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Insulin-Mediated Increases in the HDL Cholesterol/Cholesterol Ratio in Humans Craig N. Sadur and Robert H. Eckel Downloaded from http://atvb.ahajournals.org/ by guest on May 10, 2017 The role of insulin in the regulation of lipoprotein cholesterol distribution was studied in 18 human volunteers who were of normal weight, normolipemic, and glucose-tolerant. We used the euglycemic clamp technique and made comparisons with six saline-infused controls. While total cholesterol and low density lipoprotein cholesterol levels fell similarly by 6 hours in both groups (p = NS), there was a greater decrease in triglyceride levels (p < 0.02) but less decrease in high density lipoprotein cholesterol levels (p < 0.02) in the insulin-infused group. These changes resulted in both a higher high density lipoprotein cholesterol/total cholesterol ratio (p = 0.01) and a higher high density lipoprotein cholesterol/low density cholesterol ratio (p = 0.02) in the insulin-infused group. The timing of this effect of insulin implies a mechanism unrelated to high density lipoprotein turnover. Thus, insulin infusion during euglycemia appears to alter cholesterol distribution favorably in normal subjects. (Arteriosclerosis 3:339-343, July/August 1983) to examine the insulin effect in a more direct manner would be beneficial. In the present study, the euglycemic clamp technique has provided a tool for assessment of the effect of insulin on lipoprotein cholesterol distribution in human subjects. he role of insulin in the regulation of lipoprotein T cholesterol metabolism is unclear. In vivo, diseases at the extremes of serum insulin concentration have been associated with alterations in lipoprotein cholesterol distribution. In obesity, a state where serum insulin is high, there are increases in total, very low density lipoprotein (VLDL), and low density lipoprotein (LDL) cholesterol and a decrease in high density lipoprotein (HDL) cholesterol.12 Type I diabetes mellitus, where serum insulin is low, is associated with altered lipoprotein metabolism which normalizes with improved glycemic control.3"5 In fact, some studies have demonstrated increased levels of HDL cholesterol,67 HDL cholesterol/total cholesterol, or HDL cholesterol/LDL cholesterol ratios8 in insulin-treated diabetics. Although the lipoprotein alterations seen in the insulinopenic patient could have resulted from insulin deficiency alone, a direct effect of insulin on lipoprotein cholesterol metabolism has not been proven because other metabolic alterations are also corrected (e.g., ketonemia). Thus, a method Methods The study group included 18 subjects who received intravenous insulin and glucose. Each person was average in height and within 20% of ideal body weight according to the Metropolitan Life Insurance Company Standards.9 The body mass index (wt/ht2) was 2.06 x 10" 3 ± 0.05 kg/cm2 (x ± SEM). The subjects ranged in age from 20 to 45 years, x = 29.2 ± 1.4 years. There were 15 women and three men. The control group, composed of four women and two men, received intravenous infusions of 0.9% (normal) saline at approximately 100 to 150 ml/hr. They were also normal in height and weight with a body mass index of 2.17 x 10~3 ± 0.05 kg/cm2 and with an age range of 23 to 40 years, x = 29.0 ± 2.5 years. Individuals were excluded if they were taking drugs with known effects on glucose or lipid metabolism, such as oral contraceptives, other estrogenic compounds, diuretics, or other antihypertensive medications. All subjects were free of acute or chronic illnesses. Two individuals were euthyroid while taking thyroxine for full thyroid hormone replacement. Physical examinations confirmed the normal From the Division of Endocrinology, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado. Support for this work was provided by National Institutes of Health Grants AM 26356 and RR-00051 to the General Clinical Research Center at the University of Colorado Health Sciences Center. Address for reprints: Craig N. Sadur, M.D., B151, 4200 East Ninth Avenue, Denver, Colorado 80262. 