Insulin-Mediated Increases in the HDL Cholesterol/Cholesterol Ratio in Humans

Transcription

1 Insulin-Mediated Increases in the HDL Chlesterl/Chlesterl Rati in Humans Craig N. Sadur and Rbert H. Eckel The rle f insulin in the regulatin f lipprtein chlesterl distributin was studied in 18 human vlunteers wh were f nrmal weight, nrmlipemic, and glucse-tlerant. We used the euglycemic clamp technique and made cmparisns with six saline-infused cntrls. While ttal chlesterl and lw density lipprtein chlesterl levels fell similarly by 6 hurs in bth grups (p = NS), there was a greater decrease in triglyceride levels (p < 0.02) but less decrease in high density lipprtein chlesterl levels (p < 0.02) in the insulin-infused grup. These changes resulted in bth a higher high density lipprtein chlesterl/ttal chlesterl rati (p = 0.01) and a higher high density lipprtein chlesterl/lw density chlesterl rati (p = 0.02) in the insulin-infused grup. The timing f this effect f insulin implies a mechanism unrelated t high density lipprtein turnver. Thus, insulin infusin during euglycemia appears t alter chlesterl distributin favrably in nrmal subjects. (Arterisclersis 3: , July/August 1983) Dwnladed frm by n Nvember 10, 2018 The rle f insulin in the regulatin f lipprtein chlesterl metablism is unclear. In viv, diseases at the extremes f serum insulin cncentratin have been assciated with alteratins in lipprtein chlesterl distributin. In besity, a state where serum insulin is high, there are increases in ttal, very lw density lipprtein (VLDL), and lw density lipprtein (LDL) chlesterl and a decrease in high density lipprtein (HDL) chlesterl. 12 Type I diabetes mellitus, where serum insulin is lw, is assciated with altered lipprtein metablism which nrmalizes with imprved glycemic cntrl. 3 " In fact, sme studies have demnstrated increased levels f HDL chlesterl, 67 HDL chlesterl/ttal chlesterl, r HDL chlesterl/ldl chlesterl ratis 8 in insulin-treated diabetics. Althugh the lipprtein alteratins seen in the insulinpenic patient culd have resulted frm insulin deficiency alne, a direct effect f insulin n lipprtein chlesterl metablism has nt been prven because ther metablic alteratins are als crrected (e.g., ketnemia). Thus, a methd Frm the Divisin f Endcrinlgy, Department f Medicine, University f Clrad Health Sciences Center, Denver, Clrad. Supprt fr this wrk was prvided by Natinal Institutes f Health Grants AM 2636 and RR-0001 t the General Clinical Research Center at the University f Clrad Health Sciences Center. Address fr reprints: Craig N. Sadur, M.D., B11, 4200 East Ninth Avenue, Denver, Clrad t examine the insulin effect in a mre direct manner wuld be beneficial. In the present study, the euglycemic clamp technique has prvided a tl fr assessment f the effect f insulin n lipprtein chlesterl distributin in human subjects. Methds The study grup included 18 subjects wh received intravenus insulin and glucse. Each persn was average in height and within 20% f ideal bdy weight accrding t the Metrplitan Life Insurance Cmpany Standards. 9 The bdy mass index (wt/ht 2 ) was 2.06 x 10" 3 ± 0.0 kg/cm 2 (x ± SEM). The subjects ranged in age frm 20 t 4 years, x = 29.2 ± 1.4 years. There were 1 wmen and three men. The cntrl grup, cmpsed f fur wmen and tw men, received intravenus infusins f 0.9% (nrmal) saline at apprximately 100 t 10 ml/hr. They were als nrmal in height and weight with a bdy mass index f 2.17 x 10~ 3 ± 0.0 kg/cm 2 and with an age range f 23 t 40 years, x = 29.0 ± 2. years. Individuals were excluded if they were taking drugs with knwn effects n glucse r lipid metablism, such as ral cntraceptives, ther estrgenic cmpunds, diuretics, r ther antihypertensive medicatins. All subjects were free f acute r chrnic illnesses. Tw individuals were euthyrid while taking thyrxine fr full thyrid hrmne replacement. Physical examinatins cnfirmed the nrmal 339

