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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1805-1811.)
© 1995 American Heart Association, Inc.

Articles

Effect of Diabetes on Lipoprotein Size

Amareshwar T.K. Singh; David L. Rainwater; Steven M. Haffner; John L. VandeBerg; Wendy R. Shelledy; Perry H. Moore, Jr; Thomas D. Dyer

From the Department of Genetics, Southwest Foundation for Biomedical Research, and the Division of Clinical Epidemiology (S.M.H.), Department of Medicine, University of Texas Health Science Center, San Antonio, Tex.

Correspondence to David L. Rainwater, PhD, Department of Genetics, Southwest Foundation for Biomedical Research, PO Box 28147, San Antonio, TX 78228-0147.

	Abstract

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Abstract The effects of diabetes on lipoprotein particle sizes were assessed using samples from 94 subjects with non–insulin-dependent diabetes mellitus. From a larger population of nondiabetic subjects who showed normal glucose tolerance, we selected an exact match in terms of age, sex, and menopausal status. We designed a protocol to make nondenaturing gradient gels for the resolution of LDL subfractions and generated two measures of LDL size: diameter of the predominant LDL species and proportion of LDL cholesterol (LDL-C) in particles larger than 25.5 nm (large LDL-C). Similarly, we made two measures of HDL size, large HDL cholesterol (HDL-C) and large HDL–apoAI, which represent the proportion of HDL-C and apoAI, respectively, occurring on particles larger than HDL₃. In pairwise comparisons, diabetes was associated with significantly (P<.004) smaller lipoprotein particles for all measures except large HDL-C. Each of the size measures was significantly and positively correlated with each of the others, suggesting that common metabolic mechanisms influence lipoprotein particle sizes across classes of lipoproteins. In addition, each of the size measures was correlated with a variety of measures of HDL and ß-lipoprotein concentrations, which included HDL-C, LDL-C, triglycerides, and apoAI, apoB, and apoE. We used stepwise regression analyses to select from the measures of lipoprotein concentrations those independently correlated with each of the lipoprotein size measures. After adjusting for these metabolic correlates of lipoprotein size measures, we found the effect of diabetes on lipoprotein size measures was no longer significant except for a modest effect (P=.027) on large HDL–apoAI. These results suggest that diabetes alters aspects of lipoprotein metabolism that result in modification of lipoprotein particle sizes.

Key Words: electrophoresis • lipoproteins, low-density • apolipoproteins • lipoproteins, high-density • diabetes

	Introduction

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LDL particles are the major carriers of plasma cholesterol in humans. LDL exhibits substantial heterogeneity based on measures of size, density, and chemical composition.¹ Krauss and Burke² identified, with the use of analytical procedures such as gradient gel electrophoresis and density gradient ultracentrifugation, many distinct subclasses of LDL in plasma samples from healthy humans. Austin et al^{3 4} categorized LDL particles into two types, denoted the A and B patterns. The A pattern is characterized by the predominance of large, buoyant LDL particles and the B pattern by small, dense LDL particles; the point of separation between the two patterns is approximately 25.5 nm. Subjects with B-pattern LDL tended to have atherogenic lipoprotein profiles, including higher levels of triglycerides, VLDL and IDL mass, and apoB, and lower levels of apoAI and HDL cholesterol (HDL-C).^{5 6} In addition, small, dense LDL was associated with an increased risk for myocardial infarction.³ LDL subclass phenotype is influenced genetically,^{4 5 7 8} but a number of environmental factors, such as sex, age,^{6 9} diet,¹⁰ drugs,¹¹ obesity,¹² hormones,^{13 14 15} and diabetes mellitus^{16 17 18} also are reported to influence LDL size phenotype.

A similar atherogenic lipoprotein profile is observed in insulin resistance syndrome,¹⁹ or syndrome X.²⁰ Insulin resistance syndrome also features a host of other coronary heart disease risk factors, such as hypertension, obesity, body-fat distribution, and glucose tolerance. LDL from diabetic patients shows increased heterogeneity with a tendency toward smaller, B-pattern particles.^{17 18 21 22} The accumulation of B-pattern particles in diabetic subjects may be dependent on underlying changes in triglyceride metabolism.^{6 21 22} In addition, HDL levels and particle sizes are reduced in diabetic patients,^{23 24} which also may be related to elevated triglyceride concentrations.²⁵

In the present study, we developed a method for casting nondenaturing polyacrylamide gradient gels that enabled us to compare LDL particles and other lipoprotein measures in diabetic and nondiabetic Mexican Americans matched for age and sex.

