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Department of Clinical and Experimental Medicine, Federico II University Medical School, 80131 Naples; and Novo-Nordisk (M.I., G.A.C.), 00144 Rome, Italy
Address all correspondence and requests for reprints to: Dr. Angela A. Rivellese, Department of Clinical and Experimental Medicine, Federico II University Medical School, Via S. Pansini 5, 80131 Naples, Italy. E-mail: nmcd{at}unina.it
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Abstract |
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Introduction |
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We have already shown that, independently of blood glucose control, insulin therapy compared with glibenclamide is associated with greater decreases in plasma triglyceride, and large very low density lipoprotein (VLDL) subfractions, increases in the smallest VLDL particles, increases in the high density lipoprotein-2 (HDL2) fraction, and no change in total low density lipoprotein (LDL). All of these effects are accompanied by a significant decrease in the postheparin plasma hepatic lipase (HL) activity without any variation in plasma lipoprotein lipase (LPL) activity (5).
As triglyceride levels and HL activity are considered major determinants of LDL subfraction distribution (6, 7), it is possible that the reduction of triglyceride, even if small, and/or that of HL induced by sc insulin therapy can lead to a change in the distribution of LDL subfractions, despite the lack of change in total LDL.
Considering the importance of LDL subfraction distribution with the smallest particles proposed to be more atherogenic (8, 9, 10, 11, 12), the evaluation of possible effects of insulin therapy on LDL composition are important for the understanding of optimal therapy for type 2 diabetic patients. Furthermore, an examination of the effects of insulin could provide information on the regulation of LDL subfraction distribution in type 2 diabetic patients.
Therefore, the aim of this study was to evaluate the effects of both insulin and sulfonylurea therapy, at comparable levels of blood glucose control, on LDL subfractions in type 2 diabetic patients without hyperlipidemia.
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Subjects and Methods |
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A detailed description of patient selection and study design has been published (5). In summary, nine men (age, 56 ± 3 yr; body mass index, 26.5 ± 0.9 kg/m2) with noninsulin-dependent diabetes (WHO criteria) (13), normolipidemic (plasma cholesterol, 5.5 ± 0.3; plasma triglyceride, 1.3 ± 0.2 mmol/L; mean ± SEM), and in satisfactory blood glucose control while taking sulfonylureas (10–15 mg/day) participated in the study. After a 3-week wash-out period during which oral hypoglycemic drugs were discontinued, patients were randomly assigned to insulin or glibenclamide therapy for 2 months and were then crossed over to the other treatment. Insulin or glibenclamide doses were adjusted on the basis of daily blood glucose profiles performed by patients at least twice a week during the first month of each treatment. Doses (insulin, 0.25 U/kg BW·day; glibenclamide, 12.2 mg/day; average dose) were kept constant during the second month, and the target level of blood glucose control was the same for each period. At the end of each treatment period, blood samples were taken for determination of lipoprotein profile (LDL and VLDL) and lipases activities.
The study was approved by the ethical committee of Federico II University, and each patient gave informed consent to participate.
Laboratory procedures
Lipoprotein separation. Blood samples were collected at the end of each treatment period, after an overnight fast (12–14 h), by vein puncture without stasis and were allowed to clot. Serum was separated by low speed centrifugation (3000 rpm for 10 min) and added with ethylenediamine tetraacetate-Na2 (final concentration, 0.05%).
Total LDL were isolated at a density of 1.063 g/mL by sequential preparative ultracentrifugation under standard conditions (14). Three LDL subfractions of increasing density and decreasing size were separated by a discontinuous density gradient preparative ultracentrifugation procedure, according to the method described by Griffin et al. (15) and modified by Tilly-Kiesi et al. (16). Tubes were centrifuged for 24 h at 23 C in a SW40 Ti rotor (Beckman Coulter, Inc., Palo Alto, CA) at 20 C on a Centrikon T2060 ultracentrifuge (Kontrol Instruments, Zurich, Switzerland) with operating mode preselection keys set at vertical on-off. After centrifugation, the tubes were emptied from the top using an ISCO density gradient flow cell and fractionator system, a WIZ Peristaltic pump, and a Fluorinert FC-40 solution (density, 1.85 g/mL) and an absorbance detector UA-6 (ISCO, Inc., Lincoln, NE). Densities were checked by a Digital Density Meter DMA-48 (Anton Paar, Graz, Austria). Three LDL subfractions of increasing density and decreasing size (large density, 1.024–1.032 g/mL; intermediate density, 1.032–1.040 g/mL; small density, 1.040–1.060 g/mL) were collected in a volume of 1.5 mL each by a Retriever II fraction collector (ISCO, Inc.). The reproducibility of the density gradient fractionation procedure was assessed by replicate analysis on a LDL sample. The coefficients of variation for large, intermediate, and small LDL for a duplicate sample (within rotor) were 4.0%, 4.3%, and 5.1%, respectively.
