Acute hyperinsulinemia and very-low-density and low-density lipoprotein subfractions in obese subjects

¹ From the Clinical Diabetes Unit, Division of Endocrinology and Diabetology, and the Division for Treatment of Chronic Diseases, University Hospital, Geneva, and the Institute of Physiology, University Hospital, Lausanne, Switzerland.

² Supported by the Swiss National Research Foundation (grant 32.40292.94 to RWJ).

³ Address reprint requests to RW James, Clinical Diabetes Unit, Division of Endocrinology and Diabetology, University Hospital, 24, rue Micheli-du-Crest, 1211 Geneva 14, Switzerland. E-mail: Richard.James{at}hcuge.ch.

ABSTRACT  
Background: The influence of hyperinsulinemia on concentrations of lipoprotein subfractions in obese, nondiabetic persons has not been clarified.

Objective: We analyzed VLDL and LDL subfractions before and after a euglycemic, hyperinsulinemic clamp.

Design: Lipoprotein subfractions were isolated from plasma samples obtained in the basal state and after a 4-h clamp from obese patients, obese patients with type 2 diabetes, and nonobese control subjects.

Results: Hyperinsulinemia tended to reduce concentrations (: 20%) of large, triacylglycerol-rich VLDL₁ in obese patients but had a minor effect on VLDL₂ and VLDL₃. Placing obese patients into insulin-sensitive and insulin-resistant subgroups revealed distinct effects of the degree of insulin sensitivity on VLDL. VLDL₁ concentrations decreased by a mean of 38% (P < 0.05) in insulin-sensitive patients after the clamp, similar to but less marked than the decrease observed in control subjects (: 62%; P < 0.01). VLDL₁ concentrations did not change significantly after the clamp in insulin-resistant patients (and patients with type 2 diabetes), whereas VLDL₃ concentrations decreased in both groups, in contrast with the changes seen in the insulin-sensitive patients and control subjects. Acute hyperinsulinemia modified the LDL subfraction profile toward a greater prevalence of small, dense LDLs in insulin-resistant patients and patients with type 2 diabetes.

Conclusions: Insulin resistance appears to be the primary determinant of the modifications to VLDL subfraction concentrations. Our results suggest a continuum of impaired insulin action on VLDL, ranging from that in healthy persons to that in patients with type 2 diabetes, in which obese patients occupy a transition state. Insulin resistance may also play a role in detrimental modifications to the LDL profile by allowing the development of hypertriglyceridemia.

Key Words: Obesity • NIDDM • type 2 diabetes • VLDL • LDL • atherosclerosis • glucose disposal • hyperinsulinemic clamp • insulin resistance

INTRODUCTION  
Multiple modifications to plasma lipoproteins are evident in obese individuals, notably those with an accumulation of abdominal fat (1). The most frequent modifications are hypertriglyceridemia (increased VLDL) and decreased HDL cholesterol. These changes reflect in part modifications to the distribution profiles of the major lipoprotein subclasses. Such qualitative alterations are poorly defined in obese patients, although reported modifications include an increased proportion of large VLDL particles and a greater predominance of smaller, denser LDLs and HDLs (2, 3). Studies in other populations suggest that these changes are of both metabolic and pathophysiologic importance (4–6).

Insulin is considered to be of primary importance in regulating plasma VLDL concentrations (7, 8), but the precise nature of its influence remains controversial. Hypotheses include hyperinsulinemia-mediated stimulation of VLDL secretion (9) and defective down-regulation of VLDL secretion as a result of insulin resistance (7). Two studies showed down-regulation of VLDL secretion during acute hyperinsulinemia in healthy, normal-weight men (10, 11). In other studies of diabetic patients, results were discordant, with contrasting reports of VLDL down-regulation by hyperinsulinemia (12) and defective action of insulin (13). However, in neither of these studies were the subjects obese [ie, subjects had a mean body mass index (in kg/m²) <30]. One study performed in obese, nondiabetic subjects suggested partially defective action of insulin on VLDL metabolism (14). The possible effect of insulin on VLDL subfractions in obese individuals has not been analyzed. However, in healthy, normal-weight subjects, insulin action was reported to result in a preferential decrease in large, triacylglycerol-rich VLDL particles as a result of lower secretion (11).