2 340 ARTERIOSCLEROSIS VOL 3, N 4, JULY/AUGUST 1983 Dwnladed frm by n Nvember 10, 2018 health f all subjects. Each ingested 40 g/m 2 f glucse and had a nrmal 3-hur ral glucse tlerance curve, accrding t the guidelines f the Natinal Diabetes Data Grup. 10 In additin, all subjects were nrmal in tests fr: tri-idthyrnine resin uptake, ttal thyrxine, thyrid-stimulating hrmne, electrlyte panel, calcium, liver panel, phsphrus, magnesium, cmplete bld cunt, and urinalysis. Fasting serum chlesterl and triglyceride (TG) were nrmal fr all subjects. All studies were apprved by the Human Subjects Cmmittee and were perfrmed in the Clinical Research Center at the University f Clrad Health Sciences Center. Infrmed cnsent was btained frm all participants studied. Fr 2 days befre the study, each subject was fed an iscalric frmula feeding cntaining 4% carbhydrate, 40% fat, and 1% prtein. After an vernight fast, n the mrning f the euglycemic clamp study, tw intravenus lines were placed in ppsite arms f the study subjects, ne fr infusin f fluids and ne fr sampling f bld. A heating pad was placed ver the arm where the samples were btained frm venus bld. Plasma glucse was determined after plasma was immediately separated with a Beckman Micrfuge (Beckman Instruments, Incrprated, Pal Alt, Califrnia) by the glucse xidase technique using the Beckman glucse analyzer (Beckman Instruments, Incrprated, Clinical Instruments Divisin, Fullertn, Califrnia). In the insulin grup, basal plasma glucse values were determined n the mrning f the euglycemic clamp study and served as the standard, determining the varying amunts f glucse infusin needed t maintain euglycemia during the rest f the study. Althugh glucse was similarly determined thrughut the saline infusin, glucse was nt infused. Basal and serial adipse tissue bipsies (at 20,80, 180, and 360 minutes after the beginning f the infusins) were perfrmed fr the measurement f lipprtein lipase activity as previusly described. 11 Mst f these data have been published and will nt be included in the present paper. Insulin was infused int the study subjects in an expnentially decreasing manner ver the first 10 minutes and then at either 40 r 120 mu/m 2 per minute t btain a steady-state serum insulin level f 7 ± r 271 ± 27 ^U/ml, respectively. Glucse infusin was begun at 4 minutes after the start f the insulin infusin. At that pint and thrughut the study, a bld sample was drawn at least every minutes t test fr plasma glucse cncentratin in rder t determine the changes in glucse infusin rate necessary t maintain euglycemia. In additin, these subjects received saline infusins similar t thse received by the cntrl subjects. A ptassium supplement f 30 meq f carbhydrate-free KCI slutin was given t all subjects the evening preceding the study and at apprximately 1. and 3. hurs after the start f the insulin infusin t maintain nrmkalemia. TG was determined by an enzymatic methd. 12 The cefficient f variatin f TG was 8%. Ttal chlesterl was als measured enzymatically 13 with a cefficient f variatin f 4%. HDL chlesterl was measured in plasma frm which VLDL and LDL were precipitated by 4% sdium phsphtungstate. 14 The HDL chlesterl was measured in the supernatant after centrifugatin in a Beckman TJ-6 centrifuge, 100 g fr 30 minutes at 4 C. The cefficient f variatin fr HDL chlesterl, including analytical and precipitatin variatin, was 8%. LDL chlesterl was calculated frm the frmula f Friedewald, et al. 1 Serum insulin levels were measured by a duble antibdy radiimmunassay with the technique f Desbuquis and Aurbach. 16 The Mann-Whitney U test fr unpaired bservatins was used fr statistical analyses. Results Baseline Data The baseline data f bth the 18 study subjects and the six cntrl subjects are displayed in Table 1. Basal plasma glucse values in all subjects ranged frm 71 t 99 mg/dl. Basal lipid determinatins were similar in bth high-insulin (n = 7) and lw-insulin (n = 11) infusin grups. Whereas TG, HDL chlesterl, and HDL chlesterl/ttal chlesterl were similar in bth the insulin and saline cntrl grups, ttal chlesterl and LDL chlesterl were significantly higher in the cntrl grup (p < 0.0) while HDL chlesterl/ldl chlesterl was significantly lwer in the cntrl grup (p < 0.0). These differences became insignificant when the data were matched fr gender. Table 1. Baseline Data Befre Insulin r Saline Infusin Insulin Saline Age (yrs) Weight (kg) ± ±3.3 Wt/Ht 2 x 10" 3 (kg/cm 2 ) Glucse (mg/dl) Ttal chlesterl (mg/dl) LDL chlesterl (mg/dl) TG (mg/dl) HDL chlesterl (mg/dl) HDL chlesterl/ ttal chlesterl (%) 2.06 ± ± ± 74±7 6± ± ± ±11 3± HDL chlesterl/ LDL chlesterl (%) 67± Values are means ± SEM. TG = triglyceride; HDL = high density lipprtein; LDL = lw density lipprtein.

