The increased risk of atherosclerotic cardiovascular disease

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1 Absence of Acute Inhibitory Effect of Insulin on Chylomicron Production in Type Diabetes Juan-Patricio Nogueira, Marie Maraninchi, Sophie Béliard, Nadège Padilla, Laurence Duvillard, Julien Mancini, Alain Nicolay, Changting Xiao, Bernard Vialettes, Gary F. Lewis, René Valéro Objective Overproduction of intestinally derived apob-48-containing triglyceride-rich lipoproteins (TRLs) (chylomicrons) has recently been described in type diabetes, as is known for hepatic TRL-apoB-1 (very-low-density lipoprotein) production. Furthermore, insulin acutely inhibits both intestinal and hepatic TRL production, whereas this acute inhibitory effect on very-low-density lipoprotein production is blunted in type diabetes. It is not currently known whether this acute effect on chylomicron production is similarly blunted in humans with type diabetes. Methods and Results We investigated the effect of acute hyperinsulinemia on TRL metabolism in 18 type diabetic men using stable isotope methodology. Each subject underwent 1 control (saline infusion [SAL]) lipoprotein turnover study followed by a second study, under 1 of the 3 following clamp conditions: (1) hyperinsulinemic-euglycemic, () hyperinsulinemic-hyperglycemic, or (3) hyperinsulinemic-euglycemic plus intralipid and heparin. TRL-apoB-48 and TRL-apoB-1 production and clearance rates were not different between SAL and clamp and between the different clamp conditions, except for significantly lower TRL-apoB-1 clearance and production rates in hyperinsulinemiceuglycemic plus intralipid and heparin clamp compared with SAL. Conclusion This is the first demonstration in individuals with type diabetes that chylomicron production is resistant to the normal acute suppressive effect of insulin. This phenomenon may contribute to the highly prevalent dyslipidemia of type diabetes and potentially to atherosclerosis. Clinical Trial Registration URL: Unique identifier: NCT99. (Arterioscler Thromb Vasc Biol. 1;3: ) Key Words: apolipoproteins atherosclerosis diabetes mellitus fatty acids lipoproteins The increased risk of atherosclerotic cardiovascular disease associated with insulin-resistant states and type diabetes is of great public health concern. 1 The typical diabetic dyslipidemia is characterized by a number of abnormalities, including elevated plasma triglyceride (TG) levels, low high-density lipoprotein cholesterol, increased proportion of small and dense low-density lipoprotein, and postprandial hyperlipidemia. Dyslipidemia in insulin-resistant states contributes to the residual cardiovascular risk and atherosclerosis. 3 Diabetic dyslipidemia includes characteristic accumulation of triglyceride-rich lipoproteins (TRLs), which has been attributed to a combination of defective TRL removal and overproduction from liver (TRL-apoB-1 or very-low-density lipoprotein [VLDL]) 3 and from intestine (TRLapoB-48 or chylomicrons). 6 8 TRL-apoB-48 and TRL-apoB-1 have been identified as proatherogenic in type diabetes. 9 The mechanisms leading to the overproduction of TRLs in the setting of type diabetes and insulin resistance remain to be fully characterized, particularly for intestine. We have recently shown that acute elevation of plasma free fatty acids (FFA) stimulates not only hepatic but also intestinal TRL production in Syrian Golden hamsters 1 and in fed healthy humans, 11 demonstrating functional similarities between these organs in this respect. The effects of hyperglycemia on TRL metabolism remain controversial, either decreasing 1 or stimulating VLDL production. 13 Insulin acutely inhibits hepatic TRL-apoB-1 secretion in healthy humans, in vivo in animals, and in cell culture experiments, even though chronic hyperinsulinemic states, which are usually associated with insulin resistance, are characterized by chronic overproduction of VLDL. In fasting humans, this acute inhibitory effect seems to be completely or partly independent of the FFA suppressive effect of hyperinsulinemia. 