| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Clinical and Experimental Medicine (A.A.R., C.D.N., L.D.M., L.P., C.I., S.C., G.R., G.A.), Federico II University, 80131 Naples, Italy; and Department of Endocrinology and Metabolism, University of Pisa (S.D.P.), 56124 Pisa, Italy
Address all correspondence and requests for reprints to: Dr. Angela A. Rivellese, Department of Clinical and Experimental Medicine, Medical School, Federico II University, Via S. Pansini 5, 80131 Naples, Italy. E-mail: rivelles{at}unina.it.
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
A more precise characterization of postprandial lipid metabolism in type 2 diabetic patients with normotriglyceridemia and optimal blood glucose control is of clinical relevance, because postprandial lipid abnormalities may help to explain, at least in part, the increased cardiovascular risk observed in these patients.
Many factors may be involved in the genesis of postprandial lipid abnormalities in type 2 diabetic patients (1, 2, 9). An impairment in the activity of lipoprotein lipase (LPL), the key enzyme in the catabolism of both exogenous and endogenous triglyceride-rich lipoproteins, has been considered of importance (10). However, although a reduction of LPL activity in type 2 diabetic patients and in general in patients with insulin resistance, has been reported under fasting conditions, the role of LPL activity during the postprandial period has not been fully elucidated (10, 11).
Therefore, the aim of our study was to evaluate the plasma lipid response to a standard fat-rich meal, differentiating the exogenous and the endogenous lipoprotein particles, in a group of type 2 diabetic patients with optimal fasting triglyceride levels and optimal blood glucose control. A further specific aim of our study was to evaluate in depth the possible role of LPL in modulating the postprandial lipid response. This was performed by measuring the basal plasma activity of LPL throughout the entire postprandial period as well as the plasma LPL activity after heparin stimulation 6 h after the meal.
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Seven patients with type 2 diabetes mellitus (12) (five men and two women) treated with diet (n = 6) or diet and sulfonylurea (n = 1) and five nondiabetic controls (one man and four women) participated in the study. The baseline characteristics (Table 1
) of the diabetic patients and controls were: age, 49 ± 7 and 48 ± 4 yr; and body mass index, 28.3 ± 3.6 and 25.1 ± 3.6 kg/m2. All subjects had normal fasting plasma concentrations of triglyceride below 150 mg/dl (<1.7 mmol/liter) and cholesterol below 212 mg/dl (<5.5 mmol/liter) and hemoglobin A1c levels less than 6.8%.
|
3/3 genotype. Three of six diabetic patients had an apo E 2/3 genotype, and three had genotype 3/3. As basal plasma lipids and postprandial lipid responses were similar in the patients with 2/3 and 3/3 genotypes, data were pooled for statistical analysis. The study protocol was approved by the Federico II University ethical committee, and informed consent was obtained from the participants. Experimental procedures
In the morning after at least 12 h of fasting, subjects were administered a standard mixed meal consisting of a potato gateau (a pie made of mashed potato, whole milk, egg, cheese, ham, and butter). The meal, which provided 944 kcal, was composed of 31% carbohydrate, 57% fat (34% saturated fat), and 12% protein. Before the meal and over the 6 h after the meal, serial blood samples were taken for determination of plasma levels of glucose, insulin, lipids, lipoproteins, apo B-48, apo B-100, free fatty acids (FFA), and LPL activity. At the end of the study, heparin (100 U/kg body weight) was injected iv, and a blood sample was obtained after 15 min for LPL and hepatic lipase (HL) activities measurement.
