Interaction of very-low-density lipoprotein isolated from type I (insulin-dependent) diabetic subjects with human monocyte-derived macrophages

Interaction of Very-Low-Density Lipoprotein Isolated From Type I (Insulin-Dependent) Diabetic Subjects With Human Monocyte-Derived Macrophages Richard L. Klein, Timothy

J. Lyons, and Maria F. Lopes-Virella

Very-low-density lipoproteins (VLDL) (density < 1 .OD5 g/mL) were isolated from type I (insulin-dependent) diabetic patients in good to fair glycemic control and from age-, sex-, and race-matched, nondiabetic, control subjects. VLDL were incubated with human, monocyte-derived macrophages obtained from nondiabetic donors, and the rates of cellular cholesteryl ester synthesis and cholesterol accumulation were determined. VLDL isolated from diabetic patients stimulated significantly more cholesteryl ester synthesis than did VLDL isolated from control subjects (4.04 f 1 .Ol v 1.99 + 0.39 nmol “C-cholesteryl oleate synthesized/mg cell protein/20 h; mean f SEM. P < .05). The stimulation of cholesteryl ester synthesis in macrophages incubated with VLDL isolated from diabetic patients was paralleled by a significant increase in intracellular cholesteryl ester accumulation (P < .05). The increases in cholesteryl ester synthesis and accumulation in macrophages were mediated by a significant increase in the receptor mediated, high affinity degradation (2.55 2 0.23 v 2.12 2 0.20 pg degradedlmg cell protein/20 h) and accumulation (283 r 35 v 242 + 33 nglmg cell protein120 h) of ‘a51-VLDL isolated from diabetic patients compared with VLDL from control subjects. To determine if changes in VLDL apoprotein composition were responsible for the observed changes in cellular rates of cholesteryl ester synthesis and accumulation, we also examined the apoprotein composition of the VLDL from both groups. There were no significant differences between the apoproteins B. E. and C content of VLDL from both groups. We also determined the chemical composition of VLDL isolated from both groups of subjects. The free cholesterol content of VLDL isolated from diabetic patients increased significantly and, thus, also contributed to the increased rates of cellular cholesteryl ester synthesis and accumulation. In conclusion, we have shown that VLDL isolated from Type I diabetic patients interact abnormally with human macrophages. and this altered interaction may contribute to the increased prevalence of atherosclerosis in diabetes. 0 1989 by W.B. Saunders Company.

T

HERE IS AN INCREASED prevalence of atherosclerosis in patients with diabetes mellitus, and vascular disease accounts for 70% to 80% of the deaths in diabetic patients.le3 Abnormalities of lipoprotein metabolism are intimately related to the development of atherosclerosis, and several abnormalities of lipoprotein metabolism have been described in diabetic patients, mostly when the patients were in poor metabolic control. Most of these abnormalities have been observed using cultured fibroblasts.4 We have shown recently that LDL isolated from type I (insulin-dependent) diabetic subjects in fair-to-good glycemic control stimulated cholesteryl ester (CE) synthesis and accumulation in human macrophages.5 There was a significant correlation between the degree of glycosylation of low-density lipoprotein (LDL) and the increase in CE synthesis. Results from studies using LDL glycosylated in vitro were similar, and the levels of CE synthesis and accumulation were significantly greater in cells incubated with glycosylated LDL than with control LDL.6 However, no studies are available examining the interaction of very-lowdensity lipoproteins (VLDL) isolated from insulin-dependent diabetic patients with human monocyte-derived macrophages. Several investigators have reported the presence of recepFrom the Research Service. Veterans Administration Medical Center, Charleston, SC and the Department of Medicine, Medical University of South Carolina, Charleston. Supported by the Research Service of the Veterans Administration and by the National Heart, Lung and Blood Institute, Grant No. HL-30929. Address reprint requests to Richard L. Klein, PhD, Research Service (1.51). Veterans Administration Medical Center, 109 Bee Street. Charleston, SC 29403. o 1989 by W.B. Saunders Company. 0026-0495/89/381 l-001 2%03.00/O 1108