The principal aim of the present study was to analyze the effect of acute hyperinsulinemia on well-characterized VLDL subfractions (15) in obese, nondiabetic subjects. We also studied a normal-weight (control) group and obese patients with type 2 diabetes in view of the discordant results concerning diabetic patients. In addition, LDL subfractions were analyzed during a hyperinsulinemic clamp with particular reference to the prevalence of small, dense LDL particles.

SUBJECTS AND METHODS  
Subjects
Obese patients with and without type 2 diabetes were recruited from outpatient clinics at University Hospital, Geneva. Normal-weight subjects were recruited from among the personnel of the hospital. Informed consent was obtained from all participants and the study was conducted according to the requirements of the Ethical Commission of the Medical Faculty, University of Geneva.

Participants were given an oral-glucose-tolerance test (75-g glucose load) performed according to the recommendations of the World Health Organization (16) and were classified as follows: normal (fasting glucose <6.1 mmol/L, 2-h oral-glucose-tolerance test <7.8 mmol/L), glucose intolerant (fasting glucose >6.1 but <7.0 mmol/L, 2-h oral-glucose-tolerance test >7.8 but <11.1 mmol/L), and having type 2 diabetes (fasting glucose >7.0 mmol/L, 2-h oral-glucose-tolerance test >11.1 mmol/L). Of the patients with type 2 diabetes, 7 were treated by diet alone and 7 with oral, antidiabetic drugs (5 with sulfonylureas and 2 with metformin). The diabetic patients were in good metabolic control ( ± SEM glycated hemoglobin: 7.8 ± 0.2%; normal range: 3–6%). The subjects' percentages of body fat were measured as described previously (3).

Euglycemic, hyperinsulinemic clamp
Clamp studies were performed as described by De Fronzo et al (17). After subjects had fasted overnight (10–12 h), an intravenous catheter (Venflon; Viggo-Spectramid, Helsinborg, Sweden) was inserted into an antecubital vein for glucose and insulin infusion. A second catheter was placed in a contralateral vein for blood sampling and was kept patent with isotonic saline. Insulin was infused at a continuous rate of 6 mg•kg^-1•min^-1. Glucose (20%) infusion was varied to maintain a plasma concentration (measured every 5 min) of 5.0 mmol/L. Insulin concentrations during the clamp (4 h) were those measured during the last 30 min. Glucose disposal was that measured during the last 30 min.

Lipoprotein subfractions
For lipoprotein analyses, 2 blood samples were obtained before (basal) and after the hyperinsulinemic clamp (4 h). These were used to isolate 3 VLDL subfractions (VLDL₁, S_f: 100–400; VLDL₂, S_f: 60–100; and VLDL₃, S_f: 20–60) and 3 LDL subfractions (LDL₁, S_f: 12–20; LDL₂, S_f: 6–12; and LDL₃, S_f: 3–6) by cumulative flotation ultracentrifugation, as described in detail previously (15, 18). The nomenclature is that originally defined by Lindgren et al (15). VLDL₁ and VLDL₂ correspond to large particles, which some laboratories do not differentiate and refer to collectively as VLDL₁ (eg, as in reference 11). VLDL₃ corresponds to small-diameter VLDL particles and is referred to in some publications as VLDL₂. LDL₂ and LDL₃ occupy the density range attributed to LDL by conventional ultracentrifugation; LDL₂ represents particles larger in diameter and LDL₃ represents smaller, denser particles [as extensively characterized by Lindgren et al (15)]. The total protein and individual lipid (triacylglycerol, phospholipid, and free and esterified cholesterol) contents were quantified and subfraction concentrations are expressed as the sum of the concentrations of the individual components (18, 19) (interassay CVs of 5–8% depending on the subfraction).