Laboratory procedures
Lipoprotein separation. Samples were kept at 4 C before, during, and after centrifugation. To minimize proteolytic degradation of apo B, 1.0 µl 10 mM phenylmethylsulfonylfluoride (Sigma-Aldrich Chemie, Steinheim, Germany) dissolved in isopropanol and 5 µl aprotinin (Trasylol, Bayer, Leverkusen, Germany) were added for each 1 ml plasma. Fasting and postprandial lipoprotein subfractions were isolated by discontinuous density gradient ultracentrifugation according to Redgrave and Carlson (13) as slightly modified by Karpe et al. (14, 15). This method is based on a four-step density gradient consisting of plasma with a density increased to 1.10 g/ml with solid sodium bromide (0.1268 g/ml) and of three different sodium bromide solutions of decreasing density. To form the gradient, 4 ml plasma (density 1.10 g/ml) were transferred to Beckman Ultra-Clear ultracentrifuge tubes (14 x 95 mm) previously coated with a thin layer of polyvinyl alcohol to make the tubes wettable (16). Three milliliters each of density 1.065 and 1.020 g/ml and 2.8 ml of density 1.006 g/ml sodium bromide solutions were carefully layered above the plasma samples. Ultracentrifugation was carried out in a Beckman SW 40 Ti rotor on a Beckman Optima L-90K ultracentrifuge (Beckman Instruments, Inc., Fullerton, CA). Three consecutive runs were performed at 15 C and 40,000 rpm to float: Svedberg flotation unit (Sf), >400 particles (chylomicrons, 32 min); Sf 60–400 particles [large very low density lipoprotein (VLDL), 3 h 32 min]; and Sf, 20–60 particles (small VLDL, 17 h). After each run, each subfraction (1 ml) was carefully aspirated from the top of the tube, which was then refilled with 1 ml density 1.006 g/ml solution before the next spin cycle. The Sf 12–20 particles [intermediate density lipoproteins (IDL), 2.5 ml] and the Sf 0–12 particles [low density lipoproteins (LDL), 3 ml] were recovered by following aspirations after the Sf 20–60 particles had been collected. High density lipoproteins (HDL) were isolated from plasma by the phosphotungstic acid/magnesium chloride precipitation method (17).
Apo B-48 and apo B-100. Concentrations of apo B-48 and apo B-100 were analyzed in chylomicrons, large VLDL, and small VLDL fractions by SDS-PAGE (18). Briefly, delipidated aliquots of samples were dissolved in buffer and run in self-made 3.5- 20% gel gradient. Generally a 2- to 5-µl aliquot was used for the evaluation of the apo B-100 content, and a 20- to 30-µl aliquot was used for the evaluation of apo B-48. Highly concentrated fasting LDL apo B-100 was used as standard. Controls for apo B-48 and apo B-100 determinations were prepared from the concentrated nonfasting triglyceride-rich lipoprotein fraction. Six standard samples ranging from 0.125–0.750 µg apo B-100 and four control samples were included in each run. Gels were stained overnight with Coomassie Blue G-250 and destained in water solution with 7% acetic acid and 12% methanol with at least four changes of destainer over 24 h. Scanning of gels was performed on a laser densitometer (Ultroscan XL, Pharmacia-LKB Technology, Uppsala, Sweden) at 595 nm connected to a personal computer that was equipped with software providing automatic integration of apo B-48 and apo B-100 peaks; the measurement was expressed as the area under curves (Gelscan XL, Pharmacia-LKB Technology). Concentrations of apo B-48 and apo B-100 were calculated from area under curves with the standard curve fitted by linear regression. The intra- and intergel coefficients of variation were 15.7% and 12.6% for apo B-48 and 7.8% and 11.4% for apo B-100, respectively. The detection limits for apo B-48 and apo B-100 ranged between 0.01 and 0.02 mg/liter.
Plasma lipolytic activities. Postheparin blood samples for LPL and HL activities were obtained 6 h after the meal, whereas serial samples for preheparin LPL were obtained before and during the test meal. Samples were collected into tubes containing EDTA, and plasma was immediately separated by centrifugation at 4 C and stored at –80 C until analysis. Lipase activities were determined according to the method described by Nilsson-Ehle and Ekman (19), using as substrate a [3H]trioleoylglycerol emulsion stabilized by dioleoyl phosphatidyl choline. Specific measurements of LPL and HL are based on differences in pH and NaCl molarity, the presence of serum in the incubation mixture, as well as the addition of albumin to the substrate emulsion either before or after sonication. LPL activity in preheparin plasma was measured after separation by Sephadex column chromatography according to a modified procedure (20). The main modifications were that lower amounts of heparin-Sepharose gel (1 ml) and plasma sample (0.5 ml) were used, and LPL was eluted with 3 ml 1% heparin buffer. The coefficient of variation for preheparin LPL activity measurement was 10.3% intraassay and 20.3% interassay. Due to the high interassay variability, data were corrected by a standard control sample.