339 340 ARTERIOSCLEROSIS VOL 3, No 4, JULY/AUGUST 1983 Downloaded from http://atvb.ahajournals.org/ by guest on May 10, 2017 health of all subjects. Each ingested 40 g/m2 of glucose and had a normal 3-hour oral glucose tolerance curve, according to the guidelines of the National Diabetes Data Group.10 In addition, all subjects were normal in tests for: tri-iodothyronine resin uptake, total thyroxine, thyroid-stimulating hormone, electrolyte panel, calcium, liver panel, phosphorus, magnesium, complete blood count, and urinalysis. Fasting serum cholesterol and triglyceride (TG) were normal for all subjects. All studies were approved by the Human Subjects Committee and were performed in the Clinical Research Center at the University of Colorado Health Sciences Center. Informed consent was obtained from all participants studied. For 2 days before the study, each subject was fed an isocaloric formula feeding containing 45% carbohydrate, 40% fat, and 15% protein. After an overnight fast, on the morning of the euglycemic clamp study, two intravenous lines were placed in opposite arms of the study subjects, one for infusion of fluids and one for sampling of blood. A heating pad was placed over the arm where the samples were obtained from venous blood. Plasma glucose was determined after plasma was immediately separated with a Beckman Microfuge (Beckman Instruments, Incorporated, Palo Alto, California) by the glucose oxidase technique using the Beckman glucose analyzer (Beckman Instruments, Incorporated, Clinical Instruments Division, Fullerton, California). In the insulin group, basal plasma glucose values were determined on the morning of the euglycemic clamp study and served as the standard, determining the varying amounts of glucose infusion needed to maintain euglycemia during the rest of the study. Although glucose was similarly determined throughout the saline infusion, glucose was not infused. Basal and serial adipose tissue biopsies (at 20,80, 180, and 360 minutes after the beginning of the infusions) were performed for the measurement of lipoprotein lipase activity as previously described.11 Most of these data have been published and will not be included in the present paper. Insulin was infused into the study subjects in an exponentially decreasing manner over the first 10 minutes and then at either 40 or 120 mU/m2 per minute to obtain a steady-state serum insulin level of 75 ± 5 or 271 ± 27 ^U/ml, respectively. Glucose infusion was begun at 4 minutes after the start of the insulin infusion. At that point and throughout the study, a blood sample was drawn at least every 5 minutes to test for plasma glucose concentration in order to determine the changes in glucose infusion rate necessary to maintain euglycemia. In addition, these subjects received saline infusions similar to those received by the control subjects. A potassium supplement of 30 mEq of carbohydrate-free KCI solution was given to all subjects the evening preceding the study and at approximately 1.5 and 3.5 hours after the start of the insulin infusion to maintain normokalemia. TG was determined by an enzymatic method.12 The coefficient of variation of TG was 8%. Total cholesterol was also measured enzymatically13 with a coefficient of variation of 4%. HDL cholesterol was measured in plasma from which VLDL and LDL were precipitated by 4% sodium phosphotungstate.14 The HDL cholesterol was measured in the supernatant after centrifugation in a Beckman TJ-6 centrifuge, 1500 g for 30 minutes at 4°C. The coefficient of variation for HDL cholesterol, including analytical and precipitation variation, was 8%. LDL cholesterol was calculated from the formula of Friedewald, et al.15 Serum insulin levels were measured by a double antibody radioimmunoassay with the technique of Desbuquois and Aurbach.16 The Mann-Whitney U test for unpaired observations was used for statistical analyses. Results Baseline Data The baseline data of both the 18 study subjects and the six control subjects are displayed in Table 1. Basal plasma glucose values in all subjects ranged from 71 to 99 mg/dl. Basal lipid determinations were similar in both high-insulin (n = 7) and low-insulin (n = 11) infusion groups. Whereas TG, HDL cholesterol, and HDL cholesterol/total cholesterol were similar in both the insulin and saline control groups, total cholesterol and LDL cholesterol were significantly higher in the control group (p < 0.05) while HDL cholesterol/LDL cholesterol was significantly lower in the control group (p < 0.05). These differences became insignificant when the data were matched for gender. Table 1. Baseline Data Before Insulin or Saline Infusion Age (yrs) Weight (kg) Wt/Ht2 x 10" 3 (kg/cm2) Glucose (mg/dl) Total cholesterol (mg/dl) LDL cholesterol (mg/dl) TG (mg/dl) HDL cholesterol (mg/dl) HDL cholesterol/ total cholesterol (%) HDL cholesterol/ LDL cholesterol (%) Insulin Saline 29.2+1. 