Sven-Olof Olofsson

Overproduction of Very Low–Density Lipoproteins Is the Hallmark of the Dyslipidemia in the Metabolic Syndrome

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.

Overproduction of large VLDL particles is driven by increased liver fat content in man

Aims/hypothesisWe determined whether hepatic fat content and plasma adiponectin concentration regulate VLDL1 production.MethodsA multicompartment model was used to simultaneously determine the kinetic parameters of triglycerides (TGs) and apolipoprotein B (ApoB) in VLDL1 and VLDL2 after a bolus of [2H3]leucine and [2H5]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.ResultsUnivariate 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 VLDL1 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 VLDL1 in subjects with type 2 diabetes. Despite negative correlations with fasting TG levels, liver fat content, and VLDL1 TG and ApoB pool sizes, adiponectin was not linked to VLDL1 TG or ApoB production and thus was not a predictor of VLDL1 production. However, adiponectin correlated negatively with the removal rates of VLDL1 TG and ApoB.Conclusions/interpretationWe 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 VLDL1 particles and leading to the characteristic dyslipidaemia associated with type 2 diabetes.

SNARE proteins mediate fusion between cytosolic lipid droplets and are implicated in insulin sensitivity.

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), α-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 α-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.

Apolipoprotein B : a clinically important apolipoprotein which assembles atherogenic lipoproteins and promotes the development of atherosclerosis

Apolipoprotein (apo) B exists in two forms apoB100 and apoB48. ApoB100 is present on very low‐density lipoproteins (VLDL), intermediate density lipoproteins (IDL) and LDL. ApoB100 assembles VLDL particles in the liver. This process starts by the formation of a pre‐VLDL, which is retained in the cell unless converted to the triglyceride‐poor VLDL2. VLDL2 is secreted or converted to VLDL1 by a bulk lipidation in the Golgi apparatus. ApoB100 has a central role in the development of atherosclerosis. Two proteoglycan‐binding sequences in apoB100 have been identified, which are important for retaining the lipoprotein in the intima of the artery. Retention is essential for the development of the atherosclerotic lesion.

Lipid droplets as dynamic organelles connecting storage and efflux of lipids.

Neutral lipids are stored in the cytosol in so-called lipid droplets. These are dynamic organelles with neutral lipids as the core surrounded by a monolayer of amphipathic lipids (phospholipids and cholesterol) and specific proteins (PAT proteins and proteins involved in the turnover of lipids and in the formation and trafficking of the droplets). Lipid droplets are formed at microsomal membranes as primordial droplets with a diameter of 0.1-0.4 microm and increase in size by fusion. In this article, we review the assembly and fusion of lipid droplets, and the processes involved in the secretion of triglycerides. Triglycerides are secreted from cells by two principally different processes. In the mammary gland, lipid droplets interact with specific regions of the plasma membrane and bud off with an envelope consisting of the membrane, to form milk globules. In the liver and intestine, very low-density lipoproteins (VLDL) and chylomicrons are secreted by using the secretory pathway of the cell. Finally, we briefly review the importance of lipid droplets in the development of insulin resistance and atherosclerosis.

Overproduction of VLDL1 Driven by Hyperglycemia Is a Dominant Feature of Diabetic Dyslipidemia

Objective—We sought to compare the synthesis and metabolism of VLDL1 and VLDL2 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 VLDL1 and VLDL2 after a bolus injection of [2H3]leucine and [2H5]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 VLDL1 apoB and TG. Direct production of VLDL2 apoB and TG was not influenced by diabetes per se. The production rates of VLDL1 apoB and TG were closely related, as were the corresponding pool sizes. VLDL1 and VLDL2 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 VLDL1 TG production rate. Conclusion—Insulin resistance and DM2 are associated with excess hepatic production of VLDL1 particles similar in size and composition to those in nondiabetic subjects. We propose that hyperglycemia is the driving force that aggravates overproduction of VLDL1 in DM2.

The assembly and secretion of apolipoprotein B-containing lipoproteins

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.

Identification of Apo B-100 segments mediating the interaction of low density lipoproteins with arterial proteoglycans.