tors for VLDL isolated from nondiabetic, normolipemic humans on the surface of murine7-9 and human” macrophages. Receptor binding of these lipoproteins probably does little to contribute to the formation of foam cells because little, if any, cellular CE accumulation results from the interaction.*‘-‘4 The macrophage receptor responsible for binding of these VLDL is not apparent; however, a distinct receptor pathway for VLDL isolated from hypertriglyceridemic, human subjects has been shown in murine macrophages.” In contrast, the interaction of macrophages with an altered form of VLDL, &VLDL, results in the exaggerated accumulation of cellular CE.11-‘3Recent studies using humanI and murine’7r’8 macrophages suggest that uptake of /3-VLDL is mediated by the LDL receptor. Kraemer et alI9 have shown recently that VLDL isolated from type II (non-insulin-dependent) diabetic subjects are taken up more avidly by mouse peritoneal macrophages and result in increased intracellular accumulation of CE, although the macrophage receptor responsible for the increased uptake is not evident. Thus, we decided to investigate if VLDL isolated from type I diabetic subjects would interact abnormally with human macrophages and cause intracellular CE accumulation, and if that abnormal interaction, if present, resulted from an altered composition of the VLDL because of the diabetic state. MATERIALS AND METHODS

Subjects

Eleven patients with type I diabetes were recruited from the Private Diagnostic Clinic of the Medical University of South Carolina and were diagnosed clinically by the abrupt onset of dependence on injected insulin to prevent ketosis and to preserve life in accordance with the criteria established by the National Diabetes Data Group. *’ In addition, four patients were qualified for inclusion

Merabolism, Vol39,

No 11 (November), 1989: pp 1108-l

114

1109

MACROPHAGE METABOLISM OF VLDL IN DIABETES

in the Diabetes Control and Complication Trial (DCCT)” at the Medical University of South Carolina, although they did not participate in that study. For every diabetic patient, an age-, sex-, and race-matched nondiabetic control subject was recruited, and blood was drawn on the same day from each member of the matched pair. Of the 11 diabetic patients, 8 performed home blood-glucose monitoring and were asked to provide measurements of blood glucose taken four times daily (before meals) during the week before blood sampling. The mean of these values for each patient was recorded as mean home blood glucose (MHBG). Four patients had evidence of background retinopathy, and two had evidence of neuropathy. None of the patients had proteinuria. Two patients were receiving thyroxine therapy for hypothyroidism, but were clinically euthyroid; two controls were taking diuretics (furosemide or thiazide’t, and one control, a ~-blocking agent. One diabetic patient and her corresponding control were in the third trimester of pregnancy. Additional characteristics of the diabetic and control groups, including weight, age, sex, and race distribution, are shown in Table 1. A 60-mL sample of blood was collected in ethylenediaminetetraacetic acid (EDTA) (1 mg/mL blood) from all subjects after a 12- to 14-hour fast. This sample was used to isolate VLDL for metabolic studres and for the determination of VLDL composition. Fasting plasma glucose, hemoglobin A,,(HbA,,), and fasting lipid profile were determined in all subjects. HbA,, levels were significantly higher in the diabetic subjects (P < .OOl). There were no significant differences in any of the parameters of the lipid profile between normals and diabetics (Table 2). Informed consent, as approved by the Institutional Review Board for Human Research of the Medical University of South Carolina, was obtained from all subjects involved in the study. Protocol Cholesteryl ester synthesis and accumulation were measured in human monocyte-derived macrophages incubated with VLDL from the diabetic subjects and their respective controls. In addition, the VLDL isolated from several subject pairs were radiolabeled with “‘I, and the degradation of “‘1-VLDL apoproteins by human macrophages was determined. For each experiment, the monocytes used were obtained from an individual donor. Different donors were used for different experiments. Matched VLDL samples from diabetic and control subjects were always studied in the same experiment, and each VLDL sample was incubated in duplicate. Lipid composition (free and esterified cholesterol, triglycerides and phospholipids) and apoprotein composition were measured in each VLDL sample. Monocyte Isolation and Maturation Monocytes were isolated from leukapheresis specimens by counter current centrifugal elutriation. *x*~The monocyte preparations were found to be 93% pure by checking morphology on Wright’+stained cytocentrifuge preparations, 92% pure by nonspecific esterase staining, and 93% pure by observing their ability to ingest latex particles.24 The average viability of the cells was 99%. The isolated

Table 1. Clinical Characteristics

Subjects Diabetic In=

Race IS/w)

219

l/10

11)

Control (n =

sex (M/F)

*Mean f SEM (range).