	Methods

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Subjects
The San Antonio Family Heart Study is a study of risk factors for cardiovascular disease in a Mexican-American population.²⁶ We identified 94 participants from this study who were diagnosed with non–insulin-dependent diabetes mellitus using World Health Organization criteria. Among the 94 diabetic research subjects, 41 were taking no medications for diabetes at the time of the clinical visit. Of those taking medications, 31 were taking oral agents and 22 were taking insulin, either with or without oral agents. Duration of diagnosed diabetes in these subjects averaged 5.4 years (SD, 7.4; range, 0 to 31.9 years). For each diabetic individual, we identified from the same study population an unrelated nondiabetic individual who had normal glucose tolerance (ie, plasma glucose levels less than 7.77 mmol/L 2 hours after a 75.0-g glucose challenge) and who matched exactly for age, sex, and menopausal status. Procedures were approved by the Institutional Review Board of the University of Texas Health Science Center, San Antonio, and all subjects gave written informed consent. Blood samples were drawn after an overnight fast and again 2 hours after administration of a 75-g glucose load (Orangedex, Custom Laboratories). Plasma was isolated by low-speed centrifugation and stored as individual aliquots at -80°C in plastic tubing segments.²⁷ Age, sex, and menopausal status were ascertained during the clinic interview. Weight and height were measured in light clothing without shoes, and body mass index was calculated as weight divided by height squared (in kilograms per meter squared).

Chemical Analyses
In fasted samples and in those taken after glucose administration, we measured the concentrations of glucose by use of an Abbott V/P Analyzer and insulin by use of radioimmunoassay-based kits (Diagnostic Products Corp). The remaining traits were measured only in the fasting plasma samples. Plasma cholesterol and triglyceride concentrations were determined by commercially available enzyme-based assays (Boehringer Mannheim and Stanbio) with the use of a Gilford SBA-300 clinical chemistry analyzer. The dextran sulfate–Mg²⁺ precipitation procedure²⁸ was used to precipitate apoB-containing lipoproteins before quantifying HDL-C; non–HDL-C was calculated as the difference between total cholesterol and HDL-C concentrations. The interassay coefficients of variation for control products in these assays were 1.7% for cholesterol, 6.6% for HDL-C, and 3.2% for triglycerides. Apolipoprotein concentrations were measured in a commercial laboratory (Medical Research Laboratories). ApoAI and apoB concentrations were determined by nephelometry.^{29 30 31} ApoE and apoAII concentrations were determined using competitive immunoassays.^{32 33} The interassay coefficients of variation for control products in these assays were 3.5% for apoAI, 4.4% for apoAII, 2.9% for apoB, and 8.1% for apoE.

Making LDL Gels
Nondenaturing 3% to 18% polyacrylamide gradient gels (LDL gels) were cast on the basis of a modification of published protocols.³⁴ Table 1 gives the characteristics of the 3% to 18% gel gradient.

View this table: [in this window] [in a new window]   Table 1. Characteristics of the Gradient Segments Used in the Production of LDL 3% to 18% Gradient Gels

The stock solutions, which were made up in TBE buffer ([in mmol/L] Tris 90, boric acid 81.5, disodium EDTA 2.5 [pH 8.35]), included the following (in g/L): solution 1: acrylamide 172.8, bis-acrylamide (N-N'-methylene-bis-acrylamide) 7.2 (18% total, 4.0% cross-linker), and sucrose 50 (all from Bio-Rad Laboratories) and solution 2: acrylamide 28.8 and bis-acrylamide 1.2 (3% total, 4% cross-linker). After these solutions were filtered through a sintered glass funnel, they were stored at room temperature and used within 1 month. The high-limit solution was made immediately before the gradient was cast and contained 1 vol solution 1, 0.0015 vol freshly prepared ammonium persulfate (100 g/L; Bio-Rad), and 0.00025 vol 3-dimethylaminopropionitrile (Sigma Chemical Co). The low-limit solution was also made immediately before the gradient was cast and contained 1 vol solution 2, 0.0046 vol ammonium persulfate, and 0.00066 vol 3-dimethylaminopropionitrile. The gradient was generated with a Wiz dual-pump gradient controller system (Isco), mixed in an external mixing chamber, and cast into a GSC-8 gel slab casting apparatus (Pharmacia) as described.³⁴