4 57.7 + 2. 2 2.06 ± 0 . 05 84.2±1. 7 159 + 5 88±5 74±7 56±3 29.0 ±2.5 63.8±3.3 2.17±0.05 86.8 + 2.6 179±8 108 + 6 90±11 53±6 35 + 2 30 + 3 67±6 50 + 4 Values are means ± SEM. TG = triglyceride; HDL = high density lipoprotein; LDL = low density lipoprotein. INSULIN INCREASES HDL CHOLESTEROL/CHOLESTEROL Sadur and Eckel 341 Glucose Saline infusion resulted in a gradual fall in plasma glucose over the 6-hour infusion period to 92% ± 3% of basal. While insulin was infused into study subjects, plasma glucose was maintained by a variable glucose infusion and ranged throughout the study from 94% to 103% of basal value. Total Cholesterol Downloaded from http://atvb.ahajournals.org/ by guest on May 10, 2017 For total cholesterol and all remaining lipid measurements, no significant differences between insulin at low (40 mU/m2 per minute) and high (120 mU/m2 per minute) infusion rates were found. Thus, all insulin data are combined measurements for both insulin infusion rates. Although women appeared to have a greater decrease in total cholesterol by 6 hours during both the insulin infusions (men = - 1 0 ± 2 mg/dl vs women = - 2 1 ± 3 mg/dl) and the saline infusions (men = - 9 ± 8 mg/dl vs women = - 2 1 ± 5 mg/dl), these differences were not statistically significant. The combination of men and women, as shown in Figure 1 A, revealed similar 6-hour declines in both infusion groups (saline = - 1 7 ± 5 mg/dl vs insulin = - 1 9 ± 3 mg/dl, p = NS). -15 -25 5 -5 LDL Cholesterol As shown in Figure 1 B, there were no significant differences between the two groups in the decreases in LDL cholesterol levels. Trlglycerldes TG levels decreased more in the study subjects than in the controls (Figure 1 C). The p values for the differences in TG for the two groups at the 80minute, 180-minute, and 380-minute time-points were < 0.01, < 0.05, and < 0.02, respectively. There was no gender difference in basal or subsequent TG changes for either saline or insulin infusions. -15 B o -to HDL Cholesterol The change in HDL cholesterol is seen in Figure 1 D. In both study subjects and controls, there was a fall in HDL cholesterol with the infusions, but the insulin-infused group had no further decrease after the first 80 minutes. Whereas the differences between the two groups were insignificant at 80-minute and 180-minute time-points, the difference at 380 minutes was statistically significant (p < 0.02). Again, there was no gender difference between responses during insulin and saline infusions. -40 5 -5 Figure 1. Changes in total cholesterol (A), LDI cholesterol (B), triglycerides (C), and HDL cholesterol (D) are plotted by minutes during saline (•—•) and insulin (o—-o) infusions. *p s£ 0.01, t p < 0.05, tP *= 0.02. Data are mg/dl (mean ± SEM). -10 0 80 180 Mln 380 342 ARTERIOSCLEROSIS VOL 3, No 4, JULY/AUGUST 1983 Miscellaneous o aj o o Insulin-stimulated adipose tissue lipoprotein lipase failed to correlate with changes in cholesterol distribution. There was also no relationship between the fall in serum triglyceride and the changes in HDL cholesterol. In addition, the amount of glucose infused did not predict the changes observed. O "5 o O Q I o © Downloaded from http://atvb.ahajournals.org/ by guest on May 10, 2017 "5 o Discussion -2 15 10 5 2 .2 0 r 1 S -.0 80 B ISO Mln 380 Figure 2. Changes in the HDL cholesterol/total cholesterol ratio (A) and HDL cholesterol/LDL cholesterol ratio (B) are plotted by minutes during saline (•—•) and insulin (o—-o) infusions. *p s= 0.01, %p ^ 0.02. Data are shown as percentages (mean ± SEM). HDL Cholesterol/Total Cholesterol When expressed as the ratio of HDL cholesterol/ total cholesterol, insulin infusion resulted in a higher absolute increase in the ratio at 380 minutes when compared to saline infusion, 2.8 ± 1.2% vs - 1 . 2 ± 0.8%, respectively (Figure 2 A, p = 0.01). Once again, gender did not affect the changes that occurred during the infusions. In men, the 6-hour HDL cholesterol/total cholesterol ratio changed - 2 . 0 ± 0.0% with saline infusion vs 2.0 ± 1.2% with insulin; in women, the change was - 0 . 