Rita Kohen Avramoglu

Tumor Necrosis Factor-α Induces Intestinal Insulin Resistance and Stimulates the Overproduction of Intestinal Apolipoprotein B48-Containing Lipoproteins

There is growing evidence suggesting intestinal insulin resistance and overproduction of apolipoprotein (apo) B48–containing chylomicrons in insulin-resistant states. In the current study, we investigated the potential role of the inflammatory cytokine tumor necrosis factor-α (TNF-α) in the development of insulin resistance and aberrant lipoprotein metabolism in the small intestine in a Syrian golden hamster model. TNF-α infusion decreased whole-body insulin sensitivity, based on in vivo euglycemic clamp studies in chow-fed hamsters. Analysis of intestinal tissue in TNF-α–treated hamsters indicated impaired phosphorylation of insulin receptor-β, insulin receptor substrate-1, Akt, and Shc and increased phosphorylation of p38, extracellular signal–related kinase-1/2, and Jun NH2-terminal kinase. TNF-α infusion also increased intestinal production of total apoB48, triglyceride-rich lipoprotein apoB48, and serum triglyceride levels in both fasting and postprandial (fat load) states. The effects of TNF-α on plasma apoB48 levels could be blocked by the p38 inhibitor SB203580. Ex vivo experiments using freshly isolated enterocytes also showed TNF-α–induced p38 phosphorylation and intestinal apoB48 overproduction, effects that could be blocked by SB203580. Interestingly, TNF-α increased the mRNA and protein mass of intestinal microsomal triglyceride transfer protein without altering apoB mRNA levels. Enterocytes were found to have detectable levels of both TNF-α receptor types (p55 and p75), and antibodies against either of the two TNF-α receptors partially blocked the stimulatory effect of TNF-α on apoB48 production and p38 phosphorylation. In summary, these data suggest that intestinal insulin resistance can be induced in hamsters by TNF-α infusion, and it is accompanied by intestinal overproduction of apoB48-containing lipoproteins. TNF-α–induced stimulation of intestinal lipoprotein production appears to be mediated via TNF-α receptors and the p38 mitogen-activated protein kinase pathway.

Mechanisms of metabolic dyslipidemia in insulin resistant states: deregulation of hepatic and intestinal lipoprotein secretion.

The growing epidemic of the metabolic syndrome is now well recognized and there is widespread effort to understand the pathogenesis of this complex syndrome and its major metabolic consequences. One of the severe complications accompanying insulin resistant states is the hypertriglyceridemia that appears to occur largely due to overproduction of triglyceride-rich, apolipoprotein B (apoB) containing-lipoproteins. As a result, mechanisms regulating the overproduction of these atherogenic apoB-containing lipoproteins have been the focus of much investigation in recent years. Both in vitro as well as in vivo models of insulin resistance are currently being used to further our understanding of the mechanisms involved in the deregulation of lipid metabolism in insulin resistant states. Evidence from these animal models as well as human studies has identified hepatic very low density lipoprotein (VLDL) overproduction as a critical underlying factor in the development of hypertriglyceridemia and metabolic dyslipidemia. In recent years, a dietary animal model of insulin resistance, the fructose-fed hamster model developed in our laboratory, has proven invaluable in studies of the link between development of an insulin resistant state, derangement of hepatic lipoprotein metabolism, and overproduction of apoB-containing lipoproteins. Evidence from the fructose-fed hamster model now indicates oversecretion of both hepatically-derived apoB100-containing VLDL as well as intestinal apoB48-containing triglyceride-rich lipoproteins in insulin resistant states. A number of novel intracellular factors that may be involved in modulation of VLDL have also been identified. This review focuses on these recent developments and examines the hypothesis that a complex interaction among enhanced flux of free fatty acids from peripheral tissues to liver and intestine, chronic up-regulation of de novo lipogenesis by hyperinsulinemia, and attenuated insulin signaling in the liver and the intestine may be critical to lipoprotein overproduction accompanying insulin resistance.