Analytic measures
Blood glucose, insulin, and fatty acids were measured with commercially available kits as described previously (20). Lipids were measured by automated procedures with commercially available kits (19). Esterified cholesterol was calculated as follows: (total cholesterol - free cholesterol) x 1.68. Apolipoprotein (apo) A and apo B were quantified by immunoturbidometry with commercially available kits (Bayer Diagnostics, Tournai, Belgium). The interassay CVs for apo A-I and apo B were 4.7% and 5.6%, respectively.

Statistical analyses
Data are expressed as means ± SEMs. Statistical comparisons were made with paired (within group) and unpaired (between group) t tests and by analysis of variance (ANOVA). Variables with skewed distributions were logarithmically transformed. Spearman coefficients were used for correlations between continuous variables; the determinants of the decrease in VLDL during the clamp and the concentration ratios of LDL₂ to LDL₃ were analyzed by multivariate stepwise logistic regression. Analyses were performed with the STATVIEW statistical package (Version 4.0; Abacus Concepts Inc, Berkeley, CA).

RESULTS  
Demographic and clinical data for the 3 groups of subjects are given in Tables 1 and 2. Seven of the subjects in the obese group (5 men and 2 women) were glucose intolerant. The normal-weight (control) group was younger than the obese groups but there was no significant difference in mean age between the 2 obese groups. Plasma concentrations of cholesterol, triacylglycerol, and apo A-I were not significantly different among the 3 groups. In contrast, HDL-cholesterol concentrations were significantly lower (to a similar extent) in the 2 obese groups than in control subjects. Fatty acids were not significantly different between the groups, although concentrations tended to be higher in obese patients and in patients with type 2 diabetes. Hyperinsulinemia was not significantly different between groups during the clamp. However, insulin sensitivity, as defined by glucose disposal, differed significantly between the groups, with a graded decrease from the control subjects to the obese patients to the patients with type 2 diabetes.

View this table:
TABLE 1.. Demographic and clinical characteristics of control subjects, obese patients, and obese patients with type 2 diabetes¹  

View this table:
TABLE 2.. Plasma lipid concentrations of control subjects, obese patients, and obese patients with type 2 diabetes before (basal) and after the euglycemic, hyperinsulinemic clamp¹  
After the clamp, plasma triacylglycerol concentrations decreased significantly in the control subjects (Table 2). Fatty acid concentrations also decreased significantly and to a similar extent in all 3 groups. There were minor, nonsignificant decreases in total cholesterol in all 3 obese groups. A comparison (ANOVA) between groups of the changes (initial – final value) in triacylglycerol concentrations showed significantly greater changes in the control subjects (P < 0.005) and obese patients (P < 0.005) than in the diabetic patients, but no significant difference between the control and obese groups. Changes in cholesterol were also significantly different between the control and obese groups (P = 0.05) and between the control and diabetic groups (P = 0.05) but not between the obese and diabetic patients. The decrease in apo B was significantly different between obese and diabetic patients (P = 0.04) but not between control subjects and diabetic patients.

VLDL subfractions
Changes to VLDL subfraction concentrations during the hyperinsulinemic clamp are illustrated in Figure 1. There was a 20% decrease in triacylglycerol-rich VLDL₁ concentrations (NS) in obese patients whereas changes in VLDL₂ and VLDL₃ averaged <8%. There was no significant change in plasma concentrations of any of the VLDL subfractions in diabetic patients, although VLDL₃ tended to decrease (by 17%). In contrast, there were significant decreases in the VLDL₁ and VLDL₂ subfractions in control subjects but no significant changes in the smallest VLDL subfraction, VLDL₃.

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FIGURE 1. . Mean (±SEM) concentrations of VLDL₁, VLDL₂, and VLDL₃ in fasting plasma samples (basal) and in samples after a euglycemic, hyperinsulinemic clamp (clamp) from control subjects (n = 10), obese patients (n = 19), and obese patients with type 2 diabetes (n = 14). ^**,***Significantly different from basal (ANOVA): ^**P < 0.05, ^***P < 0.01.