8 ± 1.3% vs 3.0 ± 1.4%. HDL Cholesterol/LDL Cholesterol As with HDL cholesterol/total cholesterol, the study group had a statistically significant increase in the HDL cholesterol/LDL cholesterol ratio by 380 minutes after the start of the infusion (Figure 2 B, p = 0.02). In this study the euglycemic clamp technique has provided a tool for examination of the effect of insulin on lipoprotein cholesterol distribution in 18 normal subjects. Because the response was similarly affected at the two insulin infusion rates, 40 and 120 mU/ m2 per minute, the results from the two groups were combined. In both saline-infused and insulin-infused subjects, a progressive decrease in total cholesterol occurred over 380 minutes. LDL cholesterol also fell similarly in the two groups during the same time course because of a greater decrease in TG levels but a smaller decrease in HDL cholesterol levels in insulin-infused subjects. Some caution in the interpretation of the calculated LDL cholesterol data is needed because the maintenance of VLDL composition was assumed but not proven. Because substantial intravenous volumes were infused in both groups, a dilutional effect is likely. The insulin effect on trigycerides, however, was more than dilutional and has been reported previously.11 Most important, insulin administration reduced the decrease in HDL cholesterol level, particularly during the latter part of the infusion. This effect was not associated with the insulin-stimulatory effect on adipose tissue lipoprotein lipase activity, the decrease in triglyceride level, or the amount of glucose infused (data not shown). Moreover, when the data were expressed as the ratios of both HDL cholesterol/total cholesterol and HDL cholesterol/LDL cholesterol, insulin administration resulted in higher ratios than those found with normal saline infusions. Some investigators have felt that these ratios provide the most meaningful lipid assessment of antiatherogenic risk.17'18 Because the half-life of apo A-l and apo A-ll is measured in days,19 the effects of the hormone on HDL production or clearance would be unlikely explanations for the differences noted between saline and insulin administration. Possibly, an effect of insulin on cholesterol transfer between two lipoprotein particles played a role. In vitro studies have revealed that esterified cholesterol can be transferred from HDL to VLDL, an effect mediated by a specific transfer protein.20 This phenomenon also occurs with triglyceride transferred from VLDL to HDL. Both these transfers are reversible21 and are probably independent of each other.20 Although HDL cholesterol, the HDL cholesterol/total cholesterol ratio, and the HDL cholesterol/LDL cho- INSULIN INCREASES HDL CHOLESTEROL/CHOLESTEROL Downloaded from http://atvb.ahajournals.org/ by guest on May 10, 2017 lesterol ratio, did not change significantly at the 80and 180-minute time-points in the study group when compared to the control group, the 380-minute timepoints revealed significant changes with the insulin infusion, consistent with the temporal course of lipid transfers decribed in vitro. Thus, perhaps insulinmediated increases in the HDL cholesterol/total cholesterol and HDL cholesterol/LDL cholesterol ratios could be due to alterations in cholesterol transfer to or from HDL, preventing HDL cholesterol concentrations from falling during the infusion period. The evidence that insulin affects cholesterol metabolism in humans is indirect. A favorable effect of insulin on cholesterol metabolism is best exemplified by Type I diabetics who demonstrate a reduction in total cholesterol after improvement of the hypoinsulinemic state.3"5 After insulinization, lipids return to normal. In fact, higher levels of circulating free insulin concentrations found in treated Type I diabetics22'M may relate to higher levels of HDL cholesterol67 or HDL cholesterol/total cholesterol8 found in these patients. An unfavorable effect of insulin on cholesterol metabolism would have also been possible. For instance, high carbohydrate diets, which have been associated with reductions in total and LDL cholesterol2425 have also resulted in increases in VLDL 2425 and decreases in HDL cholesterol.24 In addition, obese subjects who are hyperinsulinemic have increases in total cholesterol, VLDL cholesterol, and LDL cholesterol with lower HDL cholesterol concentrations than normal-weight controls. 