3 Glucse INSULIN INCREASES HDL CHOLESTEROL/CHOLESTEROL Sadur and Eckel 341 Saline infusin resulted in a gradual fall in plasma glucse ver the 6-hur infusin perid t 92% ± 3% f basal. While insulin was infused int study subjects, plasma glucse was maintained by a variable glucse infusin and ranged thrughut the study frm 94% t 103% f basal value. Dwnladed frm by n Nvember 10, 2018 Ttal Chlesterl Fr ttal chlesterl and all remaining lipid measurements, n significant differences between insulin at lw (40 mu/m 2 per minute) and high (120 mu/m 2 per minute) infusin rates were fund. Thus, all insulin data are cmbined measurements fr bth insulin infusin rates. Althugh wmen appeared t have a greater decrease in ttal chlesterl by 6 hurs during bth the insulin infusins (men = -10 ± 2 mg/dl vs wmen = -21 ± 3 mg/dl) and the saline infusins (men = -9 ± 8 mg/dl vs wmen = -21 ± mg/dl), these differences were nt statistically significant. The cmbinatin f men and wmen, as shwn in Figure 1 A, revealed similar 6-hur declines in bth infusin grups (saline = -1 7 ± mg/dl vs insulin = -19 ± 3 mg/dl, p = NS). LDL Chlesterl As shwn in Figure 1 B, there were n significant differences between the tw grups in the decreases in LDL chlesterl levels. Trlglycerldes TG levels decreased mre in the study subjects than in the cntrls (Figure 1 C). The p values fr the differences in TG fr the tw grups at the 80- minute, 180-minute, and 380-minute time-pints were < 0.01, < 0.0, and < 0.02, respectively. There was n gender difference in basal r subsequent TG changes fr either saline r insulin infusins. HDL Chlesterl The change in HDL chlesterl is seen in Figure 1 D. In bth study subjects and cntrls, there was a fall in HDL chlesterl with the infusins, but the insulin-infused grup had n further decrease after the first 80 minutes. Whereas the differences between the tw grups were insignificant at 80-minute and 180-minute time-pints, the difference at 380 minutes was statistically significant (p < 0.02). Again, there was n gender difference between respnses during insulin and saline infusins t B Figure 1. Changes in ttal chlesterl (A), LDI chlesterl (B), triglycerides (C), and HDL chlesterl (D) are pltted by minutes during saline ( ) and insulin ( -) infusins. *p s 0.01, tp < 0.0, tp *= Data are mg/dl (mean ± SEM) Mln 380

4 342 ARTERIOSCLEROSIS VOL 3, N 4, JULY/AUGUST 1983 Dwnladed frm by n Nvember 10, 2018 aj O " O Q I " S -.0 B r 1 80 ISO Mln 380 Figure 2. Changes in the HDL chlesterl/ttal chlesterl rati (A) and HDL chlesterl/ldl chlesterl rati (B) are pltted by minutes during saline ( ) and insulin ( -) infusins. *p s= 0.01, %p ^ Data are shwn as percentages (mean ± SEM). HDL Chlesterl/Ttal Chlesterl When expressed as the rati f HDL chlesterl/ ttal chlesterl, insulin infusin resulted in a higher abslute increase in the rati at 380 minutes when cmpared t saline infusin, 2.8 ± 1.2% vs -1.2 ± 0.8%, respectively (Figure 2 A, p = 0.01). Once again, gender did nt affect the changes that ccurred during the infusins. In men, the 6-hur HDL chlesterl/ttal chlesterl rati changed -2.0 ± 0.0% with saline infusin vs 2.0 ± 1.2% with insulin; in wmen, the change was -0.8 ± 1.3% vs 3.0 ± 1.4%. HDL Chlesterl/LDL Chlesterl As with HDL chlesterl/ttal chlesterl, the study grup had a statistically significant increase in the HDL chlesterl/ldl chlesterl rati by 380 minutes after the start f the infusin (Figure 2 B, p = 0.02). Miscellaneus Insulin-stimulated adipse tissue lipprtein lipase failed t crrelate with changes in chlesterl distributin. There was als n relatinship between the fall in serum triglyceride and the changes in HDL chlesterl. In additin, the amunt f glucse infused did nt predict the changes bserved. Discussin In this study the euglycemic clamp technique has prvided a tl fr examinatin f the effect f insulin n lipprtein chlesterl distributin in 18 nrmal subjects. Because the respnse was