16,18,19 In insulin-resistant humans and animals, this acute inhibitory effect is blunted or absent. One may Received on: July 7, 11; final version accepted on: January 13, 1. From the Unité Mixte de Recherche Institut National de la Recherche Agronomique 16 (J.P.N., M.M., N.P., A.N., R.V.) and Biostatistics Research Unit (Laboratoire d Enseignement et de Recherche sur le Traitement de l Information Médicale) (J.M.), University of la Méditerranée, Marseille, France; Department of Nutrition, Metabolic Diseases, Endocrinology, Assistance Publique-Hôpitaux de Marseille, La Timone Hospital, Marseille, France (B.V., R.V.); Unité Mixte de Recherche Institut National de la Santé et de la Recherche Médicale 939, La Pitié Hospital, Paris, France (S.B.); Departments of Medicine and Physiology, Division of Endocrinology and Metabolism, University of Toronto, Canada (C.X., G.F.L.); Institut National de la Santé et de la Recherche Médicale 866, Dijon, France (L.D.). The online-only Data Supplement is available with this article at ATVBAHA /-/DC1. Correspondence to René Valéro, MD, PhD, Service de Nutrition, Maladies Métaboliques, Endocrinologie, Hôpital La Timone, 64 Rue Saint Pierre, 13 Marseille, France. rvalero@mail.ap-hm.fr 1 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at DOI: /ATVBAHA

2 14 Arterioscler Thromb Vasc Biol April 1 Table 1. Mean Baseline Demographic Characteristics and Fasting Biochemical Parameters of Patients With Type Diabetes in the Saline Condition INS GLY (n 6) INS (n 6) INS IH (n 6) Age, y BMI, kg/m Duration of diabetes, y HbA 1c,% Glucose, mmol/l Insulin, pmol/l C-peptide, nmol/l HOMA-IR Plasma FFA, mmol/l Plasma TG, mmol/l Plasma TC, mmol/l Plasma HDL-C, mmol/l Plasma LDL-C, mmol/l TRL-apoB-48, mg/dl TRL-apoB-1, mg/dl Data are means SEM. apob indicates apolipoprotein B; BMI, body mass index; HDL-C, high-density lipoprotein cholesterol; HOMA-IR, homeostasis model assessment insulin resistance; INS, hyperinsulinemic-euglycemic condition; INS GLY, hyperinsulinemic-hyperglycemic condition; INS IH, hyperinsulinemic-euglycemic plus intralipid and heparin condition; LDL-C, low-density lipoprotein cholesterol; TC, total cholesterol; TG, triglycerides; TRL, triglyceride-rich lipoprotein; FFA, free fatty acids. speculate that the blunting of this acute suppressive effect may contribute to the chronic overproduction of TRL particles. At the intestinal level, insulin has also been shown to acutely inhibit intestinal TRL-apoB-48 production of the chow-fed hamster but not of the insulin-resistant fructose-fed hamster. 3 Insulin added acutely to the medium of human fetal small intestinal cells reduces chylomicron secretion. 4 More recently, it has been shown, using methodology similar to the present study, that insulin acutely inhibits intestinal lipoprotein secretion in healthy fed humans, in part by suppressing plasma FFA. The acute effect of insulin on intestinal TRL-apoB-48 production has not been previously examined in individuals with type diabetes. In the present study, we investigated the effects of acute hyperinsulinemia on intestinal and hepatic TRL metabolism in 18 men with type diabetes in a constant fed state under conditions of euglycemia, hyperglycemia, and elevated concentrations of plasma FFA, the latter conditions designed to dissociate the direct or indirect potential effects of insulin. Methods Subjects Mean baseline demographic characteristics and fasting biochemical parameters of the 18 male type diabetic patients are outlined in Table 1. For details, please see the online-only Data Supplement. The Research Ethics Board of la Méditerranée University approved the study, and all subjects gave written informed consent. Experimental Protocol for Lipoprotein Kinetic Studies Each subject underwent separate lipoprotein kinetic studies, as described below, 4 weeks apart. In each study, following an overnight fast, an intravenous catheter was inserted into a superficial vein in each forearm, 1 for infusion and 1 for blood sampling. At 6 am on the day of the kinetic study, the patients started an hourly supplement ingestion (Nutrini Drink, Nutricia, Zoetermeer, the Netherlands: % calories from carbohydrates, 9% from proteins, 41% from fat [4.