Gradient Gel Electrophoresis
The gels were prerun in TBE buffer at 120 V for 20 minutes with a GE-2/4 electrophoretic chamber (Pharmacia). Samples were made dense with sucrose, and a volume that contained 4 µL plasma was loaded on the gels. Each gel was calibrated by use of the following: (1) Pharmacia high-molecular-weight standards containing thyroglobulin (17.0-nm diameter) and several other proteins not used for calibration, including ferritin, catalase, and lactate dehydrogenase; (2) carboxylated latex microspheres (38 nm, Duke Scientific); and (3) two bands of LDL in a lyophilized plasma sample, with diameters of 27.5 nm and 26.6 nm. Diameters of the two LDL bands in the standard were estimated by P.J. Blanche, Lawrence Berkeley Laboratory, Berkeley, Calif, using data from 20 different gels that were calibrated with standards 1 and 2 above and the locations of three consistently occurring bands of ß-lipoproteins in a plasma lipoprotein standard used in their laboratory (unpublished data, 1994). Electrophoresis was initiated by applying voltage to the chamber in the following sequence: 15 V for 15 minutes, 70 V for 20 minutes, and 125 V for 24 hours (3000 V · h). After electrophoresis was completed, the gels were presoaked in 50% ethylene glycol monoethyl ether solution (Cellosolve, Sigma) for 1 hour, stained overnight (16 hours) with 11.0 g/L Sudan black B (Sigma) in 50% ethylene glycol monoethyl ether, and then destained with multiple changes of 50% ethylene glycol monoethyl ether for a total of approximately 8 hours.³⁵ Because the molecular-weight standard proteins do not stain with Sudan black B, Coomassie brilliant blue R-250 (Sigma) was used to stain the lower part of the gel containing these proteins, and gels were destained in 50% methanol, 10% acetic acid, and 40% water.³⁶ After destaining was completed, gels were soaked in the TBE buffer to restore gel size and shape before scanning.

Densitometry and LDL Phenotyping
The gels were subjected to densitometric scanning at 632.8 nm with an LKB-Ultroscan XL laser densitometer with GELSCAN XL software. Gels were calibrated for size using the migration distance (R_f) of each standard relative to thyroglobulin; a quadratic equation in relative migration distance was fit to the natural logarithms of the diameters of the standards: ln(diameter)=C₀+C₁R_f+C₂ R_f², where C₀, C₁, and C₂ are the calibration coefficients.³⁷ We developed a computer program in house that automatically calibrated each gel, subtracted the baseline, and calculated particle diameter for the predominant peak in each sample lane. In addition, the program determined fractional absorbance for Sudan black B–stained LDL particles in five size intervals⁹ : LDL-I (26.4 to 29.0 nm), LDL-II (25.5 to 26.4 nm), LDL-III (24.2 to 25.5 nm), LDL-IV A (23.2 to 24.2 nm), and LDL-IV B (21.0 to 23.2 nm). For analyses, however, we summed fractional absorbances for LDL particles larger than 25.5 nm, and these are called large LDL cholesterol (LDL-C). Most samples were assayed twice; repeatability for the estimate of particle diameter was 92.7% (n=163) and of large LDL-C was 71.1% (n=114).