VLDL subfractions were isolated by density gradient ultracentrifugation as previously described in detail (5).
Other measurements. Total cholesterol, triglycerides, and phospholipids were assayed on serum, isolated lipoproteins, and their subfractions by enzymatic colorimetric methods (17, 18, 19, 20) using commercially available kits (Roche Molecular Biochemicals, Mannheim, Germany), adequately modified to obtain a high sensitivity at low concentrations, on a COBAS MIRA autoanalyzer (Roche, Basle, Switzerland). Quality control of lipid analysis is regularly ensured in our laboratory by the WHO Prague Reference Center (21). The recovery of the total LDL (sum of each lipid concentration in LDL subfractions as a percentage of the concentration in total LDL) was 94 ± 3.1% for cholesterol, 91 ± 2.7% for triglycerides, and 95 ± 2.2% for phospholipids without a difference between the two treatments. Coefficients of variation for lipid assays were below 3%.
LPL and HL activities were evaluated according to the method of Nilsson-Ehle (22). Briefly, blood samples were collected in tubes containing ethylenediamine tetraacetate, 15 min after iv heparin administration (100 IU/kg BW). Plasma was immediately separated by centrifugation at 4 C and stored at -25 C. Coefficient of variations were 6.3% and 2.4% (intraassay) and 8.9% and 4.6% (interassay) for LPL and HL, respectively.
Statistical analysis
Data are expressed as the mean ± SEM unless otherwise specified. Variables that were not uniformly distributed were log-transformed before statistical analysis. Statistical analysis was performed according to standard methods (23) using the Statistical Package for the Social Sciences (SPSS, Inc., Chicago, IL) software. The data from each treatment were compared using Student’s t test for paired data. Simple correlation between changes obtained with the two treatments were also performed. P < 0.05 was considered as statistically significant (two-tailed).
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Results |
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). LDL cholesterol, triglyceride, and
phospholipids as well as LDL total lipid concentration were similar
after the two treatment periods (Table 2). However, the two treatments induced
different effects on LDL subfractions (Fig. 1). Cholesterol (24.3 ± 2.0
vs. 19.6 ± 1.9 mg/dL; P < 0.05,
insulin vs. glibenclamide), phospholipids (14.8 ± 1.7
vs. 11.9 ± 1.7 mg/dL; P < 0.006), and
total lipid (44.5 ± 3.6 vs. 36.5 ± 3.7 mg/dL;
P < 0.02) concentrations of large LDL were
significantly higher at the end of the insulin therapy than at the end
of the glibenclamide period. On the other hand, the total lipid
concentration of small LDL tended to decrease (59.0 ± 9.8
vs. 76.1 ± 16.8 mg/dL; P = NS) after
insulin therapy, but this difference did not reach statistical
significance; the same happened for their lipid constituents;
cholesterol (32.2 ± 5.9 vs. 43.5 ± 10.1 mg/dL;
P = NS), triglyceride (6.3 ± 2.5 vs.
7.1 ± 2.9 mg/dL; P = NS), and phospholipids
(20.5 ± 10.7 vs. 25.5 ± 17.8 mg/dL;
P = NS; Fig. 1). Intermediate LDL did not change. The
different effect on large and small LDL subfractions led to a
significantly different distribution profile after insulin therapy
compared with that after glibenclamide. In fact, expressing each LDL
subfraction lipid concentration (cholesterol plus triglyceride plus
phospholipids) as a percentage of the total LDL lipid concentration,
there was an increase in the largest particles (25.3 ± 2.3%
vs. 20.7 ± 2.1%; P < 0.03) and, on
the other hand, a significant percent decrease in the smallest ones
(31.2 ± 3.0% vs. 38.3 ± 3.8%;
P < 0.03; Fig. 2).
Since, as reported previously (4), insulin also induced a
significant reduction in plasma triglyceride (0.9 ± 0.1
vs. 1.1 ± 0.1 mmol/L; P < 0.05),
large VLDL (26.5 ± 3.0% vs. 37.8 ± 3.4%;
P < 0.02), and HL (247.2 ± 22.3 vs.