% of total calories from saturated fat, 4.6% from monounsaturated fat, and 1% from polyunsaturated fat]). From 8 am to the end of the study (1 pm), all patients underwent an initial study with an intravenous infusion of saline (6 ml/h) (SAL). Four weeks later, the subjects were assigned to 1 of 3 hyperinsulinemic clamp conditions, with an attempt to match the 3 groups for age, body mass index, and HbA 1c. The 3 clamp conditions were (1) intravenous infusion of insulin (8 mui/m min Novorapid, Novo Nordisk, Chartres, France) and glucose % solution at a variable rate to maintain the blood glucose around. mmol/l (hyperinsulinemic-euglycemic condition [INS], n 6); () as in condition 1, but the blood glucose was maintained at approximately 11 mmol/l (hyperinsulinemic-hyperglycemic condition [INS GLY], n 6); and (3) as in condition 1, but with coinfusion of intralipid (% solution at 1 ml/h, Fresenius Kabi, Uppsala, Sweden) and heparin ( U/h, Heparin Choay, Sanofi-Aventis) to prevent the anticipated reduction in circulating FFA that occurs with hyperinsulinemia (hyperinsulinemic-euglycemic plus intralipid and heparin condition [INS IH], n 6) (Figure 1A). For 3 subjects (1 in each treatment group), we used regular human insulin (Actrapid, Novo Nordisk) instead of Novorapid to enable the measurement of plasma exogenous insulin levels throughout the clamps. Blood glucose was assessed at the bedside every to 1 minutes using an Accu-Chek Performa Analyzer (Roche Diagnostics, Meylan, France). Kinetic studies were performed in a constant fed state because apob-48 levels are too low in the fasted state to accurately assess isotopic enrichments for calculation of kinetic parameters. To achieve a constant fed state, the subjects ingested aliquots of the liquid food supplement every hour from 6 am to 1 pm, with each hourly aliquot equivalent to 1/16th of their total daily caloric needs as estimated by the use of the Harris-Benedict equation (mean caloric intake SEM: kcal). Four hours after starting to ingest the liquid formula and hours after starting infusions of saline, insulin, or insulin plus intralipid and heparin (at 1 am), subjects received a primed-constant infusion (1 mol/kg bolus followed by 1 mol kg 1 hr 1 for 1 hours) of deuterium-labeled leucine (l-[,,- H 3 ]-leucine, 99%, Cambridge Isotope Laboratories, Andover, MA) to enrich apob-48 and apob-1 for assessment of the fractional catabolic rate (FCR), the pool size (PS), and the production rate (PR) of TRL-apoB-48 and TRL-apoB-1 as previously described. 7 Blood samples were collected in the fasting state (6 am), before (1 am) and after the start of the primed-constant infusion of D3-leucine, at 1, 3,, 7, 9, 1, 11, and 1 hours. Laboratory Methods Plasma was immediately separated from blood samples at 3 rpm for 1 minutes at 4 C. TRLs were isolated by gradient ultracentrifugation as previously described. 6 Proteins in the TRL fraction were determined, 7 delipidated, and subsequently separated by SDS- PAGE, with clear separation of the apob-48 and apob-1 bands. apob-48 and apob-1 gel slices were hydrolyzed and derivatized to allow the determination of leucine isotopic enrichment, as previously described, 7 by electron impact ionization gas chromatography/mass spectrometry. Tracer-to-tracee ratios were calculated from isotopic ratios for each sample and standard enrichment curves. For details, please see the Methods section in the online-only Data Supplement. Calculation of Lipoprotein PS and Production and Clearance Rates by Compartmental Modeling A 3-compartment model was fitted to the stable isotope enrichment curves using SAAM II computer software (version 1., University of Washington, Seattle, WA) (Figure I in the online-only Data Supplement). PR was calculated using the FCR of TRL-apoB-48 or TRL-apoB-1 multiplied by PS measured over the 1 hours of the kinetic study, where PS average plasma concentration (mg/l) between 1 and 1 hours of the kinetic study (ie, from 1 am to 1 pm) plasma volume (L)/kg body weight (plasma volume was