 
Obese patients were examined in greater detail by subdividing them according to their insulin sensitivity. (Subgrouping according to glucose tolerance gave broadly similar results to those described below.) The characteristics of the insulin-sensitive and insulin-resistant patients are given in Table 3. As the defining characteristic, glucose disposal differed significantly between the 2 subgroups. Fasting blood glucose and waist-to-hip ratio also differed between the 2 subgroups. Differences in body mass index were of borderline significance (P = 0.06). Shown in Figure 2 are the VLDL subfraction concentrations before and after the hyperinsulinemic clamp. There was a significant (: 38%) decrease in plasma concentrations of the largest, most triacylglycerol-enriched subfraction (VLDL₁) in the insulin-sensitive subgroup, but no significant changes in VLDL₂ or VLDL₃. In insulin-resistant patients, concentrations of VLDL₁ and VLDL₂ did not change significantly, but concentrations of the small, dense VLDL₃ decreased (by 15%; P < 0.05).

View this table:
TABLE 3.. Demographic and clinical characteristics of obese insulin-sensitive (IS) and insulin-resistant (IR) patients¹  

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FIGURE 2. . Mean (±SEM) concentrations of VLDL₁, VLDL₂, and VLDL₃ in fasting plasma samples (basal) and in samples after a euglycemic, hyperinsulinemic clamp (clamp) from obese insulin-sensitive (IS; n = 10) and insulin-resistant (IR; n = 9) patients. ^*Significantly different from basal, P < 0.05 (ANOVA).

 
The decrease in plasma VLDL₁ concentrations (analyzed as the percentage reduction) was correlated in a highly significant, positive manner with insulin sensitivity (combined groups: r = 0.61, P < 0.0001) and the percentage decrease in fatty acids (r = 0.44, P < 0.005). When further analyzed by forward stepwise multiple regression (Table 4, model A), glucose disposal (insulin sensitivity) was the predominant determinant of the decrease in VLDL₁. Neither the decrease in fatty acids nor any indicator of obesity was significantly associated with the decrease in triacylglycerol-rich VLDL₁. This model accounted for 47.9% of the variation in the VLDL₁ concentration changes.

View this table:
TABLE 4.. Analysis by stepwise logistic regression of determinants of the decrease in VLDL₁ concentrations during hyperinsulinemia (model A) and of the variations in the basal concentration ratios of LDL₂ to LDL₃ (model B)¹  
The percentage decrease in VLDL₃ in the combined insulin-resistant and diabetic groups was significantly correlated with insulin sensitivity alone (P < 0.01) in a stepwise regression analysis. A model containing insulin sensitivity (glucose disposal), weight-to-hip ratio, and sex best explained 32% of the variations in VLDL₃ concentrations.

LDL subfractions
Analyses of changes in the lipoprotein subfractions within the LDL density range were limited to the subfractions LDL₂ and LDL₃ [representing large and small, dense LDLs, respectively, as documented by Lindgren et al (15)]. The ratio of their concentrations is an indication of the prevalence of small, dense LDLs in plasma. These ratios in plasma before and after the hyperinsulinemic clamp are shown in Figure 3. The ratio was higher in the basal plasma of control subjects and insulin-sensitive patients than in that of insulin-resistant and diabetic patients (query> ± SEM: 3.39 ± 0.46 compared with 2.17 ± 0.35 in the combined groups; P < 0.05). With acute hyperinsulinemia, the ratios remained stable in the control and insulin-sensitive subjects (3.45 ± 0.49). In contrast, the ratios decreased in the insulin-resistant and diabetic patients (1.78 ± 0.29; P < 0.05).

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FIGURE 3. . Mean (±SEM) ratios of LDL₂ to LDL₃ in fasting plasma samples (basal) and in samples after a euglycemic, hyperinsulinemic clamp (clamp) from control subjects (n = 10), obese insulin-sensitive patients (IS; n = 10), obese insulin-resistant patients (IR; n = 9), and obese patients with type 2 diabetes (n = 14). ^*Significantly different from basal in the combined IR obese and diabetic groups, P < 0.05.