The Interactions of low density llpoproteln (LDL) and apollpoproteln (apo) B-100 segments with chondroltin-6-S04 rich aortic proteoglycans aggregate (CSPG) were studied by using quantitative frontal elutlon affinity chromatography. The affinity of the agarose-CSPG was higher for LDL than for very low density llpoproteln, and high density llpoproteln was not bound. LDL from different Individuals had dissociation coefficients (Kd) from 28 to 179 nM. Experiments with tryptlc hydrolysates of apo B suggested that the capacity of LDL to bind with CSPG resides in the protein. Nine apo B-100 hydrophlllc peptldes, 12 to 26 amlno acids long, were selected, and three were found to Interact with the agarose-bound CSPG: apo B P-1 (LRKHKUDVISMY- RELLKDLSKEA, residues 4230 to 4254), apo B P-2 (RLTRKRGLKLATALSLSNK, residues 3359 to 3377), and apo B P-11 (RQVSHAKEKLTALTKK, residues 2106 to2121). These peptides competed with LDL for binding to the agarose-bound and soluble CSPG; apo B P-2 was the most effective. This correlates with Kd values: 63, 86, and 82 /iM for apo B P-2, P-1, and P-11, respectively. The peptldes shared an excess of positive-charged side chains. Apo B P-2 belongs to the lysand arg-rich, LDL-receptor domain. Apo E also binds to the agarose-proteoglycan. The results suggest that apo B regions with sequences and charge distributions analogous to those of residues 3359 to 3377, 4230 to 4254, and 2106 to 2121 are among those responsible for the Interaction of LDL with Intlma-media CSPG.

Inhibition of the Microsomal Triglyceride Transfer Protein Blocks the First Step of Apolipoprotein B Lipoprotein Assembly but Not the Addition of Bulk Core Lipids in the Second Step

The microsomal triglyceride transfer protein (MTP) is required for assembly and secretion of the lipoproteins containing apolipoprotein B (apoB): very low density lipoproteins and chylomicrons. Evidence indicates that the subclasses of these lipoproteins that contain apoB-48 are assembled in a distinct two-step process; first a relatively lipid-poor primordial lipoprotein precursor is produced, and then bulk neutral lipids are added to form the core of these spherical particles. To determine if either step is mediated by MTP, a series of clonal cell lines stably expressing apoB-53 and MTP was established in non-lipoprotein-producing HeLa cells. MTP activity in these cells was ~30%, and apoB secretion was 7-33% of that in HepG2 cells on a molar basis. Despite having robust levels of triglyceride and phospholipid synthesis, these cell lines, as exemplified by HLMB53-59, secreted >90% of the apoB-53 on relatively lipid-poor particles in the density range of 1.063-1.21 g/ml. These results suggested that coexpression of MTP and apoB only reconstituted the first but not the second step in lipoprotein assembly. To extend this observation, additional studies were carried out in McArdle RH-7777 rat hepatoma cells, in which the second step of apoB-48 lipoprotein assembly is well defined. Treatment of these cells with the MTP photoaffinity inhibitor BMS-192951 before pulse labeling with [35S]methionine/cysteine led to an 85% block of both apoB-48 and apoB-100 but not apoAI secretion, demonstrating inhibition of the first step of lipoprotein assembly. After a 30-min [35S]methioneine/cysteine pulse labeling and 120 min of chase, all of the nascent apoB-48 was observed to have a density of high density lipoproteins (1.063-1.21 g/ml), indicating that only the first step of lipoprotein assembly had occurred. The addition of oleic acid to the cell culture media activated the second step as evidenced by the conversion of the apoB-48 high density lipoproteins to very low density lipoproteins (d < 1.006 g/ml) during an extended chase period. Inactivation of MTP after completion of the first step, but before stimulation of the second step by the addition of oleic acid, did not block this conversion. Thus, inhibition of MTP did not hinder the addition of bulk core lipid to the primordial lipoprotein precursor particles, indicating that MTP is not required for the second step of apoB-48 lipoprotein assembly.

Acute suppression of VLDL1 secretion rate by insulin is associated with hepatic fat content and insulin resistance

Aims/hypothesisOverproduction of VLDL1 seems to be the central pathophysiological feature of the dyslipidaemia associated with type 2 diabetes. We explored the relationship between liver fat and suppression of VLDL1 production by insulin in participants with a broad range of liver fat content.MethodsA multicompartmental model was used to determine the kinetic parameters of apolipoprotein B and TG in VLDL1 and VLDL2 after a bolus of [2H3]leucine and [2H5]glycerol during a hyperinsulinaemic–euglycaemic clamp in 20 male participants: eight with type 2 diabetes and 12 control volunteers. The participants were divided into two groups with low or high liver fat. All participants with diabetes were in the high liver-fat group.ResultsThe results showed a rapid drop in VLDL1-apolipoprotein B and -triacylglycerol secretion in participants with low liver fat during the insulin infusion. In contrast, participants with high liver fat showed no significant change in VLDL1 secretion. The VLDL1 suppression following insulin infusion correlated with the suppression of NEFA, and the ability of insulin to suppress the plasma NEFA was impaired in participants with high liver fat. A novel finding was an inverse response between VLDL1 and VLDL2 secretion in participants with low liver fat: VLDL1 secretion decreased acutely after insulin infusion whereas VLDL2 secretion increased.Conclusions/interpretationInsulin downregulates VLDL1 secretion and increases VLDL2 secretion in participants with low liver fat but fails to suppress VLDL1 secretion in participants with high liver fat, resulting in overproduction of VLDL1. Thus, liver fat is associated with lack of VLDL1 suppression in response to insulin.