Hepatic Microsomal Triglyceride Transfer Protein Research Articles

Abstract 1051: Endothelial CEPT1 Impacts Aortic Atherosclerosis And Hepatic Lipoprotein Metabolism

Background: Circulating lipids that contribute to the development of atherosclerosis are derived from very low-density lipoprotein (VLDL), produced in liver by a process requiring microsomal triglyceride transfer protein (MTTP). Since choline ethanolamine phosphotransferase 1 (CEPT1) generates phospholipids critical for membrane composition and signaling, we hypothesized that Cept1 deficiency reduces hepatic MTTP and aortic atherosclerosis. Methods: Conditional endothelial cell (EC)-specific knockdown of Cept1 ( Cept1 fl/fl Cre+ ) was performed in ApoE -/- mice maintained on a 42% high-fat diet for 12 weeks, serum lipids were measured, and aorta, aortic roots, and liver tissue were harvested (Figure, A). In addition, EC-hepatocyte (HepG2 cells) signaling was evaluated using an in vitro co-culture assay, and Mttp gene expression was assessed in HepG2 cells, cultured with and without ECs treated with Cept1 siRNA (Figure, B and C). Results: Compared to ApoE -/- mice, Cept1 fl/fl Cre+ ApoE -/- mice had significantly reduced serum cholesterol (P < .05; Figure, D) and triglyceride (P < .01; Figure, E), and reduced atherosclerotic lesions of the aorta and aortic root (P < .01 and P < .05, respectively; Figure, F-I). Cept1 fl/fl Cre+ ApoE -/- mice also demonstrated more severe hepatic steatosis and numerically greater content of liver lipid droplets (Figure, J and K). Cept1 knockdown in ECs co-cultured with HepG2 cells resulted in a significant decrease in Mttp expression (P < .01; Figure, L). Conclusion: Our findings suggest that deficiency of endothelial Cept1 significantly reduces aortic atheroprogression. This may, in part, be due to a reduction in serum lipid levels via inhibition of hepatocyte MTTP driven by signals from endothelial cells. These findings open a new avenue of exploration regarding the influence of EC signaling on hepatic lipid metabolism and its impact on hyperlipidemia and atherosclerotic cardiovascular diseases.

Dietary egg white protein hydrolysate improves orotic acid-induced fatty liver in rats by promoting hepatic phospholipid synthesis and microsomal triglyceride transfer protein expression

We investigated the effects of egg white protein hydrolysates (EWH) on orotic acid (OA)-induced nonalcoholic fatty liver (NAFL) in rats. Effects of the egg white protein (EWP) and EWH were also compared. Four groups of male Sprague-Dawley rats were separately fed AIN-76-based diets, supplemented with 20% casein for control, or with 1% OA, together with either 20% casein (OA), 20% EWP, or 20% EWH, respectively, for 3 d (developing stage) and 14 d (developed stage). In both feeding periods, animals from the OA group showed higher accumulation hepatic triacylglycerol (TAG) compared with those from the control group. In the 14-d experiment, dietary EWP and EWH significantly reduced the hepatic TAG levels. Intake of EWP reduced liver fat in OA-fed rats by 61%, while EWH reduced it by 92%. In addition, EWH restored the OA-induced high serum-TAG level to that seen in the control group. The 3 d experiment showed that consumption of EWH improved the expression of hepatic MTP, that was reduced by OA, without changing Mttp gene expression. It also increased the hepatic synthesis of PC and PE by enhancing the transcription of Pcyt1 and Pemt genes. Inclusion of EWP and EWH in the diet improves the OA-induced NAFL. EWH reduces the liver TAG better than EWP, and works more rapidly. Dietary EWH ameliorates OA-induced NAFL by promoting the secretion of hepatic TAG.