3 Nogueira et al Insulin Effect on Chylomicron in Type Diabetes 141 A 6am 8am 1am CONTROL STUDY Saline (6 ml/h) (SAL) and HYPERINSULINEMIC STUDY Insulin 8 mui/m.min+ glucose (%). mmol/l (INS) or Insulin 8 mui/m.min+ glucose (%) 11 mmol/l (INS+GLY) or Insulin 8 mui/m.min+ glucose (%). mmol/l + intralipid (%, 1ml/h)+ heparin ( U/h) (INS+IH) FFA ( mmol/l) 1pm F B CLAMP-INS+GLY CLAMP-INS CLAMP-INS+IH Time (h) Nutrini Drink (hourly) Glucose (mmol/l) C corrected in relation to weight). 8 For details, please see the Methods section in the online-only Data Supplement. TRL-apoB-48 and TRL-apoB-1 tracer-to-tracee ratios versus time are presented in Figure IIA to IID in the online-only Data Supplement. Statistical Analysis Statistical analysis was performed using SPSS (version 17., SPSS, Chicago, IL) software. Results are presented as means SEM. The mean values of the parameters and the statistical comparison between studies were calculated during the 1-hour kinetic studies (ie, from 1 am to 1 pm, the time period of deuterated leucine infusion). The Wilcoxon paired test was used to study changes between SAL and clamp conditions, and the Kruskal-Wallis test was used for comparisons among the 3 groups in the SAL condition and in clamp conditions. For all the analyses, P. was considered significant. Results D3-Leucine bolus +primed-constant infusion 4 CLAMP-INS+GLY CLAMP-INS CLAMP-INS+IH F Time (h) Fasting Plasma Lipids, Biochemical Characteristics, and TRL Composition of Patients At baseline (fasting state of the SAL condition), the 3 treatment groups (INS, INS GLY, and INS IH) were well matched, with no significant difference in demographic characteristics or fasting biochemical parameters (Table 1). Concentrations of TG, FFA, Insulin, C-Peptide, and Glucose in Plasma and of TG, apob-48, and apob-1 in TRL Fraction When we compared the 3 groups (INS, INS IH, and INS GLY) in the clamp conditions, plasma FFA concentration was higher in INS IH compared with INS or INS GLY (P.1 for both), plasma glucose concentration was higher in Insulin (pmol/l) F D CLAMP-INS+GLY CLAMP-INS CLAMP-INS+IH Time (h) Figure 1. A, Study protocol. B to D, Plasma free fatty acids (FFA) (B), glucose (C) and insulin (D) levels in fasting (F) and over the time course of the kinetic study (from 1 am to 1 pm) during the hyperinsulinemic clamps (CLAMP) for the 3 groups of patients with type diabetes (INS, INS IH, INS GLY, as defined in A). Data are means SEM, except for insulin levels measured only in 1 patient per group. *P.1 for plasma FFA levels between INS IH and INS or INS GLY; *P.1 for plasma glucose levels between INS GLY and INS or INS IH. INS GLY compared with INS or INS IH (P.1 for both), and plasma insulin concentration was not different among the 3 groups (Figure 1B 1D). Plasma C-peptide and glucose infusion rate were higher in INS GLY compared with INS or INS IH (P.1 for both). There were no differences in plasma TG, TRL-TG, TRL-apoB-48, and TRL-apoB-1 levels among the 3 groups. For details, please see Table I and Figure IIIA to IIID in the online-only Data Supplement. When we compared SAL and clamp conditions within each treatment group (INS, INS IH or INS GLY), we found in INS that plasma glucose and C-peptide concentrations decreased significantly in clamp versus SAL (P.1 for both), whereas plasma FFA, plasma TG, TRL-TG, TRL-apoB-48, and TRLapoB-1 concentrations did not change significantly. In INS GLY, TRL-TG, TRL-apoB-48, and TRL-apoB-1 concentrations decreased significantly in clamp versus SAL (P. for all), whereas plasma glucose, TG, FFA, and C-peptide concentrations did not change significantly. In INS IH, plasma glucose and C-peptide concentrations decreased significantly, whereas plasma FFA increased significantly in clamp versus SAL (P.1 for all) and plasma TG, TRL-TG, TRL-apoB-48 and TRL-apoB-1 concentrations did not change significantly. For details, please see Table I and Figure IIIA to IIID in the online-only Data Supplement. Effect of Acute Hyperinsulinemia on TRL-apoB-48 and TRL-apoB-1 PS, FCR, and PR There were no differences in TRL-apoB-48 or TRL-apoB- 1 PS, FCR, and PR between SAL and clamp conditions for