Other measurements. Total cholesterol and triglyceride concentrations were assayed in plasma and isolated lipoprotein fractions by enzymatic colorimetric methods (Roche, Mannheim, Germany) on an autoanalyzer Cobas Mira (ABX Diagnostics, Montpellier, France). Blood glucose was measured by standard enzymatic methods, and hemoglobin A1c by HPLC (reference values, 4.3–5.9%). Plasma nonesterified fatty acid concentrations were analyzed by enzymatic colorimetric method (Wako Chemicals GmbH, Neuss, Germany). Plasma insulin concentrations were measured by RIA (Technogenetics, Milan, Italy).
Statistical analysis
Data are expressed as the mean ± SEM unless otherwise stated. Postprandial incremental area was calculated by the trapezoidal method as the area under the curve above the baseline fasting value. Differences between diabetic patients and control subjects were evaluated by t test for independent samples. Differences between the two groups at single time points after the meal were first evaluated by ANOVA for repeated measures. Two-tailed tests were used, and P < 0.05 was considered statistically significant. Variables not normally distributed were analyzed by nonparametric tests (Mann-Whitney U test for independent samples). Statistical analysis was performed according to standard methods using the Statistical Package for Social Sciences software (SPSS/PC, SPSS, Inc., Chicago, IL).
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
As expected, blood glucose concentrations were higher in the diabetic patients at fasting and remained higher during the postprandial period in comparison with the control group (Fig. 1).
|
). The insulin incremental area, in fact, was significantly higher in diabetic patients (6.6 ± 2.5 vs. 11.9 ± 2.5 µU/ml·h; 39.6 ± 15 vs. 71.8 ± 18 pmol/liter·h; P < 0.02).
FFA levels were similarly reduced in the diabetic patients and the control subjects 2 h after the test meal; thereafter, they tended to increase in both groups with unexpected lower values in diabetic patients, even if the difference was small and not statistically significant (Fig. 1).
Lipids and lipoproteins
Whole plasma. After the test meal, plasma triglycerides increased in both groups of subjects, reaching a peak after 3 h in the controls and after 5 h in the diabetic patients. During the postprandial period, triglyceride concentrations at each time point were higher in diabetic patients than in controls, and the difference was more evident in the late postprandial phase (P < 0.05 at 4, 5, and 6 h). The triglyceride incremental area was increased in diabetic patients vs. controls (408 ± 69 vs. 330 ± 108 mg/dl·h; 4.61 ± 0.78 vs. 3.73 ± 1.23 mmol/liter·h), but the difference was not statistically significant.
Plasma cholesterol, instead, tended to decrease after the meal without any difference between diabetic patients and control subjects; the incremental area was –15.8 ± 13.5 mg/dl·h (–0.41 ± 0.35 mmol/liter·h) in diabetic patients and –27.4 ± 13.5 mg/dl·h (–0.71 ± 0.35 mmol/liter·h) in controls (P =NS).
Chylomicrons.
The postmeal triglyceride response in chylomicrons behaved similarly in type 2 diabetic patients and in controls during the first 3 h, but for a 1-h peak delay in diabetics. Six hours after the meal, triglyceride levels were significantly higher compared with control values (P < 0.05; Fig. 2). Before the test meal the cholesterol content of the chylomicron fraction was significantly higher in diabetic patients (P < 0.05; Fig. 2). Similarly, the response to the meal was higher in diabetic patients, especially in the second half of the postprandial curve (P < 0.05 vs. controls at 4 h). The cholesterol incremental area was almost doubled in diabetic patients compared with controls (6.9 ± 1.9 vs. 4.2 ± 1.9 mg/dl·h; 0.18 ± 0.05 vs. 0.11 ± 0.05 mmol/liter·h), but the difference did not reach statistical significance (Table 2).
|
|
and, as incremental areas, in Table 2. The response to the meal was small and in any case similar between diabetic patients and control subjects.
Large VLDL.