Membrane-bound sn-1,2-diacylglycerols explain the dissociation of hepatic insulin resistance from hepatic steatosis in MTTP knockout mice

Microsomal triglyceride transfer protein (MTTP) deficiency results in a syndrome of hypolipidemia and accelerated NAFLD. Animal models of decreased hepatic MTTP activity have revealed an unexplained dissociation between hepatic steatosis and hepatic insulin resistance. Here, we performed comprehensive metabolic phenotyping of liver-specific MTTP knockout (L-Mttp−/−) mice and age-weight matched wild-type control mice. Young (10–12-week-old) L-Mttp−/− mice exhibited hepatic steatosis and increased DAG content; however, the increase in hepatic DAG content was partitioned to the lipid droplet and was not increased in the plasma membrane. Young L-Mttp−/− mice also manifested normal hepatic insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps, no PKCε activation, and normal hepatic insulin signaling from the insulin receptor through AKT Ser/Thr kinase. In contrast, aged (10-month-old) L-Mttp−/− mice exhibited glucose intolerance and hepatic insulin resistance along with an increase in hepatic plasma membrane sn-1,2-DAG content and PKCε activation. Treatment with a functionally liver-targeted mitochondrial uncoupler protected the aged L-Mttp−/− mice against the development of hepatic steatosis, increased plasma membrane sn-1,2-DAG content, PKCε activation, and hepatic insulin resistance. Furthermore, increased hepatic insulin sensitivity in the aged controlled-release mitochondrial protonophore-treated L-Mttp−/− mice was not associated with any reductions in hepatic ceramide content. Taken together, these data demonstrate that differences in the intracellular compartmentation of sn-1,2-DAGs in the lipid droplet versus plasma membrane explains the dissociation of NAFLD/lipid-induced hepatic insulin resistance in young L-Mttp−/− mice as well as the development of lipid-induced hepatic insulin resistance in aged L-Mttp−/− mice.

Reduced Serum Cholesterol and Triglyceride Levels in a Choline-Deficient L-Amino Acid-Defined High-Fat Diet (CDAHFD)-Induced Mouse Model of Non-alcoholic Steatohepatitis (NASH).

Non-alcoholic fatty liver disease (NAFLD) or non-alcoholic seatohepatitis (NASH) is one of the major health problems world wide, because of increased abdominal obesity. To date, specific and effective medications to treat or prevent NAFLD/NASH have not been established. To identify appropriate molecular targets for that purpose, suitable animal models of NAFLD/NASH have been explored. A choline-deficient amino acid-defined high fat diet (CDAHFD)-induced mouse model of NASH has been developed. However, its relevance to human NASH, including serum lipid profiles, have not been clearly defined. In this study, we have revealed that mice fed CDAHFD showed significantly lowerd serum total cholesterol and triglyceride (TG) levels, in addition to reduced body weight (BW). Furthermore, hepatic microsomal triglyceride transfer protein (MTP) expression was significantly downregulated in CDAHFD-fed mice. Thus, the current CDAHFD-fed mouse model has points that are distinct from human NAFLD/NASH, in general, which is based upon abdominal obesity.

Association between hepatic steatosis and MTP gene –493G/T polymorphism in the patients with HCV genotype 1 infection

AimHepatitis C virus (HCV) affects approximately 250 million people worldwide. If patients are untreated, 80% of patients with chronic HCV develop liver failure, liver cirrhosis (LC), and hepatocellular carcinoma (HCC). HCV genotype 1 is the most prevalent among the infected individuals with HCV. Hepatic steatosis is known as accumulation of lipid molecules in hepatocytes, and its prevalence is approximately 55% in CHC infection. The reason of HCV-related hepatic steatosis in CHC infection is mainly HCV core protein. HCV core protein inhibits activities of microsomal triglyceride transfer protein (MTP) which is a lipid transfer protein expressed in the liver. The –493G/T polymorphism in the promoter region of MTP gene has been associated with HCV-related hepatic steatosis. This polymorphism in MTP gene influences MTP mRNA expression, therefore which might also affect lipid transfer. We evaluated the association between MTP gene polymorphism and the risk of HCV genotype 1-related hepatic steatosis. MethodsIn the current study, MTP gene polymorphism was explored in 144 biopsy-proven chronic HCV genotype 1 patients by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. ResultsThe results showed that there were no any difference between the steatosis and the non-steatosis groups for the allele and genotype frequencies of the -493G/T polymorphism (P > .05). Moreover, MTP genotypes (GG vs. TG + TT) were not associated with BMI, fibrosis stages and the levels of biochemical parameters. Additionally, there were statistically significant differences in the biochemical parameters including triglyceride, total cholesterol, LDL, VLDL levels between the two groups (P < .05). ConclusionsIn conclusion, the current study demonstrates for the first time that MTP gene -493G/T polymorphism has not a major effect on the risk of HCV genotype 1-related hepatic steatosis in Turkish population. Further studies are imperative to clarify the association of this polymorphism with HCV genotype 1 infection in HCV-related hepatic steatosis.