4 14 Arterioscler Thromb Vasc Biol April 1 Table. Mean Kinetic Parameters in the 3 Groups of Patients With Type Diabetes (INS, INS IH, INS GLY) in the SAL and Clamp Conditions Parameters INS GLY (n 6) INS (n 6) INS IH (n 6) PS-apoB-48-SAL, mg PS-apoB-48-clamp, mg * FCR-apoB-48-SAL, pools/d FCR-apoB-48-clamp, pools/d PR-apoB-48-SAL, mg kg 1 d 1 PR-apoB-48-clamp, mg kg 1 d 1 PS-apoB-1-SAL, mg PS-apoB-1-clamp, mg FCR-apoB-1-SAL, pools/d FCR-apoB-1-clamp, * pools/d PR-apoB-1-SAL, mg kg 1 d 1 PR-apoB-1-clamp, mg kg 1 d * Means SEM data are for the duration of the 1-h kinetic studies after the infusion of D3-leucine (from 1 am to 1 pm). Data were compared for each group between SAL and clamp conditions and in SAL or clamp conditions between INS and INS GLY, between INS IH and INS GLY, and between INS and INS IH. apob indicates apolipoprotein B; FCR, fractional catabolic rate; INS, hyperinsulinemic-euglycemic condition; FFA, free fatty acids; INS GLY, hyperinsulinemic-hyperglycemic condition; INS IH, hyperinsulinemic-euglycemic plus intralipid and heparin condition; PR, production rate; PS, pool size; SAL, saline. *P.. the 3 groups (INS, INS GLY, and INS IH) except for a significant decrease in FCR and PR of TRL-apoB-1 in the INS IH group in clamp versus SAL conditions (P. for both) and with the exception that TRL-apoB-48 PS was significantly lower in clamp versus SAL in INS GLY (P.). This reduction in TRL-apoB-48 PS was explained by a nonsignificant trend toward a % reduction in PR (P.64) and a % increase in FCR (P.6) of TRL apob-48 in this group (Table and Figure A D; Tables II and III in the online-only Data Supplement). We found no difference in TRL-apoB-48 or TRL-apoB- 1 PS, FCR, and PR among the 3 groups (INS, INS GLY, and INS IH) in SAL and clamp conditions (Table ). Discussion We have shown for the first time in humans that the acute inhibitory effect of insulin on intestinal TRL-apoB-48 production, recently shown in healthy humans using a similar method, is blunted in patients with type diabetes. Our findings are in keeping with the results of an animal study showing that intestinal lipoprotein production of chow-fed hamsters but not insulin-resistant fructose-fed hamsters was responsive to the acute inhibitory effect of insulin. 3 The intestinal insulin insensitivity is accompanied by impaired insulin signaling that has been shown ex vivo in enterocytes Fractional Catabolic Rate (pools/day) Fractional Catabolic Rate (pools/day) 1 1 TRL-apoB-48 SAL CLAMP INS+GLY INS INS+IH 1 1 A C TRL-apoB-1 SAL CLAMP Production Rate (mg.kg -1.day -1) Production Rate (mg.kg -1.day -1) TRL-apoB-48 INS+GLY INS INS+IH TRL-apoB-1 INS+GLY INS INS+IH INS+GLY INS INS+IH Figure. Triglyceride-rich lipoprotein (TRL)-apolipoprotein B (apob)-48 fractional catabolic rate (A) and production rate (B) and TRL-apoB-1 fractional catabolic rate (C) and production rate (D) in saline (SAL) and hyperinsulinemic clamp (CLAMP) conditions for the 3 groups of patients with type diabetes (hyperinsulinemic-euglycemic condition [INS], hyperinsulinemiceuglycemic plus intralipid and heparin condition [INS IH], and hyperinsulinemic-hyperglycemic condition [INS GLY]). Data are means SEM. *P.. of fructose-fed hamsters. 3 The consequences of the insulin signaling defect at the level of the enterocyte are an increase in de novo lipogenesis and chylomicron assembly and secretion. 3,9,3 We also confirmed the results of several studies in animal models of insulin resistance,31,3 in type diabetic and obese, insulin-resistant human subjects, 1 showing that the acute inhibitory effect of insulin on hepatic TRL-apoB- 1 production is blunted. The kinetic parameters of our study are different (lower PS and PR and higher FCR) with regard to those of the only kinetic study on TRL-apoB-48 metabolism in type diabetic subjects versus controls, 8 but several differences between the studies may explain these discrepancies. The diabetic population in that study was more insulin resistant (mean HOMA-IR:.6 versus 4.4) and was frankly more hypertriglyceridemic (mean fasting plasma TG levels: 4.6 versus. mmol/l) than our diabetic population. The diet was solid and ingested every half-hour versus liquid and hourly in our study. TRL-apoB-48 levels were measured using analytic SDS-PAGE versus ELISA in our study. To calculate the PS, we corrected the plasma volume in relation to weight. Oral hypoglycemic drugs were only metformin, sulfonylurea, or both in our study, but 3 patients received a thiazolidinedione in the other study, and the majority of our patients received lipid-lowering drugs. We found no difference in the kinetic parameters of TRL-apoB-48 and TRL-apoB-1 between SAL and clamp regardless of their lipid-lowering therapy (statin, fibrate, or no treatment) but a significant increase in TRL-apoB-1 PR in the fibrate group in SAL (Table IV in the online-only Data Supplement). Our study was not designed to assess the effect of lipid-lowering drugs. Those treated with the various lipid-modifying agents were distributed equally among the 3 groups, the medications were continued unchanged throughout the study, and each subject served as his own control. Although we cannot completely rule out a potential confounding effect of these drugs on the main findings of the present study, it is unlikely that the absence of insulin-mediated suppression of TRL-apoB-48 or B D

5 Nogueira et al Insulin Effect on Chylomicron in Type Diabetes 143 apob-1 production would have occurred as a result of the fact that the patients enrolled in the present study were taking lipid-lowering therapies. An acute elevation of plasma FFA has previously been shown to stimulate TRL-apoB-48 production in healthy humans 11 and in chow-fed hamsters, 1 with no stimulatory effect of FFA seen in insulin-resistant hamsters that already overproduce TRLapoB These findings are in keeping with our observation, in that a marked elevation of plasma FFA in the clamp-ins IH study failed to significantly stimulate TRL-apoB-48 PR compared with SAL, although there was a nonsignificant trend toward an increase in TRL-apoB-48 PR. FFA may stimulate intestinal TRL-apoB-48 assembly and secretion by impairing insulin signaling or by increasing the pool of intracellular fatty acids incorporated into secreted lipoprotein particles. 33 In the present study, we obtained no efficient additive suppression of plasma FFA under any of our clamp conditions compared with SAL, which could partly explain the inability of insulin to acutely suppress TRL-apoB-48 production. Impaired clearance of intestinal TRL-apoB-48 and hepatic TRL-apoB-1 has been shown in patients with type diabetes compared with controls, 8 possibly related to the postprandial impairment of lipoprotein lipase stimulation that normally occurs in nondiabetic subjects. 34,3 In our study and similar studies in healthy humans, there was no clear acute impairment in TRL-apoB-48 FCR when insulin or FFA concentrations were experimentally raised. 11, The INS IH group showed a parallel decrease of FCR and PR of hepatic TRL-apoB-1 without any change in the PS in clamp versus SAL. The reduction of FCR could be explained by the impeded effect of intralipid infusion on normal lipoprotein lipase-mediated lipolysis reflecting the competition between intralipid (chylomicron-like TRLs) and VLDL for the same lipolytic pathway, previously described in healthy young men, 36 or by the reduction in TRL-apoB-1 PR decreasing substrate for lipoprotein lipase-mediated lipolysis. The acute role of plasma FFA in the TRL-apoB-1 production is still being debated. Whereas it has been reported that acute elevation of plasma FFA stimulates TRL-apoB-1 production in healthy fasted and fed humans, 11,16 the similar increase of plasma FFA (-fold) by heparin infusion did not lead to a rise in VLDL production in the same population. 37 Moreover, the reduction of plasma FFA with acipimox decreased the PR of VLDL1, increased the PR of VLDL fraction, and produced no change in total VLDL PR. 18 The explanation could be that FFA availability regulates the production of VLDL in the longer term but not acutely because the use of cytosolic TGs, rather than plasma FFA, as a direct source of VLDL provides a buffer mechanism. 37 The reduction of TRL-apoB-1 FCR in INS IH could reduce the TRL-TG source taken up by the liver and directly available for VLDL production and may explain the decrease of TRL-apoB-1 PR. Recently, it has been shown in vivo (McA- RH7777 cells) and in vivo in mice, a parabolic effect on apob-1 secretion, with moderate FFA exposure increasing apob-1 secretion, whereas greater lipid loading inhibited apob-1 in a duration-dependent manner (the incubation of McA-RH7777 cells for 16 hours with 1 mg/dl intralipid stimulated apob- 1 secretion, whereas mg/dl inhibited it and had no effect on apob-48 secretion). This decrease effect was linked to the induction of endoplasmic reticulum stress increasing apob-1 degradation through both proteasomal and nonproteasomal pathways, worsening hepatic steatosis. 