HDL Size Phenotypes
HDL size phenotypes were measured as described before.³⁸ Briefly, lipoproteins in plasma were separated on the basis of size with nondenaturing 3% to 31% polyacrylamide gradient gels.³⁴ To detect size distributions of apoAI, we transferred proteins to nitrocellulose paper electrophoretically, and we used immunoblotting procedures with a radioiodinated secondary antibody to measure the distributions of apoAI.^{38 39} Locations of radioactivity were detected by use of autoradiography and quantified by use of densitometry with an LKB-Ultroscan laser densitometer with GSXL software. The distributions of cholesteryl esters among the HDL subclasses were detected by staining with Sudan black B and measured by densitometry.^{40 41} HDL absorbance profiles were analyzed by fitting curves representing the generally accepted HDL subclasses, as suggested previously.⁴² Curves were fit to HDL_3c (7.2 to 7.7 nm), HDL_3b (7.8 to 8.2 nm), HDL_3a (8.2 to 8.8 nm), HDL_2a (8.8 to 9.7 nm), HDL_2b (9.7 to 12.9 nm), and HDL₁ (ie, HDL particles larger than 12.9 nm). The component curves gave a summed absorbance profile that very closely matched each observed absorbance profile; the average correlation coefficient value, r², was .9977 (SD, ±.0016; n=186) for apoAI and .9891 (SD, ±.0191; n=188) for cholesteryl esters. The fractional absorbances for HDL₁, HDL_2b, and HDL_2a were summed to calculate the percent of apoAI or cholesteryl esters on HDL particles larger than those of HDL_3, and they are called large HDL-apoAI and large HDL-C, respectively.

Statistical Methods
Statistical procedures were conducted using a software package (Manugistics). To reduce skewness, apoE and triglyceride concentrations were transformed to their natural logarithms before analyses.

	Results

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Comparisons of Diabetic and Nondiabetic Subjects
The average age of subjects in the study group was 49.4 years (range, 25 to 81 years), and there were nearly twice as many women as men (61 versus 33). Although the study group subjects were somewhat older, the characteristics of subjects in the study group matched the characteristics of the parent population (61% female; mean age, 40.1 years; range, 15 to 94 years; n=1203). Table 2 shows other characteristics of the study population. As we expected, the diabetic individuals had significantly higher levels of fasting insulin and glucose and of glucose 2 hours after a 75-g glucose load. None of the lipoprotein measures were significantly different between the two groups with the exception of the concentrations of triglycerides and apoE, both of which were higher in the diabetic group. When these analyses were repeated for female and male subjects separately, we found that the same variables were significant, except that there was no longer a significant effect of diabetes for body mass index and apoE in male subjects (data not shown).

View this table: [in this window] [in a new window]   Table 2. Characteristics of Diabetic and Nondiabetic Subjects Matched for Age, Sex, and Menopausal Status

Effects of Diabetes on LDL Size Measures
Table 3 shows significant differences (by paired t test) between the two groups for two measures of LDL size. Diabetic individuals had smaller particles (mean, 25.8 nm) and relatively less stain in particles larger than 25.5 nm (mean, 58.6%) than did the age- and sex-matched nondiabetic individuals (means were 26.2 nm and 67.7%, respectively). Fig 1 shows frequency histograms of LDL peak diameters for the nondiabetic and diabetic groups, and Fig 2 shows the effect of diabetes on fractional distributions of cholesteryl esters among size-resolved LDL subfractions. When LDL particles from male and female subjects were analyzed separately, the trends were the same, but there was a significant effect of diabetes only for female subjects (Table 3).

View this table: [in this window] [in a new window]   Table 3. Effect of Diabetes on Four Measures of Lipoprotein Size for Sex- and Age-Matched Sample Subjects1

View larger version (17K): [in this window] [in a new window]   Figure 1. Frequency histograms for LDL particle diameter distributions in nondiabetic and diabetic individuals. LDL subclasses in plasma were resolved on the basis of size by use of nondenaturing gradient gel electrophoresis and stained with Sudan black B. LDL peak migrations relative to those of standards were used to estimate predominant particle diameters.

View larger version (23K): [in this window] [in a new window]   Figure 2. Graph showing the effect of diabetes on distribution of cholesteryl esters among LDL subclasses. LDL particles in plasma were resolved on the basis of size as in Fig 1. Presented are means and SEM for fractional absorbance for each of five LDL subclasses in nondiabetic (hatched bars) and diabetic (solid bars) subjects.