Triglycerides, cholesterol, apo B-48, and apo B-100 in the large VLDL are shown in Fig. 3 and as incremental areas in Table 2. Triglycerides and cholesterol were higher at fasting in diabetic patients compared with controls and remained higher during the entire postprandial period; the difference was more evident in the last part of the curve, with still increasing values in the diabetic patients even 6 h after the meal. The incremental areas for both triglycerides and cholesterol were more than doubled in diabetic patients compared with controls. The concentrations of apo B-48 and apo B-100 in large VLDL were also higher in the diabetic patients compared with the controls at different time points of the postprandial curve. The apo B-100 incremental area was markedly higher in the diabetic patients compared with the controls (65.5 ± 11.5 vs. 12.4 ± 1.8 mg/liter·h; P < 0.05). Equally higher was apo B-48 incremental area (7.08 ± 2.65 vs. 1.17 ± 0.88 mg/liter·h; P < 0.05).
|
. There were no differences between diabetic patients and control subjects except for apo B-48, whose postprandial incremental area was double in diabetic patients compared with controls (1.77 ± 0.88 vs. 0.88 ± 0.44 mg/liter·h), but the difference was not statistically significant.
IDL components (cholesterol, triglycerides, apo B-48, and apo B-100) tended to decrease after the meal without significant differences between diabetic patients and controls except for apo B-48 (Table 2). In fact, the apo B-48 incremental area was significantly less negative in diabetic patients compared with controls (P < 0.05; Table 2). No significant differences in LDL and HDL lipid responses to the meal were observed between the two groups (Table 2).
Lipolytic enzymes
Plasma postheparin LPL activity, evaluated 6 h after the meal, was very similar in the diabetic and the control subjects (76 ± 6 vs. 80 ± 12 mU/liter, respectively). Plasma postheparin hepatic lipase activity was instead significantly higher in the diabetic patients compared with the controls (257 ± 23 vs. 153 ± 22 mU/liter; P < 0.05).
As shown in Fig. 4, preheparin LPL plasma activity, obtained without heparin stimulation, was higher in diabetic patients, significantly so at the end of the test meal.
|
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Our data on exogenous lipoproteins are in agreement with those reported by Chen et al. (3), who found in a group of type 2 diabetic patients, similar to our subjects with regard to optimal blood glucose control and fasting normotriglyceridemia, a significant increase in retinyl palmitate in the nonchylomicronemic triglyceride-rich fraction. Our results are, instead, in contrast with data presented by Lewis et al. (7), who found postprandial lipid abnormalities only in the type 2 diabetic patients with fasting hypertriglyceridemia (serum triglycerides, >150 mg/dl; >1.7 mmol/liter).
Furthermore, our diabetic patients, although normotriglyceridemic in the fasting condition, were also characterized in the postprandial period by a significant increase in the number of endogenous large VLDL particles, as reported in other studies (4, 5)
In healthy individuals insulin secretion increases in the postprandial phase, leading to a reduction in the secretion of VLDL by the liver (2). This allows normal handling of the intestinally derived lipoproteins, which share a common catabolic pathway through LPL with liver-derived lipoproteins. In our type 2 diabetic patients, even with fasting normotriglyceridemia, there was, instead, a substantial increase in the endogenous VLDL fraction during the postprandial phase, as indicated by the significantly higher incremental area of apo B-100 in large VLDL. As postprandial insulin response in our diabetic patients was higher compared with controls, it is likely that the increase in endogenous large VLDL is explained by a reduced inhibitory effect of insulin on VLDL secretion, as shown in the fasting condition (22).
At odds with data reported in the literature (4, 5), the increase in chylomicron apo B-48 after the meal was not particularly evident in either diabetic or controls subjects. As people from southern Europe are characterized by an earlier apo B-48 postprandial response (23), it is possible that by taking our first sample 2 h after the meal, we missed an earlier apo B-48 increase. In any case, at variance with other studies, postprandial chylomicron apo B-48 levels in our diabetic patients were not different from those in control subjects. Our patients had a mild type 2 diabetes in optimal blood glucose control, whereas increased synthesis of chylomicrons has been reported in diabetic patients with unsatisfactory blood glucose control (24).
The triglyceride and especially the cholesterol content of chylomicrons were increased in our diabetic patients, especially in the late postprandial phase. It is of note that the cholesterol content remained significantly higher in the postabsorptive period as shown by the significantly higher levels observed in the fasting condition before the test meal. This enrichment in cholesterol of chylomicrons, which could be due to a higher activity of cholesterol ester transfer protein in the diabetic patients (2), may give more atherogenic properties to these large particles (8).