33 f 3* (23 to 53)

219 11)

Age (Vr)

l/IO

34 * 3 (25 to 491

monocytes were transferred to 35mm tissue culture dishes (1 x lo6 cells/dish) and incubated in a specially formulated medium at 37“C in a humidified incubator (5% CO, atmosphere) as described previously.’ The medium was removed and replaced with fresh medium every 3 days to induce maturation. Monocytes were incubated for 8 days before metabolic experiments were begun. VLDL Isolation and Composition VLDL (density < 1.006 g/mL) were isolated from plasma at 10°C by ultracentrifugation at 60,000 rpm for 18 hours in a Beckman 60 Ti rotor (Beckman Instruments, Inc, Palo Alto, CA). The floating VLDL was removed after tube-slicing, and this VLDL solution was washed through a layer of saline solution by ultracentrifugation at 10°C in a SW 41 rotor (Beckman) spun at 40,000 rpm for 24 hours. The floating VLDL were isolated after slicing the tube and were dialysed against 0.9% NaCl, 0.01% weight per volume (wt/vol) ethylenediaminetetraacetic acid (EDTA), pH 7.4. Salt solutions used to adjust solvent densities also contained 0.1% (wt/vol) EDTA, pH 7.4. The VLDL preparations were sterilized by passage through a 0.2 pm filter (Gelman Sciences, Ann Arbor, MI) and stored at 4OC. Samples of isolated VLDL were extracted with chloroform/ methanol (2:l) (vol:v01).*~Their free and total cholesterol contents were determined by gas chromatography as described previously.4 The triglyceride content of isolated VLDL was measured using a semi-automated method standardized by the Lipid Research Clinics Program.26 The phosphorus content of phospholipids was assayed by the method of Bartlett.*’ Protein was determined by the Lowry method,28 using bovine serum albumin (Sigma Chemical Company, St Louis, MO) as a standard. The apoprotein composition of VLDL isolated from both groups was examined using quantitative immunoelectrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). VLDL apoprotein B concentrations were determined using immunoelectrophoresis, with LDL (1.030 < density < 1.050) as the standard.4 We have standardized this assay through our participation in an international collaborative study initiated by the Centers for Disease Control (CDC) and sponsored by the Standardization Committee of the International Union of Immunological Societies (IUIS)?9,‘o Our results compare well with those reported by other investigators with an average coefficient of variation for the assay of less than 9%. The relative proportions of apoprotein E and apoprotein C were estimated by densitometric scanning of apo-VLDL proteins separated by electrophoresis using 12% polyacrylamide gels. VLDL (100 pg) was delipidated three times with 50 volumes of ice-cold ethanoldiethylether (3:1, vol/vol), washed with ether, dried under a stream of nitrogen and solubilized in 0.1 mol/L Tris-HCl, pH 6.7 containing 1% SDS and 5% j3-mercaptoethanol. The samples were heated for 5 minutes at 100°C before loading onto the gels. After electrophoresis (Beckman),“,‘* the gels were fixed and stained overnight in a solution of 50% methanol, 7.5% acetic acid, and 0.1% Coomassie blue G-250. Gels were destained in a solution of 7.5% acetic acid and 5% methanol. The destained gels were scanned in a Beckman scanning densitometer. Densitometric scanning was validated and its

of Diabetic and Control Subjects

BodyMass Index 24.1

+ 1.2

(16.1 to 28.5) 25.0

+ 1.4

118.6 to 33.91

Durationof Diabetes IV) 16 + 3 (5 to 33) -

Insulin (‘J/kg) 0.79

+ 0.13

(0.39 to 1.90)

1110

KLEIN, LYONS, AND LOPES-VIRELLA

Table 2.

Subjects Diabetic

FPG (mg/dL) 238

f 32’

(77 to 432) Control

81 *2$

Parameters MHBG kng/dL) 180 i 20T (88to271) -

(70to91)

of Glycemic Control and Plasma Lipid Levels in Diabetic and Control Groups LipoproteinCholesterol(mg/cU

PlasmaCholesterol (mg/dL)

PlasmaTG Img/dL1

VLDL

183 f 12

103 f 13

32 + 10

(8.5 to 10.5)

(150 to 288)

(47 to 188)

5.6 zt O.l$

192 * 12

114 + 15

(5.3 to 6.0)

(141 to 280)

Hb&, (%) 8.5 + 0.5

(67to211)

(6 to 93) 31 k6 (5 to 57)

LDL 107 f

HDL 11

(65 to 154)