38 We also showed that acute elevation of blood glucose together with hyperinsulinemia did not affect TRL-apoB-48 or TRLapoB-1 FCR or PR. However, we found in INS GLY a significant decrease in TRL-apoB-48 PS in clamp versus SAL. This decrease in TRL-apoB-48 PS could be due to a tendency toward an increase in TRL-apoB-48 FCR, a decrease in TRLapoB-48 PR that associated with a tendency to an increase in TRL-apoB-1 FCR ( 3%) could explain the significant decrease of TRL-apoB-48, TRL-apoB-1, and TRL-TG in INS GLY. The role of glucose on TRL metabolism remains debated. In a short-term (not acute) study, a reduction in postprandial chylomicrons has been shown after the improvement of glycemic control in type diabetes using intensified dietary intervention, drugs, or insulin. 39 Whereas VLDL-TG was shown to be decreased in lean men and women and in obese men but not obese women in hyperinsulinemic-hyperglycemic conditions, 1 another study has found that hyperglycemia is a driving force in the overproduction of VLDL1 but not VLDL in type diabetes. 13 Both studies were conducted in a fasting state. 1,13 In a study with a design similar to the present study except performed in the fasting state and with a shorter duration of the hyperinsulinemic clamp in type diabetes, VLDL1 and VLDL PR and FCR were comparable regardless of euglycemic or hyperglycemic conditions, except a significant reduction in VLDL PR under hyperglycemic compared with saline conditions. Two human studies have recently shown improvement in postprandial lipemia after acute intravenous administration of glucagon-like peptide 1 in healthy subjects 4 or after 4 weeks of treatment with a dipeptidyl peptidase IV inhibitor in patients with type diabetes. 41 In our study, C-peptide was not effectively suppressed in INS GLY during hyperinsulinemic clamp, but we are not able to dissociate the role of hyperglycemia or the glucosedependent insulinotropic effect of glucagon-like peptide 1 on this endogenous insulin secretion in our constant-fed condition. In conclusion, the present report has extended previous findings in animal models of insulin resistance to humans with type diabetes and also from the liver to the intestine, demonstrating that the acute inhibitory effect of insulin on intestinal TRL-apoB-48 production is absent in patients with type diabetes. We speculate that the absence of additive insulin-mediated suppression of plasma FFA in these patients may have partly accounted for this lack of insulin-mediated suppression of TRL-apo-B48 secretion, but we found in parallel an acute inhibitory effect of high plasma FFA levels on hepatic TRL-apoB-1 production. This resistance to insulin s acute suppressive effect on both intestinal TRLapoB-48 (shown in this report) and hepatic TRL-apoB-1 (shown by others previously ) production in type diabetes may contribute to the highly prevalent dyslipidemia of type diabetes and potentially to atherosclerosis. Acknowledgments We thank Roselyne Barone (UMR INRA 16, University of la Méditerranée, Marseille, France), the Assistance Publique-Hôpitaux de Marseille (Promoter), and the Centre Investigation Clinique for technical assistance.

6 144 Arterioscler Thromb Vasc Biol April 1 Sources of Funding This work was supported by a Assistance Publique-Hôpitaux de Marseille Régional grant (PHRC-7), Société Francophone du Diabète (SFD), Lilly, Novo Nordisk, and Servier. None. Disclosures References 1. Almdal T, Scharling H, Jensen JS, Vestergaard H. The independent effect of type diabetes mellitus on ischemic heart disease, stroke, and death: a population-based study of 13, men and women with years of follow-up. Arch Intern Med. 4;164: Ginsberg HN. Insulin resistance and cardiovascular disease. J Clin Invest. ;16: Adiels M, Olofsson SO, Taskinen MR, Boren J. Overproduction of very low-density lipoproteins is the hallmark of the dyslipidemia in the metabolic syndrome. Arterioscler Thromb Vasc Biol. 8;8: Alipour A, Elte JW, van Zaanen HC, Rietveld AP, Castro Cabezas M. Novel aspects of postprandial lipemia in relation to atherosclerosis. Atheroscler Suppl. 8;9: Proctor SD, Vine DF, Mamo JC. 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