Together with the increase in the number of hepaticderived large VLDL, our diabetic patients were characterized by an increase in the number of chylomicron remnants, as shown by the increase in the apo B-48 incremental areas of large and small VLDL as well as by the lesser decrease in IDL apo B-48. These results may be due to different mechanisms, i.e. a deficit in LPL activity and/or an impairment in the hepatic uptake of these particles (10, 25) as well as increased secretion of smaller chylomicron particles. We tried to evaluate more in depth the possible role of lipolytic enzymes, measuring both the activity of postheparin LPL 6 h after the meal and the basal preheparin activity of LPL throughout the entire postprandial period. The postheparin levels were very similar between diabetic patients and control subjects, whereas LPL activity without heparin stimulation was, if anything, increased in the diabetic patients.
The exact meaning of preheparin LPL is not completely clear, and few studies have addressed this issue (26, 27). Considering preheparin activity as a good index of LPL activity, our results exclude that an absolute deficit of LPL was present during the postprandial phase in the diabetic patients. However, a relative deficit of LPL in the postprandial phase cannot be excluded; LPL activity, although increased in the diabetic patients, may have not been sufficiently high to overcome the burden of both the increased number of hepatic-derived VLDL and the appearance in the circulation of intestinally derived particles. Therefore, it is possible to conclude that, at least in this group of diabetic patients with perfectly normal fasting triglyceride levels, reduced LPL activity does not seem to play a primary role in the pathogenesis of postprandial lipid abnormalities. This is also supported by recent data reported by Eriksson et al. (28). On the other hand, a more relevant impairment of LPL activity at the adipose tissue level may not be completely excluded (11).
Hepatic lipase activity in postheparin plasma 6 h after meal ingestion was higher in the diabetic subjects. An increase in this enzyme in the fasting condition has already been shown in type 2 diabetic patients and other states of insulin resistance (2) and has been related to the excess of small, dense LDL. The increased postprandial HL activity observed in this study may indicate that the deleterious effects of increased HL activity are also operating in the postprandial condition.
In conclusion, our group of type 2 diabetic patients with normotriglyceridemia and optimal blood glucose control during the postprandial period was mainly characterized by an increase in the liver-derived VLDL particles. This increase may be due to the insulin resistance typical of these patients, which could act through a reduction in the inhibitory effect of insulin on the hepatic synthesis of VLDL apo B-100 (22). In addition, in our diabetic patients there was a postprandial increase in chylomicron remnants as well as an enrichment in chylomicron cholesterol also in the fasting condition. According to experimental data (8), all of these abnormalities may increase the cardiovascular risk of these patients, who would otherwise be considered at low cardiovascular risk if only their fasting lipid levels were evaluated.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Abbreviations: apo, Apolipoprotein; FFA, free fatty acid; HDL, high-density lipoprotein; HL, hepatic lipase; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; LPL, lipoprotein lipase; Sf, Svedberg flotation unit; VLDL, very LDL.
Received October 7, 2003.
Accepted February 12, 2004.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  S. S Lund, L. Tarnow, M. Frandsen, U. M Smidt, O. Pedersen, H.-H. Parving, and A. A Vaag Impact of metformin versus the prandial insulin secretagogue, repaglinide, on fasting and postprandial glucose and lipid responses in non-obese patients with type 2 diabetes Eur. J. Endocrinol., January 1, 2008; 158(1): 35 - 46. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Shah, B. Adams-Huet, L. Brinkley, S. M. Grundy, and A. Garg Lipid, Glycemic, and Insulin Responses to Meals Rich in Saturated, cis-Monounsaturated, and Polyunsaturated (n-3 and n-6) Fatty Acids in Subjects With Type 2 Diabetes Diabetes Care, December 1, 2007; 30(12): 2993 - 2998. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. L. Isley, J. M. Miles, B. W. Patterson, and W. S. Harris The effect of high-dose simvastatin on triglyceride-rich lipoprotein metabolism in patients with type 2 diabetes mellitus J. Lipid Res., January 1, 2006; 47(1): 193 - 200. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Annuzzi, C. De Natale, C. Iovine, L. Patti, L. Di Marino, S. Coppola, S. Del Prato, G. Riccardi, and A. A. Rivellese Insulin Resistance Is Independently Associated With Postprandial Alterations of Triglyceride-Rich Lipoproteins in Type 2 Diabetes Mellitus Arterioscler. Thromb. Vasc. Biol., December 1, 2004; 24(12): 2397 - 2402. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