Mechanisms of glucosamine-induced suppression of the hepatic assembly and secretion of apolipoprotein B-100-containing lipoproteins

Glucosamine-induced endoplasmic reticulum (ER) stress was recently shown to specifically reduce apolipoprotein B-100 (apoB-100) secretion by enhancing the proteasomal degradation of apoB-100. Here, we examined the mechanisms linking glucosamine-induced ER stress and apoB-lipoprotein biogenesis. Trypsin sensitivity studies suggested glucosamine-induced changes in apoB-100 conformation. Endoglycosidase H studies of newly synthesized apoB-100 revealed glucosamine induced N-linked glycosylation defects resulting in reduced apoB-100 secretion. We also examined glucosamine-induced changes in VLDL assembly and secretion. A dose-dependent (1–10 mM glucosamine) reduction was observed in VLDL-apoB-100 secretion in primary hepatocytes (24.2–67.3%) and rat McA-RH7777 cells (23.2–89.5%). Glucosamine also inhibited the assembly of larger VLDL-, LDL-, and intermediate density lipoprotein-apoB-100 but did not affect smaller HDL-sized apoB-100 particles. Glucosamine treatment during the chase period (posttranslational) led to a 24% reduction in apoB-100 secretion (P < 0.01; n = 4) and promoted post-ER apoB degradation. However, the contribution of post-ER apoB-100 degradation appeared to be quantitatively minor. Interestingly, the glucosamine-induced posttranslational reduction in apoB-100 secretion could be partially prevented by treatment with desferrioxamine or vitamin E. Together, these data suggest that cotranslational glucosamine treatment may cause defects in apoB-100 N-linked glycosylation and folding, resulting in enhanced proteasomal degradation. Posttranslationally, glucosamine may interfere with the assembly process of apoB lipoproteins, leading to post-ER degradation via nonproteasomal pathways.

Hepatic Regulation of Apolipoprotein B

Abbreviations: ACAT: Acyl CoA:cholesterol acyltransferase; apoB: Apolipoprotein B; COP: coat protein complex; ER: endoplasmic reticulum; ERAD: ERassociated degradation; FFA: free fatty acid; hsp70: heat shock protein 70; hsp90: heat shock protein 90; HDL: high density lipoprotein; LDL: low density lipoprotein; MTP: microsomal triglyceride transfer protein; pCMB: p-chloromercuribenzoate; PTP-1B: protein tyrosine phosphatase 1B; SREBP: sterol regulatory element binding protein; UTR: untranslated region; VLDL: very low density lipoprotein.

Phosphatase and Tensin Homolog (PTEN) Regulates Hepatic Lipogenesis, Microsomal Triglyceride Transfer Protein, and the Secretion of Apolipoprotein B-Containing Lipoproteins

Hepatic apolipoprotein B (apoB) lipoprotein production is metabolically regulated via the phosphoinositide 3‐kinase cascade; however, the role of the key negative regulator of this pathway, the tumor suppressor phosphatase with tensin homology (PTEN), is unknown. Here, we demonstrate that hepatic protein levels of apoB100 and microsomal triglyceride transfer protein (MTP) are significantly down‐regulated (73% and 36%, respectively) in the liver of PTEN liver‐specific knockout (KO) mice, and this is accompanied by increased triglyceride (TG) accumulation and lipogenic gene expression, and reduced hepatic apoB secretion in freshly isolated hepatocytes. MTP protein mass and lipid transfer activity were also significantly reduced in liver of PTEN KO mice. Overexpression of the dominant negative mutant PTEN C/S124 (adenovirus expressing PTEN C/S mutant [AdPTENC/S]) possessing constitutive phospoinositide 3‐kinase activity in HepG2 cells led to significant reductions in both secreted apoB100 and cellular MTP mass (76% and 34%, respectively), and increased messenger RNA (mRNA) levels of sterol regulatory element binding protein 1c (SREBP‐1c), fatty acid synthase (FAS), and acetyl‐CoA carboxylase (ACC). Reduced apoB100 secretion induced by AdPTENC/S was associated with increased degradation of newly‐synthesized cellular apoB100, in a lactacystin‐sensitive manner, suggesting enhanced proteasomal degradation. AdPTENC/S also reduced apoB‐lipoprotein production in McA‐RH7777 and primary hamster hepatocytes. Our findings suggest a link between PTEN expression and hepatic production of apoB‐containing lipoproteins. We postulate that perturbations in PTEN not only may influence hepatic insulin signaling and hepatic lipogenesis, but also may alter hepatic apoB‐lipoprotein production and the MTP stability. On loss of PTEN activity, increased lipid substrate availability in the face of reduced hepatic lipoprotein production capacity can rapidly lead to hepatosteatosis and fatty liver. (HEPATOLOGY 2008;48:1799–1809.)