Effects of Diabetes on HDL Size Measures
HDL size phenotypes were estimated as the percent of stain in HDL particles larger than HDL₃. Diabetic individuals tended to have a smaller proportion of apoAI (39.1%) and of cholesteryl esters (44.8%) in the larger particles than did nondiabetic individuals (42.3% and 46.0%, respectively). This was a significant difference (P=.003) only for the anti-apoAI–stained particles (Table 3). Fig 3 presents the effect of diabetes on mean distributions of apoAI among HDL size classes for nondiabetic and diabetic subjects. There remained a significant effect of diabetes on apoAI distributions, but not cholesteryl ester distributions, when tested in female and male subjects separately (Table 3). Thus, diabetes was associated with relatively smaller HDL and LDL particles.

View larger version (23K): [in this window] [in a new window]   Figure 3. Effect of diabetes on distribution of apoAI among HDL subclasses. HDL subclasses in plasma were resolved on the basis of size by use of nondenaturing gradient gel electrophoresis, as described in the text. Presented are means and SEM for fractional absorbance for each of six HDL subclasses in nondiabetic (hatched bars) and diabetic (solid bars) subjects.

Intercorrelation of Lipoprotein Size Measures
Table 4 presents univariate correlations of lipoprotein measures, both of concentration and of size, for the four size measures. All but six were significantly correlated at the P<.001 level. The lipoprotein concentration measures showed patterns of correlation that were similar for each of the size measures. Concentration measures of ß-lipoproteins were negatively correlated with the four size measures. In each case, triglycerides were the strongest correlate, followed in general by apoE, apoB, and non–HDL-C. Concentration measures of HDL were positively correlated with the four size measures. In each case, HDL-C was the strongest correlate, followed by apoAI; apoAII concentrations were not significantly correlated with any of the size measures in the univariate analyses. The lipoprotein size variables were strongly intercorrelated (Table 4). Although correlations were stronger for the two measures of LDL and the two measures of HDL, all correlations were positive and significant. The pattern of the correlations in Table 4 was almost exactly repeated, in terms of sign and rank order, in the two subsets of diabetic and nondiabetic individuals (data not shown).

View this table: [in this window] [in a new window]   Table 4. Univariate Tests for Correlations With Four Lipoprotein Size Variables

Independent Effects of Diabetes on Lipoprotein Size Measures
We used stepwise regression analyses to select lipoprotein concentration measures that were significantly correlated with each of the lipoprotein size variables. Multiple regression analyses that included the effects of the significant correlates showed diabetes had a modest (P=.027) effect on the proportion of larger HDL particles stained for apoAI. However, diabetes had no independent effect on any of the other lipoprotein size variables after adjustment for the effects of correlated lipoprotein concentration measures (Table 5).

View this table: [in this window] [in a new window]   Table 5. Effect of Diabetes on Lipoprotein Size Variables After Adjustment for Effects of Correlated Lipoprotein Concentration Measures

	Discussion

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To understand the effect of diabetes on measures of lipoprotein phenotypes, we compared pairs of diabetic and nondiabetic individuals from the San Antonio Family Heart Study who were matched for age, sex, and menopausal status. Compared with nondiabetic populations, patients with diabetes have a twofold higher risk for cardiovascular disease.⁴³ The common occurrence of risk factors in diabetic patients such as hypertension and obesity, in addition to age, smoking, and hypercholesterolemia, does not completely explain the increased morbidity and mortality from cardiovascular disease.⁴⁴ Among other factors that could contribute, alterations in lipoprotein composition and concentration may be among the most important.

Different sizes of LDL have been resolved with the application of gradient gel electrophoresis.^{2 6} We adapted published protocols for casting nondenaturing gradient gels to separate on the basis of size LDL particles in plasma samples. We found that diabetic subjects had significantly smaller dominant LDL peak diameters as analyzed by paired t test as well as by ANOVA. Similarly, diabetic subjects had a lower fraction of cholesteryl esters in LDL particles larger than 25.5 nm. When each sex was considered separately, diabetic subjects had relatively smaller particles and less large LDL-C than nondiabetic subjects, although the differences were significant only for female subjects. In their population-based study, Haffner et al¹⁶ reported a greater effect of diabetes on LDL size in women than men. Although in general women are at lower risk for cardiovascular disease than men, it has been demonstrated in some studies^{43 45} that women are prone to a greater incidence of cardiovascular disease associated with non–insulin-dependent diabetes mellitus, suggesting a greater adverse influence of diabetes on lipoproteins in women.⁴⁶