similarly affected at the tw insulin infusin rates, 40 and 120 mu/ m 2 per minute, the results frm the tw grups were cmbined. In bth saline-infused and insulin-infused subjects, a prgressive decrease in ttal chlesterl ccurred ver 380 minutes. LDL chlesterl als fell similarly in the tw grups during the same time curse because f a greater decrease in TG levels but a smaller decrease in HDL chlesterl levels in insulin-infused subjects. Sme cautin in the interpretatin f the calculated LDL chlesterl data is needed because the maintenance f VLDL cmpsitin was assumed but nt prven. Because substantial intravenus vlumes were infused in bth grups, a dilutinal effect is likely. The insulin effect n trigycerides, hwever, was mre than dilutinal and has been reprted previusly. 11 Mst imprtant, insulin administratin reduced the decrease in HDL chlesterl level, particularly during the latter part f the infusin. This effect was nt assciated with the insulin-stimulatry effect n adipse tissue lipprtein lipase activity, the decrease in triglyceride level, r the amunt f glucse infused (data nt shwn). Mrever, when the data were expressed as the ratis f bth HDL chlesterl/ttal chlesterl and HDL chlesterl/ldl chlesterl, insulin administratin resulted in higher ratis than thse fund with nrmal saline infusins. Sme investigatrs have felt that these ratis prvide the mst meaningful lipid assessment f antiathergenic risk. 17 ' 18 Because the half-life f ap A-l and ap A-ll is measured in days, 19 the effects f the hrmne n HDL prductin r clearance wuld be unlikely explanatins fr the differences nted between saline and insulin administratin. Pssibly, an effect f insulin n chlesterl transfer between tw lipprtein particles played a rle. In vitr studies have revealed that esterified chlesterl can be transferred frm HDL t VLDL, an effect mediated by a specific transfer prtein. 20 This phenmenn als ccurs with triglyceride transferred frm VLDL t HDL. Bth these transfers are reversible 21 and are prbably independent f each ther. 20 Althugh HDL chlesterl, the HDL chlesterl/ttal chlesterl rati, and the HDL chlesterl/ldl ch-

5 INSULIN INCREASES HDL CHOLESTEROL/CHOLESTEROL Sadur and Eckel 343 Dwnladed frm by n Nvember 10, 2018 lesterl rati, did nt change significantly at the 80- and 180-minute time-pints in the study grup when cmpared t the cntrl grup, the 380-minute timepints revealed significant changes with the insulin infusin, cnsistent with the tempral curse f lipid transfers decribed in vitr. Thus, perhaps insulinmediated increases in the HDL chlesterl/ttal chlesterl and HDL chlesterl/ldl chlesterl ratis culd be due t alteratins in chlesterl transfer t r frm HDL, preventing HDL chlesterl cncentratins frm falling during the infusin perid. The evidence that insulin affects chlesterl metablism in humans is indirect. A favrable effect f insulin n chlesterl metablism is best exemplified by Type I diabetics wh demnstrate a reductin in ttal chlesterl after imprvement f the hypinsulinemic state. 3 " After insulinizatin, lipids return t nrmal. In fact, higher levels f circulating free insulin cncentratins fund in treated Type I diabetics 22 ' M may relate t higher levels f HDL chlesterl 67 r HDL chlesterl/ttal chlesterl 8 fund in these patients. An unfavrable effect f insulin n chlesterl metablism wuld have als been pssible. Fr instance, high carbhydrate diets, which have been assciated with reductins in ttal and LDL chlesterl 242 have als resulted in increases in VLDL 242 and decreases in HDL chlesterl. 24 In additin, bese subjects wh are hyperinsulinemic have increases in ttal chlesterl, VLDL chlesterl, and LDL chlesterl with lwer HDL chlesterl cncentratins than nrmal-weight cntrls. 