51 *3 (40 to 63)

114 + 9

48 i 3

(86 to 164)

(36 to 68)

Abbreviations: FPG, fasting plasma glucose; MHBG, mean home blood glucose: TG, triglycerides; HDL, high-density lipoproteins. *Mean f SEM (range). TMHBG obtained from eight patients only. They reported an average of 24 blood glucose readings in 7 days. $P < .Ol Ycontrol.

reproducibility was assessed by the scanning linearity observed with gels loaded with different amounts of protein. The concentrations of apoproteins E and C in VLDL were estimated using the following calculations. The concentration of non-apoprotein B proteins (apoproteins E and C) was calculated as the difference between the total protein concentration of VLDL (apoproteins B, E, and C) minus the concentration of apoprotein B in VLDL as determined by immunoelectrophoresis. The relative concentrations in VLDL of apoproteins E and C were estimated by distributing the concentration of the non-apoprotein B proteins in VLDL in proportion to the relative densitometric areas of apoproteins E and C determined by scanning the polyacrylamide gels. Incorporation of (I-“Cl-Oleate

Into Cholesteryl Esters

Cholesteryl ester synthesis was determined after incubation of monocyte-derived macrophages for 20 hours, at 37% with serumfree medium (SFM) containing 0.2 mmol “C-oleate/2.5 mg bovine serum albumin and 100 pg VLDL isolated from diabetic and respective control subjects. After maturation into macrophages, the cells were washed with sterile phosphate-buffered saline (PBS) and were incubated in 1 mL SFM containing ‘%-oleate complexed with bovine serum albumin4 and VLDL from diabetic or control subjects as described previously. The cells were incubated for 20 hours at 37OC, harvested with a rubber policeman using 1 mL PBS, and rinsed with 2 mL of the same solution. The harvesting procedure was repeated twice to minimize cell loss. The cells were pelleted by centrifugation (1,000 x g for 30 minutes), resuspended in 10 mL PBS and centrifuged again to remove any traces of medium. Lipids in the cell pellet were extracted with chloroform/methanol (2:l) (vol/v01).*~Cholesteryl-[‘4C]-oleate was isolated by thin-layer chromatography of the lipid extracts on silica gel plates developed in a solvent system of petroleum ether/ethyl ether/acetic acid (80:20:2) (vol/vol/vol). Lipids were visualized with I, vapor, and the spots that co-migrated with a cholesteryl oleate standard were marked and scraped into scintillation vials after the total disappearance of color. Scintillation fluid (ScintiPrep, Fisher Scientific Co, Fairlawn, NJ) was added and the samples counted in a liquid scintillation counter. Correction for procedural losses was made by adding ‘H-cholesteryl oleate as an internal standard to the chloroform/methanol extraction mixture. After the lipid extraction, the cell pellet was solubilized with 1 mol/L NaOH, and the protein content was determined.” Measurement of Free and Esterijed Macrophages

Cholesterol Content in

To perform these experiments, 3 x lo6 monocytes were plated in 60-mm culture dishes and matured into macrophages as described previously. After maturation into macrophages, the medium was removed, and the cells were washed with PBS to remove any trace of medium containing whole human serum. Serum-free medium containing 100 pg/mL VLDL isolated from normal or diabetic subjects was added to each culture, and the cells were then incubated for 48