Oleate-mediated stimulation of microsomal triglyceride transfer protein (MTP) gene promoter: implications for hepatic MTP overexpression in insulin resistance.

Hepatic lipoprotein overproduction in a fructose-fed hamster model of insulin resistance was previously shown to be associated with a significant elevation of intracellular mass of microsomal triglyceride transfer protein (MTP) and elevated plasma levels of free fatty acids (FFA). Here, we further establish that fructose feeding and development of an insulin resistant state result in higher levels of MTP mRNA, protein mass, and lipid transfer activity. MTP protein mass was increased in fructose-fed hamster hepatocytes to 161 +/- 35.8% of control (p < 0.05), while MTP mRNA levels and MTP lipid transfer activity were increased to 147.5 +/- 30.8% (p < 0.05) and 177.5 +/- 14.5% (p < 0.05) of control levels, respectively. To identify underlying mechanisms, we also investigated the potential link between enhanced FFA flux and hepatic MTP gene expression. Direct modulation of MTP gene transcription by fatty acids was investigated by transfecting HepG2 cells with a reporter (luciferase) construct containing various base pair regions of the human MTP promoter including pMTP124 (with the sterol response element (SRE)), pMTP116, pMTP109 and pMTP100 (no SRE), and pMTP124SREKO (SRE sequences mutated). Treatment of HepG2 cells with oleic acid (360 muM) significantly increased luciferase activities in cells transfected with pMTP124 (136.6 +/- 11.0%, p < 0.05) and pMTP124SREKO (153.9 +/- 11.1%, p < 0.01) compared with control. Luciferase activity was also increased in a time and dose-dependent manner in the presence of oleic acid when transfected with pMTP124SREKO but not pMTP109 and pMTP100. Furthermore, long-term oleic acid treatment of HepG2 cells (10 days) induced higher levels of MTP mRNA (p < 0.05) confirming transcriptional stimulation of the MTP gene by oleic acid. In contrast, palmitate, arachidonic acid or linoleic acid did not significantly stimulate pMTP124 or pMTP124SREKO luciferase activity (p > 0.05). These data demonstrate that (1) MTP gene transcription may be directly up-regulated by oleic acid; (2) up-regulation of MTP gene transcription by oleic acid is SRE sequence independent; and (3) the sequence -116 to -109 in the MTP promoter region is essential for oleic acid-mediated stimulation. Stimulation of MTP gene expression may be a novel mechanism by which certain FFAs can induce hepatic lipoprotein secretion in insulin resistant states.

Early Development of Calcific Aortic Valve Disease and Left Ventricular Hypertrophy in a Mouse Model of Combined Dyslipidemia and Type 2 Diabetes Mellitus

Objective— This study aimed to determine the potential impact of type 2 diabetes mellitus on left ventricular dysfunction and the development of calcified aortic valve disease using a dyslipidemic mouse model prone to developing type 2 diabetes mellitus. Approach and Results— When compared with nondiabetic LDLr−/−/ApoB100/100, diabetic LDLr−/−/ApoB100/100/IGF-II mice exhibited similar dyslipidemia and obesity but developed type 2 diabetes mellitus when fed a high-fat/sucrose/cholesterol diet for 6 months. LDLr−/−/ApoB100/100/IGF-II mice showed left ventricular hypertrophy versus C57BL6 but not LDLr−/−/ApoB100/100 mice. Transthoracic echocardiography revealed significant reductions in both left ventricular systolic fractional shortening and diastolic function in high-fat/sucrose/cholesterol fed LDLr−/−/ApoB100/100/IGF-II mice when compared with LDLr−/−/ApoB100/100. Importantly, we found that peak aortic jet velocity was significantly increased in LDLr−/−/ApoB100/100/IGF-II mice versus LDLr−/−/ApoB100/100 animals on the high-fat/sucrose/cholesterol diet. Microtomography scans and Alizarin red staining indicated calcification in the aortic valves, whereas electron microscopy and energy dispersive x-ray spectroscopy further revealed mineralization of the aortic leaflets and the presence of inflammatory infiltrates in diabetic mice. Studies showed upregulation of hypertrophic genes (anp, bnp, b-mhc) in myocardial tissues and of osteogenic genes (spp1, bglap, runx2) in aortic tissues of diabetic mice. Conclusions— We have established the diabetes mellitus –prone LDLr−/−/ApoB100/100/IGF-II mouse as a new model of calcified aortic valve disease. Our results are consistent with the growing body of clinical evidence that the dysmetabolic state of type 2 diabetes mellitus contributes to early mineralization of the aortic valve and calcified aortic valve disease pathogenesis.

