Aims/hypothesis: We determined whether hepatic fat content and plasma adiponectin concentration regulate VLDL 1 production. Methods: A multicompartment model was used to simultaneously determine the kinetic parameters of triglycerides (TGs) and apolipoprotein B (ApoB) in VLDL 1 and VLDL 2 after a bolus of [ 2 H 3 ]leucine and [ 2 H 5 ]glycerol in ten men with type 2 diabetes and in 18 non-diabetic men. Liver fat content was determined by proton spectroscopy and intra-abdominal fat content by MRI. Results: Univariate regression analysis showed that liver fat content, intra-abdominal fat volume, plasma glucose, insulin and HOMA-IR (homeostasis model assessment of insulin resistance) correlated with VLDL 1 TG and ApoB production. However, only liver fat and plasma glucose were significant in multiple regression models, emphasising the critical role of substrate fluxes and lipid availability in the liver as the driving force for overproduction of VLDL 1 in subjects with type 2 diabetes. Despite negative correlations with fasting TG levels, liver fat content, and VLDL 1 TG and ApoB pool sizes, adiponectin was not linked to VLDL 1 TG or ApoB production and thus was not a predictor of VLDL 1 production. However, adiponectin correlated negatively with the removal rates of VLDL 1 TG and ApoB. Conclusions/interpretation: We propose that the metabolic effect of insulin resistance, partly mediated by depressed plasma adiponectin levels, increases fatty acid flux from adipose tissue to the liver and induces the accumulation of fat in the liver. Elevated plasma glucose can further increase hepatic fat content through multiple pathways, resulting in overproduction of VLDL 1 particles and leading to the characteristic dyslipidaemia associated with type 2 diabetes.

Insulin resistance is a key feature of the metabolic syndrome and often progresses to type 2 diabetes. Both insulin resistance and type 2 diabetes are characterized by dyslipidemia, which is an important and common risk factor for cardiovascular disease. Diabetic dyslipidemia is a cluster of potentially atherogenic lipid and lipoprotein abnormalities that are metabolically interrelated. Recent evidence suggests that a fundamental defect is an overproduction of large very low–density lipoprotein (VLDL) particles, which initiates a sequence of lipoprotein changes, resulting in higher levels of remnant particles, smaller LDL, and lower levels of high-density liporotein (HDL) cholesterol. These atherogenic lipid abnormalities precede the diagnosis of type 2 diabetes by several years, and it is thus important to elucidate the mechanisms involved in the overproduction of large VLDL particles. Here, we review the pathophysiology of VLDL biosynthesis and metabolism in the metabolic syndrome. We also review recent research investigating the relation between hepatic accumulation of lipids and insulin resistance, and sources of fatty acids for liver fat and VLDL biosynthesis. Finally, we briefly discuss current treatments for lipid management of dyslipidemia and potential future therapeutic targets.

The accumulation of cytosolic lipid droplets in muscle and liver cells has been linked to the development of insulin resistance and type 2 diabetes. Such droplets are formed as small structures that increase in size through fusion, a process that is dependent on intact microtubules and the motor protein dynein. Approximately 15% of all droplets are involved in fusion processes at a given time. Here, we show that lipid droplets are associated with proteins involved in fusion processes in the cell: NSF (N-ethylmaleimide-sensitive-factor), alpha-SNAP (soluble NSF attachment protein) and the SNAREs (SNAP receptors), SNAP23 (synaptosomal-associated protein of 23 kDa), syntaxin-5 and VAMP4 (vesicle-associated membrane protein 4). Knockdown of the genes for SNAP23, syntaxin-5 or VAMP4, or microinjection of a dominant-negative mutant of alpha-SNAP, decreases the rate of fusion and the size of the lipid droplets. Thus, the SNARE system seems to have an important role in lipid droplet fusion. We also show that oleic acid treatment decreases the insulin sensitivity of heart muscle cells, and this sensitivity is completely restored by transfection with SNAP23. Thus, SNAP23 might be a link between insulin sensitivity and the inflow of fatty acids to the cell.

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Objective-We sought to compare the synthesis and metabolism of VLDL 1 and VLDL 2 in patients with type 2 diabetes mellitus (DM2) and nondiabetic subjects. Methods and Results-We used a novel multicompartmental model to simultaneously determine the kinetics of apolipoprotein (apo) B and triglyceride (TG) in VLDL 1 and VLDL 2 after a bolus injection of [ 2 H 3 ]leucine and [ 2 H 5 ]glycerol and to follow the catabolism and transfer of the lipoprotein particles. Our results show that the overproduction of VLDL particles in DM2 is explained by enhanced secretion of VLDL 1 apoB and TG. Direct production of VLDL 2 apoB and TG was not influenced by diabetes per se. The production rates of VLDL 1 apoB and TG were closely related, as were the corresponding pool sizes. VLDL 1 and VLDL 2 compositions did not differ in subjects with DM2 and controls, and the TG to apoB ratio of newly synthesized particles was very similar in the 2 groups. Plasma glucose, insulin, and free fatty acids together explained 55% of the variation in VLDL 1 TG production rate. Conclusion-Insulin resistance and DM2 are associated with excess hepatic production of VLDL 1 particles similar in size and composition to those in nondiabetic subjects. We propose that hyperglycemia is the driving force that aggravates overproduction of VLDL 1 in DM2. Key Words: diabetes Ⅲ dyslipidemia Ⅲ VLDL Ⅲ apolipoprotein B Ⅲ triglycerides Ⅲ compartmental modeling Ⅲ kinetics Ⅲ stable isotope B y 2025, Ͼ300 million people worldwide will have type 2 diabetes mellitus (DM2). Because atherosclerosis is an important complication of DM2, this will contribute significantly to an expected increase in cardiovascular disease worldwide. 1 One important cardiovascular risk factor associated with DM2 is a dyslipidemia characterized by high levels of triglyceride (TG)-rich VLDL, low levels of HDL cholesterol, small, dense LDL, and impaired and prolonged postprandial hyperlipidemia. 2 These abnormalities are present for years before DM2 is diagnosed clinically.The discovery of heterogeneity within the major lipoprotein classes (VLDL, LDL, and HDL) has opened new avenues to identify specific perturbations of diabetic dyslipidemia. 3 VLDL particles secreted from the liver vary in size and composition and can be classified by their density (0.94 to 1.06 g/mL), diameter (20 to 75 nm), and flotation [Svedberg flotation rate (Sf) 20 to 400]. VLDL can be separated into 2 main classes: large, buoyant VLDL 1 particles (Sf 60 to 400) and small, dense VLDL 2 particles (Sf 20 to 60). VLDL 1 particles contain more TG than VLDL 2 particles and are rich in apolipoprotein (apo) CIII and apoE. 4 Large VLDL 1 particles are the major subclass of endogenous TG-rich lipoproteins and seem to be the major determinant of the plasma TG concentration in normolipidemic subjects. 5 Although elevation of plasma TG is a consistent feature of diabetic dyslipidemia, little attention has focused on the VLDL subclass distribution in DM2. However, emerging data indicate a higher increase of VLDL 1 particles than of VL...