hours. After the incubation, the medium was removed and the cells harvested, and cellular lipids were extracted as previously described. Free and total cholesterol in cell lipid extracts were assayed by gas chromatography as previously describerL4 For assay of total cholesterol, the chloroform extracts were evaporated to dryness and the residue hydrolyzed by Ishikawa’s method.‘3 Esterified cholesterol levels were obtained by subtracting free cholesterol levels from total cholesterol levels. Stigmasterol was used as an internal standard. LDL Degradation and Accumulation Studies Aliquots of VLDL isolated from six of the diabetic and control subject pairs were radiolabeled with “‘1 as described previously.4 Macrophages were incubated with lipoprotein-deficient serum (LPDS) (5 mg protein/ml SFM) for 24 hours. The medium was then removed and replaced with 1 mL medium containing LPDS at the same concentration and ‘*‘I-VLDL (10 wg/mL) with or without nonradiolabeled VLDL (250 rg/mL). The proteolytic degradation of ‘2SI-labelled lipoproteins by human macrophages was measured by assaying the amount of “‘1-trichloroacetic acid (TCA)-soluble (noniodide) material formed by the cells and excreted into the culture medium.r4 Rates of total VLDL degradation were determined in incubations containing only “‘1-VLDL and rates of nonspecific degradation were determined in parallel incubations containing a 25-fold excess of nonradiolabeled VLDL. High-affinity degradation rates were calculated as the difference between total and nonspecific degradation rates. Corrections were made for the small amounts of ‘251-labe.lledacid-soluble material that was found in parallel incubations without cells. The intracellular accumulation of %VLDL was determined after removing the medium and washing the cultures as described above. The cells were then dissolved in 1 mL of 1 N NaOH, and the amount of ‘*‘I radioactivity associated with the cells was determined. An aliquot of the cells was taken for determination of cellular protein content as described previously. The data are measurements of ‘251-VLDL that is both surface bound and internalized. Other Methods Home plasma glucose measurements were obtained and recorded by the patients using Dextrostix and a Glucometer (Ames Division, Miles Laboratories, Inc, Elkhart, IN). Plasma glucose was assayed by the glucose oxidase method, as adapted for use in the Beckman glucose analyzer. ” HbA,, was measured by isoelectric focusing of erythrocyte hemolysates in a gradient of pH 6 to 8.r6 HDL was isolated from whole plasma by precipitating VLDL and LDL with sodium phosphotungstate/magnesium chloride as described.” Total cholesterol and triglyceride were measured in whole plasma and lipoprotein fractions by the semiautomated methods standardized by the Lipid Research Clinics Program.26 Lipoprotein-deficient serum was prepared after the ultracentrifugation of plasma at a density of 1.25 g/mL as described previously.4

1111

MACROPHAGE METABOLISM OF VLDL IN DIABETES

Table 3. Cholesterol

Content

of Human Macrophsges

Table 4. Chemical Composition

incubated

With VLDL Isolated From Diabetic Patients and Control Subjects CellularCholesterol(rg/mg cellprotein) Subjwzts

Free

Ratioof Constituents(mass/mass)

Esterified

subjects

ProIFC

Diabetic

65.0

+ 3.9’

16.5 * 1.3t

Diabetic

2.2 + 0.1

Control

64.5

t 4.1

11.1 t 1.3

Control

2.5 f 0.1t

*Mean

of VLDL Isolated From Diabetic

and Control Subjects

GE/Pro l

TG/Pro

PLlPro

1.3 f. 0.2

5.6 + 0.3

2.0 * 0.2

1.2 * 0.2

6.6 + 0.7

2.0 f 0.3

Abbreviations: Pro, protein; FC, free cholesterol; CE. esterified choles-

+ SEM of triplicate determinations on each of four pairs of

terol x 1.7; TG, triglyceride: PL, phospholipid.

subjects. tP < .05 vcontrol.

greater for VLDL isolated from diabetic patients compared with control subjects, and averaged 283 f 35 and 242 + 33 ng accumulated/mg cell protein/20 h (P < .05), respectively. We also determined if changes in VLDL lipid composition, in addition to increased rates of VLDL degradation, may have contributed to the observed increase in rates of CE synthesis and accumulation in macrophages incubated with VLDL from diabetic subjects. As shown in Table 4, there was a significant increase in the free cholesterol content of VLDL isolated from diabetic patients compared with control subjects. To determine if changes in the apoprotein composition of VLDL were responsible for the changes in VLDL degradation rates, we analyzed the apoprotein composition of VLDL isolated from eight pairs of subjects from whom sufficient material was available. The percentage of total VLDL protein present as apoprotein B was similar for VLDL isolated from both groups; the results are shown in Table 5. There also were no significant differences in the percentage composition of apoproteins E and C in VLDL or in the relative amounts of the individual apoproteins of VLDL isolated from either group.