Hepatocyte‐specific Ptpn6 deletion promotes hepatic lipid accretion, but reduces NAFLD in diet‐induced obesity: Potential role of PPARγ

Hepatocyte‐specific Shp1 knockout mice (Ptpn6H‐KO) are protected from hepatic insulin resistance evoked by high‐fat diet (HFD) feeding for 8 weeks. Unexpectedly, we report herein that Ptpn6H‐KO mice fed an HFD for up to 16 weeks are still protected from insulin resistance, but are more prone to hepatic steatosis, as compared with their HFD‐fed Ptpn6f/f counterparts. The livers from HFD‐fed Ptpn6H‐KO mice displayed 1) augmented lipogenesis, marked by increased expression of several hepatic genes involved in fatty acid biosynthesis, 2) elevated postprandial fatty acid uptake, and 3) significantly reduced lipid export with enhanced degradation of apolipoprotein B (ApoB). Despite more extensive hepatic steatosis, the inflammatory profile of the HFD‐fed Ptpn6H‐KO liver was similar (8 weeks) or even improved (16 weeks) as compared to their HFD‐fed Ptpn6f/f littermates, along with reduced hepatocellular damage as revealed by serum levels of hepatic enzymes. Interestingly, comparative microarray analysis revealed a significant up‐regulation of peroxisome proliferator‐activated receptor gamma (PPARγ) gene expression, confirmed by quantitative polymerase chain reaction. Elevated PPARγ nuclear activity also was observed and found to be directly regulated by Shp1 in a cell‐autonomous manner. Conclusion: These findings highlight a novel role for hepatocyte Shp1 in the regulation of PPARγ and hepatic lipid metabolism. Shp1 deficiency prevents the development of severe hepatic inflammation and hepatocellular damage in steatotic livers, presenting hepatocyte Shp1 as a potential novel mediator of nonalcoholic fatty liver diseases in obesity. (Hepatology 2014;59:1803–1815)

Lipoprotein Metabolism in Insulin-Resistant States

The incidence of insulin-resistant states, such as type 2 diabetes and obesity, has been rapidly increasing in both adult and pediatric populations worldwide. A major complication of insulin resistance is an atherogenic dyslipidemia that contributes to a significantly higher risk of atherosclerosis and cardiovascular disease. The most fundamental defect in these patients is resistance to cellular actions of insulin, particularly resistance to insulin-stimulated glucose uptake. Insulin insensitivity appears to cause hyperinsulinemia, enhanced hepatic gluconeogenesis and glucose output, reduced suppression of lipolysis in adipose tissue leading to a high free fatty acid (FFA) flux, and increased very low-density lipoprotein (VLDL) secretion causing hypertriglyceridemia and reduced plasma levels of high-density lipoprotein (HDL) cholesterol. Although the link between insulin resistance and dysregulation of lipoprotein metabolism is well established, a significant gap of knowledge exists regarding the underlying cellular and molecular mechanisms. Genetic and diet-induced animal models of insulin resistance have been recently employed to delineate the mechanistic link between perturbations in insulin-signaling pathways and dysregulation of hepatic lipid and lipoprotein metabolism in insulinresistant states. A series of important and novel observations have been made and published in recent years that will be summarized in this chapter. The critical role of key phosphatases and protein kinases that mediate the signaling changes leading to dysregulation of hepatic lipogenesis and VLDL overproduction will be discussed. Emerging evidence suggests that insulin resistance and its associated metabolic dyslipidemia result from perturbations in key molecules of the insulin-signaling pathway, including overexpression of phosphatases, protein tyrosine phosphatase 1B (PTP-1B) and phosphatase and tensin homolog (PTEN), downregulation of the phosphatidylinositol-3-kinase (PI-3-K) pathway and basal activation of the mitogen-activated protein (MAP) kinase cascade, leading to a state of mixed hepatic insulin resistance and sensitivity. These signaling changes in turn cause an increased expression of sterol regulatory elementbinding protein (SREBP) 1c, induction of de novo lipogenesis and higher activity of microsomal triglyceride transfer protein (MTP), which together with high exogenous FFA flux collectively stimulates the hepatic production of apolipoprotein B (apoB)containing VLDL particles.