 
Stepwise logistic regression models were constructed to analyze determinants of the basal ratio of LDL₂ to LDL₃. As shown in Table 4, model B, the predominant factor influencing the prevalence of small LDL particles was the plasma triacylglycerol concentration. This corresponds to observations made by numerous groups, including our laboratory (21–23), of an increased prevalence of small, dense LDLs with a rise in plasma triacylglycerol. HDL cholesterol was also independently associated with LDL size. The model accounted for 39.8% of the variation in the ratio of LDL₂ to LDL₃. Neither glucose disposal (as a measure of insulin resistance) nor plasma fatty acids were independent predictors of LDL size.

DISCUSSION  
The present investigation is the first to show the consequences of acute hyperinsulinemia at the lipoprotein subfraction level and the modulating effect of insulin resistance in obese, nondiabetic subjects. Concentrations of large, triacylglycerol-rich VLDL particles decreased in obese patients, but this decrease was limited to those patients who retained a degree of insulin sensitivity. VLDL₁ concentrations did not change significantly in insulin-resistant obese patients. Results in the insulin-sensitive obese patients resembled those in the control subjects except that there was a lesser degree of down-regulation of VLDL₁ concentrations and no effect on VLDL₂ concentrations in the patients; ie, effects were limited to the largest, most triacylglycerol-rich VLDL particles. The data for the obese insulin-resistant subjects resembled those for obese patients with type 2 diabetes. Thus, it appears that even in insulin-sensitive obese subjects (who are admittedly more insulin-resistant than control subjects), the action of insulin is somewhat compromised.

In an earlier study, Lewis et al (14) reported a significant decrease in total VLDL triacylglycerol in obese, glucose-tolerant women during acute hyperinsulinemia and a lesser, nonsignificant decrease in total VLDL apo B. Their observation of a decrease in the ratio of VLDL triacylglycerol to apo B led them to propose that VLDL size was decreased. We show that this decrease in the ratio is in fact due to the influence of insulin on a defined VLDL subfraction.

Our results in the control subjects agree with those of Malmström et al (11), who showed that a hyperinsulinemic, euglycemic clamp leads to lower concentrations of triacylglycerol-rich VLDLs as a result of decreased secretion of the lipoprotein. Our results also confirm earlier studies showing down-regulation of total VLDL by insulin in healthy subjects (10, 12). Our data from patients with type 2 diabetes support the contention by Malmström et al (13) of impaired insulin action in diabetic patients. This contrasts with the conclusions of a preceding study (12) that reported down-regulation of VLDL in patients with type 2 diabetes. The reasons for this discrepancy are not clear, although patient selection may be a factor. Notably, our patients were similar to those of Malmström et al (13) in the degree of hypertriglyceridemia (: <2.0 mmol/L) compared with the substantially raised concentrations (: 3.5 mmol/L) reported in the study by Cummings et al (12).

In contrast with the lack of effect of insulin on large VLDLs, we observed a significant decrease in small VLDL particles in the combined insulin-resistant and diabetic groups during acute hyperinsulinemia. This is a novel observation that substantiates indications of a tendency toward reduced synthesis of small VLDL particles in patients with type 2 diabetes as noted by Malmström et al (13). This finding may also explain the discrepant results mentioned above. In their study, Cummings et al (12) did not subfraction VLDL to define more precisely the size of the particles sensitive to hyperinsulinemia. It is possible that small VLDL particles were responsible for the decrease they reported, especially because no change in the ratio of VLDL triacylglycerol to apo B was observed, which would argue against a decrease in large, triacylglycerol-rich VLDLs.