The assembly of lipoproteins containing apolipoprotein B is a complex process that occurs in the lumen of the secretory pathway. The process consists of two relatively well-identified steps. In the first step, two VLDL precursors are formed simultaneously and independently: an apolipoprotein B-containing VLDL precursor (a partially lipidated apolipoprotein B) and a VLDL-sized lipid droplet that lacks apolipoprotein B. In the second step, these two precursors fuse to form a mature VLDL particle. The apolipoprotein B-containing VLDL precursor is formed during the translation and concomitant translocation of the protein to the lumen of the endoplasmic reticulum. The VLDL precursor is completed shortly after the protein is fully synthesized. The process is dependent on the microsomal triglyceride transfer protein (MTP). Although the mechanism by which the lipid droplets are formed is unknown, recent observations indicate that the process is dependent on MTP. The fusion of the two precursors is not dependent on MTP, but the mechanism remains to be elucidated. The conversion of the apolipoprotein B-containing precursor to VLDL seems to be dependent on the ADP ribosylation factor 1 (ARF 1) and its activation of phospholipase D. During their assembly, nascent apolipoprotein B chains undergo quality control and are sorted to degradation. Such sorting, which occurs cotranslationally during the formation of the apolipoprotein B-containing precursor, involves cytosolic chaperons and ubiquitination that targets apolipoprotein B to proteasomal degradation. Other levels of sorting occur in the secretory pathway. Thus, lysosomal enzymes are involved as well as the LDL receptor.

Objectives-Adipocyte differentiation-related protein (ADRP)-containing lipid droplets have an essential role in the development of insulin resistance and atherosclerosis. Such droplets form in a cell-free system with a diameter of 0.1 to 0.4 m, while the droplets present in cells vary in size, from small to very large, suggesting that the droplets can increase in size after being assembled. We have addressed this possibility. Methods and Results-Experiments in NIH 3T3 cells demonstrated that the lipid droplets could increase in size independentlyof triglyceride biosynthesis. NIH 3T3 cells were either microinjected with ADRP-GFP (green fluorescent protein) or stained with Nile Red and followed by confocal microscopy and time-lapse recordings. The results showed that lipid droplets formed complexes with each other, with a volume equal to the sum of the merging particles. The formation of complexes could be inhibited by the nocodazole-induced depolymerization of the microtubules; thus, the process is dependent on microtubules.The presence of dynein on ADRP-containing droplets supports a role for this motor protein. Key Words: lipid droplets Ⅲ lipid droplet fusion Ⅲ adipocyte differentiation protein Ⅲ microtubules C ytosolic lipid droplets are organelles involved in the storage and turnover of triglycerides and cholesterol esters. Excessive accumulation of triglycerides, particularly in the liver and skeletal muscle, is associated with metabolic disorders such as insulin resistance and type 2 diabetes, 1,2 with a strongly increased risk of cardiovascular diseases. Moreover, the accumulation of lipid droplets in macrophages is a key feature of both early and late stages of the atherosclerotic lesion. 3 The PAT domain protein 4,5 ADRP (adipocyte differentiation-related protein) both promotes the formation of lipid droplets 6 (L.A., P.B., M.R., J.E., Marchesan D, Magnusson B, Ruiz M, P.H., Asp L, M.A.F., J.B., S.-O. O., unpublished data, 2005) and is an important constituent of the lipid droplets formed in liver, muscles, 8 and macrophages. 9 Thus, ADRP-containing lipid droplets are important in the pathogenesis of these diseases. In addition, lipid droplets have been proposed to have a central role in the inflammatory response, 10 which may further link the structures to the development of both insulin resistance and atherosclerosis. 11 Phospholipase D (PLD), which catalyzes the conversion of phosphatidylcholine to phosphatidic acid, promotes the assembly of ADRP-containing lipid droplets from intracellular membranes 12 (L.A., P.B., M.R., J.E., Marchesan D, Magnusson B, , unpublished data, 2005). The newly formed ("primordial") droplets have a diameter of 0.1 to 0.4 m, 12 whereas the mature droplets present in a cell can be more than 10 to 50ϫ larger (even larger in adipocytes) and vary considerable in size, indicating that the primordial lipid droplets increase in size after being formed. This increase appears to be an important step in the process leading to the accumulation of cytosolic lipid droplets in the ce...

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