Statistical Analysis Statistical analysis was performed using the mean paired Student’s t test and the Wilcoxon-signed-rank test to compare differences between paired data. 38 All results are expressed as mean f

SEM. RESULTS

VLDL isolated from diabetic subjects stimulated significantly more (P c .05) CE synthesis in human macrophages than VLDL isolated from control subjects. Rates of CE synthesis averaged 4.04 + 1.01 and 1.99 + 0.39 nmol “‘C-cholesteryl oleate synthesized/mg cell protein/20 h in macrophages incubated with VLDL isolated from diabetic and control subjects, respectively. Rates of CE synthesis in cells not incubated with lipoproteins averaged 0.57 + 0.07 nmol “C-cholesteryl oleate/mg/20 h. To determine if the enhancement in CE synthesis by macrophages incubated with VLDL isolated from diabetic subjects was paralleled by an intracellular accumulation of cholesterol, we determined the free and ester&d cholesterol contents of macrophages incubated with 100 bg/mL VLDL protein for 20 hours. There was no significant difference in the free cholesterol content of the cells when incubated with VLDL isolated from diabetic or control subjects (Table 3). In contrast, the esterified cholesterol content of cells incubated with VLDL from diabetic subjects increased significantly (P < .05). To investigate if the increase in rates of CE synthesis and accumulation observed in macrophages incubated with VLDL isolated from diabetic patients resulted from increased VLDL uptake, we investigated the interaction of VLDL with macrophage lipoprotein receptors by determining the rates of degradation of “‘I-control and ‘251-diabetic VLDL. The rate of receptor-mediated degradation was significantly greater for VLDL isolated from diabetic patients compared with control subjects, and averaged 2.55 f 0.23 and 2.12 f 0.20 rg degraded/mg cell protein/20 h (P < .05), respectively. Rates of nonspecific degradation were similar and averaged 0.44 + 0.05 and 0.46 + 0.06 pg/mg cell protein/20 h for control subjects and diabetic patients, respectively. The rate of receptor-mediated accumulation was also significantly Table 5. Apoprotein

Composition

DISCUSSION

The results of the present study suggest that the metabolic behavior of VLDL is significantly altered in type I diabetic subjects. Human macrophages synthesized and accumulated significantly more CE when incubated with VLDL isolated from type I diabetic subjects compared with VLDL isolated from control subjects. These results are of greater importance because they suggest the presence of an alteration in lipoprotein metabolism in type I diabetics, even in patients who are in relatively good glycemic control and whose plasma lipid and lipoprotein levels are normal. The observed increases in rates of CE synthesis and accumulation in macrophages incubated with VLDL isolated from diabetic patients were associated with both an increased uptake and degradation by macrophages of the VLDL isolated from diabetic patients and with a significantly increased free-cholesterol content of the VLDL.

of VLDL Isolated From Diabetic and Control Subjects

Percent subjects

Apo B

npoE

RatioOf Constituents(Mass/Mass) ApoC

Diabetic

50.8

i 2.5*

8.4 f 0.9

41.2

+ 0.9

Control

51.1

k 1.8

10.4 2 1.1

38.4

+ 2.1

B/E

B/C

5.9 * 0.1 4.9 k 0.1

1.2 f 0.1

5.3 rt 0.6

1.3 k 0.1

4.2 + 0.6

Abbreviations: Apo B. B: apolipoprotein B: Apo E, E: apolipoprotein E; Apo C, C: apolipoprotein C.

C/E

1112

Previous studies have reported increased cholesterol in VLDL isolated from both type I and type II diabetic patients.‘9s39 Cholesterol-enriched VLDL isolated from type II diabetic patients were degraded more rapidly by mouse peritoneal macrophages compared with VLDL isolated from nondiabetic, control subjects. I9 The apoprotein composition of the VLDL was analyzed by SDS-PAGE to identify factors responsible for the increased degradation by macrophages of the VLDL isolated from the diabetic patients. We also analyzed the VLDL from both groups of subjects in a similar manner and, in agreement with Kraemer et a1,19found no significant differences in the apoprotein composition of VLDL isolated from both groups. However, the results of Kraemer et al and the present study (Table 5) indicated that there was a proportional decrease in the apoprotein E content of the VLDL isolated from diabetic patients compared with VLDL isolated from control subjects, whereas the apoprotein C content was enriched, although neither difference was statistically significant. Gabor et a14’ have identified similar changes in the apoprotein composition of VLDL isolated from a group of randomly selected diabetic patients. These changes in the apoprotein composition of VLDL from diabetic patients, although subtle, may significantly influence the binding of the VLDL particle to macrophage lipoprotein receptors, Lipoprotein binding to cell receptors is modulated by the relative amounts of apoprotein E to apoprotein C on the particle.4’v42The proportions of VLDL apoproteins change as a function of the size of the VLDL particle. 43,MThus, subtle differences in the size, and therefore apoprotein composition, of VLDL particles isolated from diabetic patients may have influenced the interaction of the particles with macrophage lipoprotein receptors. The affinity of VLDL binding to macrophage lipoprotein receptors also may have been altered due to glycosylation of the VLDL apoproteins. The apoproteins of VLDL isolated from diabetic subjects are more glycosylated than those of VLDL isolated from nondiabetic subjects.45 The apoproteins E and C of VLDL isolated from diabetic patients are more glycosylated than apoprotein B in the particles. Presumably, this results from the transfer of apoproteins E and C to VLDL from HDL, where they had resided for considerably longer periods of time. As a result, these apoproteins are glycosylated to a greater extent than the nonexchangeable apoprotein B in VLDL, which has a much shorter half-life in plasma. Alternatively, these apoproteins may be more susceptible to nonenzymatic glycosylation than apoprotein B. It is probable that the apoproteins E and C of VLDL isolated from the diabetic patients involved in this study were more glycosylated than those of VLDL from control subjects because LDL isolated from a comparable group of diabetic patients was significantly more glycosylated than that of control subjects.5 The effect of increased glycosylation of VLDL apoproteins on the interaction of VLDL with human macrophage lipoprotein receptors is unknown; however, we have shown previously that the degradation by macrophages of LDL isolated from type I diabetic patients is significantly increased compared with LDL isolated from control subjects5 and that uptake of this LDL may be mediated by a low-affinity, high-capacity receptor on the macrophage.6