Our study appears to place obese subjects between the extremes represented by healthy subjects and diabetic patients with respect to VLDL and insulin action. The modulatory influence appears to be that of insulin resistance, not obesity, as indicated by the multivariate analysis. Previous reports postulated such a modulatory effect of insulin resistance on insulin action (7, 14, 24, 25). Thus, despite equivalent reductions in plasma fatty acids in obese insulin-resistant and diabetic patients, concentrations of large VLDLs were not affected. It can be argued that obese insulin-resistant and diabetic patients have more important reservoirs of those hepatic triacylglycerol pools that contribute primarily to VLDL triacylglycerol synthesis (26, 27) and that these reservoirs were not exhausted during the hyperinsulinemic clamp. However, in the present study, down-regulation of VLDL1 was observed in the similarly obese patients who retained a measure of insulin sensitivity. Nevertheless, fatty acid availability can attenuate the effect of insulin, as shown in healthy subjects (10, 28). These considerations do not, however, alter the potential clinical consequences of impaired, prompt down-regulation of hepatic VLDLs, which can cause exaggerated postprandial lipemia as a result of competition between VLDL and intestinally derived lipoproteins. Impaired down-regulation of hepatic VLDL can also lead to delayed catabolism of particularly atherogenic lipoprotein particles [chylomicron remnants (29–31)] and establish conditions whereby detrimental compositional or structural changes to LDL and HDL are facilitated by accelerated triacylglycerol–cholesteryl ester exchange (32–34).

The reduction in VLDL₃ in the obese insulin-resistant and diabetic patients is an intriguing observation. In contrast with large VLDL, which is linked to triacylglycerol metabolism, small VLDL is suggested to be more closely associated with cholesterol metabolism (35). Cholesterol synthesis is reportedly enhanced in diabetic patients (36, 37) and in a previous study we noted a higher concentration of free cholesterol in lipoprotein subfractions of obese subjects (3). It is therefore possible that reduced VLDL₃ concentrations reflect an effect of acute hyperinsulinemia on cholesterol synthesis.

Acute hyperinsulinemia had a subtle effect on the distribution of LDL subfractions. The general tendency for an increased prevalence of small, dense LDL evident in basal plasma samples from insulin-resistant and diabetic patients was intensified after the clamp, with a significant decrease in the ratio of LDL₂ to LDL₃. Previous studies related LDL size to insulin resistance (38, 39), but our results suggest that this link is not independent of plasma triacylglycerol concentrations. This agrees with other reports (23, 39, 40) and suggests that it is the hypertriglyceridemia of insulin resistance that drives the modifications to LDL size rather than insulin resistance per se. Thus, one can envisage a physiologic scenario in which postprandial hypertriglyceridemia, exaggerated notably in type 2 diabetes, will favor enrichment of LDL with triacylglycerol while the concurrent hyperinsulinemia stimulates lipases [presumably hepatic lipase (41, 42)] thought to be responsible for modifying LDL size. Given the extensive data indicating the atherogenic potential of small, dense LDLs (5, 43, 44), the lower ratio of LDL₂ to LDL₃ after the clamp in the present study represents a deterioration of the LDL profile. We were unable to measure lipoprotein lipase or hepatic lipase activity in the present study. However, previous studies using stable-isotope techniques to differentiate effects on VLDL synthesis and catabolism during hyperinsulinemia did not observe any significant modifications of VLDL catabolic rates (13, 36). This is consistent with other studies suggesting that skeletal muscle lipoprotein lipase is primarily involved in triacylglycerol hydrolysis (45). Insulin treatment does not modify muscle lipase in diabetes although it increases adipose tissue lipoprotein lipase (46, 47).

The results of the present study are consistent with a continuum of impaired insulin action on VLDL, ranging from that in healthy subjects to that in patients with type 2 diabetes, with obese patients in a transition phase. Insulin resistance is the principle modulating factor in this continuum. Our study confirms the divergent action of insulin on large and small VLDL particles, which is consistent with the metabolic differences between these particles, and identifies a potential effect of insulin on small VLDL particles. Finally, the present study shows detrimental changes to the LDL subfraction profile during hyperinsulinemia in patients perhaps predisposed by ineffective insulin action at the VLDL level.

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