12 Thus, although the present study was a short-term one and involved a nonphysiologic administration of insulin, we observed an antiatherogenic influence of insulin on lipoprotein cholesterol distribution in normal human subjects. This effect was predominantly on HDL cholesterol and suggests that mechanisms independent from lipoprotein turnover may be important in mediating this phenomenon. Acknowledgments We sincerely thank Orville G. Kolterman for insulin radioimmunoassays; Debbie Gwinner, Deborah J. Krinitzsky, and Judy Prasad for technical assistance; and Joni Foti for secretarial assistance. References 1. Garrison RJ, Wilson PW, Castelll WP, Felnlelb M, Kannel WB, McNamara PM. Obesity and lipoprotein cholesterol in the Framingham Offspring Study. Metabolism 1980;29: 1053-1060 2. Sadur CN, Eckel RH. Insulin-mediated lipoprotein metabolism in obesity. Diabetes 1982;31:156A 3. Pletrl A, Dunn FL, Raskin P. The effect of improved diabetic control on plasma lipid and lipoprotein levels. Diabetes 1980;29:1001-1005 Index Terms: insulin • euglycemic clamp Sadur and Eckel 343 4. Sosenko JM, Breslow JL, Mlettlnen OL, Gabbay KH. Myperglycemia and plasma lipid levels. N Engl J Med 1980; 302:650-654 5. Eckel RH, McLean E, Alters JJ, Cheung MC, Blerman EL. Plasma lipids and microangiopatny in insulin-dependent diabetes mellitus. Diabetes Care 1981;4:447-453 6. Nikklla EA, Hormlla P. Serum lipids and lipoproteins in insulin-treated diabetes. Demonstration of increased high density lipoprotein concentrations. Diabetes 1978;27:1078-1086 7. Klujber L, Molnar D, Kardos M, Jaszai V, Soltesz GY, Mestyan J. Metabolic control, glycosylated haemoglobin and high density lipoprotein cholesterol in diabetic children. Eur J Pediatr 1979; 132:289-297 8. Eckel RH, Albers JJ, Cheung MC, Wahl PW, Llndgren FT, Blerman EL. High density lipoprotein composition In insulindependent diabetes mellitus. Diabetes 1981 ;30:132-138 9. Metropolitan Lite Insurance Company. New weight standards for men and women. Statistical Bulletin 1959,40:1-4 10. National Diabetes Data Group. Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance. Diabetes 1979;28:1039-1057 11. Sadur CN, Eckel RH. Insulin stimulation of adipose tissue lipoprotein lipase: Use of the euglycemlc clamp technique. J Clin Invest 1982;69:1119-1125 12. Stavropoulos WS, Crouch RD. A new colorimetric procedure for the determination of serum triglycerides (abstr). Clin Chem 1974;20:857 13. Richmond W. Preparation and properties of a cholesterol oxidase from norcadia Sp. and its application to the enzymatic assay of total cholesterol in serum. Clin Chem 1973;19:1350-1356 14 Lop«s-Vlrella MF, Stone P, Ellis S, Colwell J A. Cholesterol determinations in high-density lipoproteins separated by three different methods. Clin Chem 1977;23:882-884 15. Frtedewald WT, Levy Rl, Fredrlckson DS. Estimation of the concentration of tow-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972:18:499-502 16. Desbuquols B, Aurbach GD. Use of the polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoassays. J Clin Endocrinol Metab 1971,33:732738 17. Burslem J, Schonfeld G, Howald MA, Weldman SW, Miller JP. Plasma apoprotein and lipoprotein lipid levels in vegetarians. Metabolism 1978;27:711-719 18. Wltztum J, Schonfeld G. High density lipoproteins. Diabetes 1979;28:326-336 19. Blum CB, Levy Rl, Elsenberg S, Hall M III, Goebel RH, Bercnan M. High density lipoprotein metabolism in man. J Clin Invest 1977;60:795-807 20. Hopkins GJ, Barter PJ. Transfers of esterified cholesterol and triglycende between high-density and very low-density lipoproteins: In vitro studies of rabbits and humans. Metabolism 1980;29:546-550 21. Barter PJ, Lally Jl. In vitro exchanges of esterified cholesterol between serum lipoproteins fractions: Studies of humans and rabbits. Metabolism 1979;28:230-236 22. Hedlng LG. Determination of total serum insulin (IRI) in insulin-treated diabetic patients. Diabetologia 1972;8:260-266 23. Rasmussen SM, Hedlng LG, Parbst E. Serum IRI in insulintreated diabetics during a 24-hour period. Diabetologia 1975; 11:151-158 24. Falko JM, Schonfeld G, Wltztum JL, Kolar JB, Salmon P. Effects of short-term high carbohydrate, fat-free diet on plasma levels of apo c-ll and apo c-lll and on apo c subspecies in human plasma lipoproteins. Metabolism 1980:29:654-661 25. Ginsberg HN, Le N-A, Mellsh J, Steinberg D, Brown WV. Effect of a high carbohydrate diet on apoprotein-B catabolism in man. Metabolism 1981:30:347-353 • HDL cholesterol • cholesterol • triglycende Insulin-mediated increases in the HDL cholesterol/cholesterol ratio in humans. C N Sadur and R H Eckel Downloaded from http://atvb.ahajournals.org/ by guest on May 10, 2017 Arterioscler Thromb Vasc Biol. 1983;3:339-343 doi: 10.1161/01.ATV.3.4.339 Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1983 American Heart Association, Inc. All rights reserved. Print ISSN: 1079-5642. 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