Diabetes is marked by characteristic alterations in lipoprotein levels, including an elevation of triglycerides and VLDL and a decreased HDL concentration.⁴⁷ In addition, it appears that small and dense LDL subclass patterns are more common in diabetic patients, as seen in the present study and other studies.^{16 21 22 48 49} This observation is important because of the association of smaller LDL particles and the LDL subclass pattern B phenotype with increased risk of developing cardiovascular disease.⁴ Abbott et al⁵⁰ demonstrated that abnormalities in lipoproteins have a physiological link with the action of insulin. Alterations in the action and concentrations of insulin play a crucial role in the regulation of lipoprotein metabolism in non–insulin-dependent diabetes mellitus. Barakat et al²² demonstrated a correlation of both insulin and triglyceride concentrations with LDL particle size. They reported that with a decrease in the levels of plasma insulin and triglyceride, LDL particle sizes may revert to the normal state, suggesting that such a change might contribute to a reduction in risk for cardiovascular disease in patients with non–insulin-dependent diabetes mellitus. Haffner et al⁵¹ and Ferrannini et al⁵² provided evidence that the contributions of compensatory hyperinsulinemia and insulin resistance are a major cause of diabetic dyslipidemia. Yet to be resolved are the mechanisms that modify LDL size distribution phenotype in diabetic patients. A possible explanation is that the small, dense LDL results directly from the remodeling of certain VLDL subfractions and are genetically determined.^{5 53} The actions of lipid transfer proteins may be another possibility.^{54 55 56} Facilitated transfer of triglycerides into LDL might take place in the event of hypertriglyceridemia; the subsequent hydrolysis of triglycerides, probably by hepatic lipase,⁵⁷ may give rise to smaller and denser LDL particles.

In addition to differences in LDL particle sizes, we found that diabetic individuals tended to have a smaller proportion of large HDL particles stained for apoAI (ie, larger than HDL₃) than did nondiabetic subjects. Although the proportion of large HDL-C was lower in diabetic than nondiabetic subjects, the difference was not significant. This result suggests compositional differences among HDL size subfractions and is consistent with the findings of Taylor et al⁵⁸ and Joven et al.⁵⁹ All the lipoprotein size measures in the present study were significantly and positively intercorrelated, confirming a previous observation.³⁸ The lipoprotein size measures also were negatively correlated with measures of ß-lipoprotein concentrations and positively correlated with measures of HDL concentrations. These extensive intercorrelations suggest that the different measures reflect common metabolic processes. To determine whether diabetes had a specific effect on lipoprotein size, we first identified by stepwise regression analysis all the lipoprotein concentration correlates for each of the four measures of particle size. These correlates were then incorporated into multiple regression models to test whether diabetes had a significant effect on particle size after adjustment for the metabolic correlates. We found diabetes had no significant effect on any LDL measure and only a modest (P=.027) effect on HDL size as shown by staining for apoAI. Therefore, although we detected dramatic effects of diabetes on several measures of lipoprotein sizes, the data suggest the effects are on general metabolic processes rather than being specific to particle size.

Several mechanisms may be involved in determining the lipoprotein profile of a diabetic patient. An understanding of how a variation in genes affects the expression of characteristic lipoprotein abnormalities in diabetes is just beginning to evolve. Meanwhile, it is reasonable to believe that mechanisms such as variations in the genes coding for apolipoproteins or for the enzymes involved in lipolytic processes may mediate abnormalities in the metabolism of lipoproteins in the diabetic patient.

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein HDL = high-density lipoprotein HDL-C = high-density lipoprotein cholesterol IDL = intermediate-density lipoprotein LDL = low-density lipoprotein LDL-C = low-density lipoprotein cholesterol VLDL = very-low-density lipoprotein<\/.>

	Acknowledgments

 
We thank Allen Ford and Jane VandeBerg for technical assistance and Patricia J. Blanche, Lawrence Berkeley Laboratory, Berkeley, Calif, for determining the diameters of the two LDL bands in the lyophilized human plasma standard. This work was supported by National Health Institutes grant HL-45522.

Received June 13, 1995; accepted August 25, 1995.

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