KLEIN, LYONS, AND LOPES-VIRELLA

Macrophages in culture secrete lipoprotein lipase into the culture medium,46-4* and the lipolytic action of this lipase may have contributed to the altered interaction of VLDL from diabetic patients with cell lipoprotein receptors. Lipolysis of triglyceride-rich lipoproteins by macrophage lipoprotein lipase accelerates the saturable uptake of VLDL by the macrophage.’ The VLDL isolated from diabetic patients were relatively more enriched in apoprotein C compared with VLDL from control subjects (Table 5). Apoprotein C-III is an inhibitor of lipoprotein lipase, whereas apoprotein C-II is an activator.49 The ratio of apoprotein C-III to apoprotein C-II in VLDL is altered in patients with type II diabetes.““,50 Apoprotein analysis in the present study cannot assess changes in the distribution of the C apoproteins; however, the increased apoprotein C content of VLDL from the diabetic patients may have increased the suitability of these VLDL as a substrate for macrophage lipoprotein lipase and thus accelerated the uptake of the particle by macrophage receptors. Human macrophages in culture also secrete apoprotein Es’ in addition to lipoprotein lipase. Recent evidence indicates that changes in apoprotein B expression are required for the binding of apoprotein E to VLDL.52 The surface expression of VLDL apoprotein B epitopes is modified during lipolysis of the VLDL by lipoprotein lipase, and this modified VLDL becomes competent to bind apoprotein E. Thus, diabetic VLDL with their increased apoprotein C content may be more susceptible to lipolysis by macrophage lipoprotein lipase, which would permit the binding of apoprotein E, also of macrophage origin. This macrophage-modified VLDL may then be a more suitable ligand for macrophage lipoprotein receptors, which would result in the observed increased rate of degradation of VLDL isolated from diabetic patients. Clearly, more studies are necessary to investigate these possibilities. In conclusion, we have shown that VLDL isolated from Type I diabetic patients in good to fair glycemic control interact abnormally with human macrophages. This altered interaction results in increased rates of cellular cholesteryl ester synthesis and accumulation. These changes are associated with both an increased rate of degradation by macrophages of VLDL isolated from the diabetic patients and with an increase in the free cholesterol content of these particles. These changes in the cholesterol metabolism of human monocyte-derived macrophages by VLDL isolated from Type I diabetic patients are not of the magnitude reported when these cells are incubated with chemically modified LDL or /3-VLDL. However, this study does show that VLDL isolated from Type I diabetic patients, in addition to LDL,’ may contribute to the increased prevalence of atherosclerosis in diabetes. ACKNOWLEDGMENT

We acknowledge Dr Henry C. Stevenson of the National Cancer Institute, Frederick, MD for graciously contributing monocytes for the conduct of these studies. We thank Larry Long and Virginia Waldrop for their excellent technical assistance and Donna Marvin for skillfully typing the manuscript.

MACROPHAGE METABOLISM OF MOL

IN DIABETES

1113

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