12 Thus, althugh the present study was a shrt-term ne and invlved a nnphysilgic administratin f insulin, we bserved an antiathergenic influence f insulin n lipprtein chlesterl distributin in nrmal human subjects. This effect was predminantly n HDL chlesterl and suggests that mechanisms independent frm lipprtein turnver may be imprtant in mediating this phenmenn. Acknwledgments We sincerely thank Orville G. Klterman fr insulin radiimmunassays; Debbie Gwinner, Debrah J. Krinitzsky, and Judy Prasad fr technical assistance; and Jni Fti fr secretarial assistance. References 1. Garrisn RJ, Wilsn PW, Castelll WP, Felnlelb M, Kannel WB, McNamara PM. Obesity and lipprtein chlesterl in the Framingham Offspring Study. Metablism 1980;29: Sadur CN, Eckel RH. Insulin-mediated lipprtein metablism in besity. Diabetes 1982;31:16A 3. Pletrl A, Dunn FL, Raskin P. The effect f imprved diabetic cntrl n plasma lipid and lipprtein levels. Diabetes 1980;29: Ssenk JM, Breslw JL, Mlettlnen OL, Gabbay KH. Myperglycemia and plasma lipid levels. N Engl J Med 1980; 302: Eckel RH, McLean E, Alters JJ, Cheung MC, Blerman EL. Plasma lipids and micrangipatny in insulin-dependent diabetes mellitus. Diabetes Care 1981;4: Nikklla EA, Hrmlla P. Serum lipids and lipprteins in insulin-treated diabetes. Demnstratin f increased high density lipprtein cncentratins. Diabetes 1978;27: Klujber L, Mlnar D, Kards M, Jaszai V, Sltesz GY, Mestyan J. Metablic cntrl, glycsylated haemglbin and high density lipprtein chlesterl in diabetic children. Eur J Pediatr 1979; 132: Eckel RH, Albers JJ, Cheung MC, Wahl PW, Llndgren FT, Blerman EL. High density lipprtein cmpsitin In insulindependent diabetes mellitus. Diabetes 1981 ;30: Metrplitan Lite Insurance Cmpany. New weight standards fr men and wmen. Statistical Bulletin 199,40: Natinal Diabetes Data Grup. Classificatin and diagnsis f diabetes mellitus and ther categries f glucse intlerance. Diabetes 1979;28: Sadur CN, Eckel RH. Insulin stimulatin f adipse tissue lipprtein lipase: Use f the euglycemlc clamp technique. J Clin Invest 1982;69: Stavrpuls WS, Cruch RD. A new clrimetric prcedure fr the determinatin f serum triglycerides (abstr). Clin Chem 1974;20: Richmnd W. Preparatin and prperties f a chlesterl xidase frm nrcadia Sp. and its applicatin t the enzymatic assay f ttal chlesterl in serum. Clin Chem 1973;19: Lp«s-Vlrella MF, Stne P, Ellis S, Clwell J A. Chlesterl determinatins in high-density lipprteins separated by three different methds. Clin Chem 1977;23: Frtedewald WT, Levy Rl, Fredrlcksn DS. Estimatin f the cncentratin f tw-density lipprtein chlesterl in plasma, withut use f the preparative ultracentrifuge. Clin Chem 1972:18: Desbuquls B, Aurbach GD. Use f the plyethylene glycl t separate free and antibdy-bund peptide hrmnes in radiimmunassays. J Clin Endcrinl Metab 1971,33: Burslem J, Schnfeld G, Hwald MA, Weldman SW, Miller JP. Plasma apprtein and lipprtein lipid levels in vegetarians. Metablism 1978;27: Wltztum J, Schnfeld G. High density lipprteins. Diabetes 1979;28: Blum CB, Levy Rl, Elsenberg S, Hall M III, Gebel RH, Bercnan M. High density lipprtein metablism in man. J Clin Invest 1977;60: Hpkins GJ, Barter PJ. Transfers f esterified chlesterl and triglycende between high-density and very lw-density lipprteins: In vitr studies f rabbits and humans. Metablism 1980;29: Barter PJ, Lally Jl. In vitr exchanges f esterified chlesterl between serum lipprteins fractins: Studies f humans and rabbits. Metablism 1979;28: Hedlng LG. Determinatin f ttal serum insulin (IRI) in insulin-treated diabetic patients. Diabetlgia 1972;8: Rasmussen SM, Hedlng LG, Parbst E. Serum IRI in insulintreated diabetics during a 24-hur perid. Diabetlgia 197; 11: Falk JM, Schnfeld G, Wltztum JL, Klar JB, Salmn P. Effects f shrt-term high carbhydrate, fat-free diet n plasma levels f ap c-ll and ap c-lll and n ap c subspecies in human plasma lipprteins. Metablism 1980:29: Ginsberg HN, Le N-A, Mellsh J, Steinberg D, Brwn WV. Effect f a high carbhydrate diet n apprtein-b catablism in man. Metablism 1981:30: Index Terms: insulin euglycemic clamp HDL chlesterl chlesterl triglycende