263.5 ± 22.6 mU/mL; P < 0.05; all important
determinants of the LDL subfraction profile), we sought to determine
possible correlations between the above changes and those in LDL
subfractions, all expressed as percent variations between values after
insulin and glibenclamide treatments. The percent change in HL after
insulin therapy was directly associated with the decrease in the
smallest LDL (r = 0.69; P < 0.05) and, inversely,
with the increase in the largest ones (r = -0.79;
P < 0.02). The percent change in plasma triglyceride
was not significantly related to any of the changes observed in the LDL
subfraction profile, whereas decreases in the largest VLDL subfraction
during insulin therapy correlated positively with decreases in the
small LDL particles (r = 0.67; P < 0.04; Fig. 3).
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Discussion |
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The data from our study are based on a small number of patients; these findings need, therefore, to be extended to other groups of diabetic patients. However, all of the patients were normolipidemic, and they have quite low triglyceride levels (average, 1.3 mmol/L), which are generally considered to be associated with a low proportion of small LDL (24). Therefore, it is possible to hypothesize that in patients with higher levels of plasma triglyceride and a preponderance of small LDL particles, as is the case of most type 2 diabetic patients with unsatisfactory blood glucose control, the effects of insulin on LDL subfraction distribution might be much more pronounced. A more relevant modification in LDL subfraction distribution could easily also induce a change in LDL size, which represents the mean particle diameter of the major LDL subclass. This, in fact, has been demonstrated previously when type 2 diabetic patients with poor glycemic control had a decrease in small LDL subfractions after 3 months of optimized blood glucose control with insulin (25).
The results obtained in this study also provide information on the control of LDL subfractions. The distribution profile of LDL subfractions is the result of complex metabolic mechanisms, whose principal determinants are 1) plasma triglyceride levels and VLDL particles, especially the largest ones, which regulate, together with the cholesterol ester transfer protein activity, the formation of large LDL, more or less enriched in triglyceride (6, 24, 26, 27); and 2) the activity of lipolytic enzymes, especially HL, which hydrolyze triglyceride of large LDL, converting them into smaller and denser particles (28). In our study two factors were associated with changes in the distribution of LDL subfractions obtained after insulin therapy. On the one hand, changes in the percentage of large VLDL were significantly associated with the reduction in small LDL, which means that the greater the decrease in large VLDL with insulin, the greatest the reduction in the small LDL particles (r = 0.67; P < 0.04). Also, the percent variation in HL with insulin was positively correlated with small LDL changes, which means that the larger decrease in HL was associated with greatest reduction in the small LDL (r = 0.69; P < 0.05). Thus, insulin therapy causes two complementary metabolic changes. It reduces the larger VLDL concentration; therefore, a smaller amount of large, triglyceride-enriched LDL was synthesized, and as a consequence, a lower amount of small LDL was produced. At the same time, the decrease in HL activity obtained with insulin therapy reduced the conversion of large LDL to the smallest ones, with a decrease in the latter and an increase in the former. Moreover, our results suggest that even minimal variations in HL and VLDL are able to change significantly the LDL subfraction pattern. Finally, they indicate that both changes in large VLDL concentration and HL activity are important even when plasma triglyceride levels are below 1.5 mmol/L, which is considered by some authors the threshold triglyceride level above which there is a rapid increase in the formation of small LDL particles (6, 26). Kinetic studies have shown that acute insulin infusion reduces the synthesis of large VLDL in healthy subjects (29). Our data suggest that chronically insulin also acts to reduce these particles. Moreover, insulin administered sc may induce a relative hepatic hypoinsulinization, which explains the reduction in HL found in type 1 and type 2 diabetic patients receiving insulin therapy (5, 30).
In our study the changes in total triglycerides were not significantly associated with changes in LDL subfraction distribution. This is at variance with data present in the literature (28) and might be due to the low triglyceride levels of our patients and to the high variability of triglyceride measurement (31).
In conclusion, sc insulin therapy compared with sulfonylureas, independently of variations in blood glucose control, induces an improvement in LDL subfraction distribution with a shift toward a less atherogenic profile (decrease in small dense LDL) in type 2 diabetic patients even with low levels of plasma triglycerides.
The effect of sc insulin therapy on the LDL subfraction profile is the opposite of that on VLDL subfractions, inasmuch as an increase in the smallest, more atherogenic particles has been shown (5) for the latter. However, this possible negative effect may be negligible from a clinical point of view when optimal blood glucose control is reached (32).
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Acknowledgments |
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Footnotes |
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Received April 4, 2000.
Revised July 11, 2000.
Accepted July 14, 2000.
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