.4 T 4.6 24.8 T 7.0 300 20.6 T 3.3 17.6 T 3.4 23.2 T 4.9 + Values are means — SD B. Linear Trend in the Cumulative Profile Oral Glucose Load of the Normals and Diabetics (NIDDM) 154 Hoo.o vu was mHouucoo HmEuo: Eouw uamuommfip >Hocmofiwflcwflm No.0 vn ma mm M mcmoE mum mosam> qm.o M mq.o mm.oH M MH.NOH m©.o M N0.H mm.m M On.0n HHHIU om< . I . | . | . . .I . . l . I Q no H + cs o mm HH + no on cm 0 + as 0 mm w + ma NM HH 0 o < om.q M qw.al oq.mm M 0H.NNH mm.N M oo.H mm.wm M m©.mmfi HH1< oa¢ mm.q M mm.q 0H.moH M ofi.owm Hm.mH M oo.m wq.qu M qw.mmoa Hlm oa< *mm.~ M Ho.H1 c.0m M mm.ooH 0N.N M mw.H| om.¢q M ma.mwN H030 do: o.m M HH.m1 mm.OHN M Nq.mmfia wN.HH M Hq.NI oq.mqa M mm.mmafi Houoummaono m¢.HH M am.o «©.HN© M «m.amqa mm.mfi M om.m ww.qHH M mw.wqm wmuwpouzawflufi ¢H.m¢ M wq.dm mN.mmm M NN.mom mo.m M mm.Nm qw.aoH M mo.~om :HHDmcH «kmfi.ma M em.Hw om.ma© M oo.omcH qm.NH M mm.mH mm.HoH M ma.a0n mwousau wGMwamm | N VCwHH HNQCHA wfiflfiwmmm I N UCTHH Hmwfifld OHQMHHN> Gus: wowuwnmfio ucowcwaom cflandelcoz maouucoo HmEMoz mammoum w>HumH=EDU map cH mummy smocflq Him anmH 155 Table B-2 Normal Controls Vs Non-Insulin Dependent Diabetics T Test of the Linear Trend Variables P Glucose 0.024 Insulin 0.267 Triglycerides 0.031 Apo A—I 0.295 Apo A—II 0.30 Apo C—II 0.017 Apo C-III 0.009 Cholesterol 0.30 HDL Cholesterol 0.001 156 Om I mammE mum mTSHm> + Nw.o M MN.o mq.HH M om.Nw Ho.H M 00. oq.mH M Hm.moH HHHIU oa< . I . . I . . I .I . I . I m NN H + co 0 Nu N + wN mm mm o + «n Mm NH + om Hm HH 0 o < mm.N M mq.N N©.©H M mo.me HH.¢ M no.MI Hm.om M mm.©NH HHI< om< mn.w M @m.©l mo.NHH M HN.Nww Nm.q M Hm.q mH.Nm M Nm.mHoH HI< oa< ©¢.N M 0N.I on.mq M NH.moH ON.N M mn.HI ow.oq M Hm.0mH HOSU Ham «m.N M oo.m mw.NwN M wc.HNNH mo.w M qN.MI mm.MMN M mo.HHHH HoumummHoso 0H.mH M oH.wH a¢.NQN M ww.mmNH ow.NH M qo.m Hw.mqm M oo.mmoH mmmfiumohHwHHH mo.NN M mm.mm mm.©wH M mm.NHq mq.mq M mq.mm 0N.N©m M w¢.mwm aHHDwaH Ho.Hm M mm.wm H©.me M oo.meH wN.m M OH.ww ow.N©m M mm.mme omoous mCHHmmmm I x vcmuH HmoGHH oGHmemm I x wcoue Hmmst oHanHm> Hansv N sonam Amuse a serum moHuoano ucowaoaon CHHchHIcoz mo N %©:um m> H mmsum mlm mHan 157 Table B—4 Study 1 Vs Study 2 of the Non—Insulin Dependent Diabetics T Test of the Linear Trend Variables P Glucose 0.255 Insulin 0.206 Triglycerides 0.255 Apo A—I 0.030 Apo A-II 0.30 Apo C—II 0.30 vApo C—III 0.020 HDL Cholesterol 0.30 Cholesterol 0.162 C. Non-Insulin Dependent Diabetics: Fasting Plasma Glucose, Insulin, Lipids, HDL Cholesterol, Apolipoproteins A—I, A-II, C—II and C—III Levels of Those on Diet, on Insulin and Oral Hypoglycemic Agents Table C-l Variables Measured in NIDDM Men (n=50) On Diet On Insulin On OHA N 24 17 9 Age 63.1 T 5.0 63.3 T 5.7 62.3 T 5.0 Height 172.4 T 7.0 172.7 T 5.5 178.3 T 6.1 Weight 88.6 T 12.6 79.8 T 9.9 97.9 T 20.4 BWI 1.2 T 0.2 1.1 T 0.2 1.2 T 0.2 + + + Glucose (mg/d1) 174.5 - 55.5 187.3 — 67.6 182.9 - 57.5 Glyco Hb (2) 10.2 T 2.0* 11.9 T 2.1* 11.1 T 2.5 + + + HDL Chol (mg/d1) 43.5 - 14.7 42.2 - 13.6 38.0 - 16.2 Total Chol (mg/d1)212.8 T 60.2 208.7 T 44.5 197.3 T 41.7 + Total TG (mg/d1) 150.2 T 83.5** 173.5 T 102.3 216.9 - 96.5** Apo A—I (mg/d1) 173.0 T 37.3 175.9 T 36.6 155.5 T 35.4 Apo A—II (mg/d1) 47.1 T 13.0 47.2 T 11.1 43.4 T 7.4 Apo C—II (mg/d1) 10.2 T 3.5 10.2 T 3.7 11.7 T 2.6 Apo C-III (mg/d1) 16.2 T 4.3 16.1 T 4.4 18.3 T 3.7 + Values are means - SD *P =<<0.01 different from men on insulin **P =