ATP binding cassette family A protein 1 determines hexosylceramide and sphingomyelin levels in human and mouse plasma

Sphingolipids, including ceramide, SM, and hexosylceramide (HxCer), are carried in the plasma by lipoproteins. They are possible markers of metabolic diseases, but little is known about their control. We previously showed that microsomal triglyceride transfer protein (MTP) is critical to determine plasma ceramide and SM, but not HxCer, levels. In human plasma and mouse models, we examined possible HxCer-modulating pathways, including the role of ABCA1 in determining sphingolipid plasma concentrations. Compared with control samples, plasma from patients with Tangier disease (deficient in ABCA1) had significantly lower HxCer (-69%) and SM (-40%) levels. Similarly, mice deficient in hepatic and intestinal ABCA1 had significantly reduced HxCer (-79%) and SM (-85%) levels. Tissue-specific ablation studies revealed that hepatic ABCA1 determines plasma HxCer and SM levels; that ablation of MTP and ABCA1 in the liver and intestine reduces plasma HxCer, SM, and ceramide levels; and that hepatic and intestinal MTP contribute to plasma ceramide levels, whereas only hepatic MTP modulates plasma SM levels. These results identify the contribution of ABCA1 to plasma SM and HxCer levels and suggest that MTP and ABCA1 are critical determinants of plasma sphingolipid levels.

Hepatic NPC1L1 promotes hyperlipidemia in LDL receptor deficient mice

Background and aimsNiemann-Pick C1-like1 (NPC1L1), a crucial cholesterol absorption receptor expressed in human intestine and liver. But in mouse it is only expressed in intestine. Previous studies elucidated that expression of human NPC1L1 in mouse liver led to increase of plasma cholesterol due to activation of absorption from bile. However, hepatic NPC1L1 function was not elucidated in LDL receptor deficient mouse (LDLR−/−) in which LDL was a main lipoprotein as in human. Methods and resultsL1-Tg/LDLR−/− mouse was created by crossing liver-specific NPC1L1 transgenic mouse (L1-Tg) with LDLR−/−. L1-Tg/LDLR−/− mice developed hyperlipidemia when fed with atherogenic diet (AD) containing 0.2% cholesterol for 21days. Compared with control mice, biliary cholesterol level in L1-Tg/LDLR−/− mice was significantly lower, plasma cholesterol level was significantly higher in L1-Tg/LDLR−/− mice under both chow diet and AD feeding. New finding in this study is augmentations of plasma TAG L1-Tg/LDLR−/− mice fed with AD. Results were shown that very low density lipoprotein (VLDL) secretion was elevated in L1-Tg/LDLR−/− mice after AD fed. The increase of VLDL secretion was further confirmed by higher expression of hepatic triacylglycerol hydrolase (TGH) and microsomal triglyceride transfer protein (MTP). ConclusionL1-Tg/LDLR−/− mouse is a humanized model to study cholesterol absorption and transportation. The results obtained from L1-Tg/LDLR−/− mouse indicated no feedback mechanism to inhibit NPC1L1 function in liver and hepatic expression of NPC1L1 correlated with VLDL secretion in hypercholesterolemia state.

ChREBP Rather Than SHP Regulates Hepatic VLDL Secretion.

The regulation of hepatic very-low-density lipoprotein (VLDL) secretion plays an important role in the pathogenesis of dyslipidemia and fatty liver diseases. VLDL is controlled by hepatic microsomal triglyceride transfer protein (MTTP). Mttp is regulated by carbohydrate response element binding protein (ChREBP) and small heterodimer partner (SHP). However, it is unclear whether both coordinately regulate Mttp expression and VLDL secretion. Here, adenoviral overexpression of ChREBP and SHP in rat primary hepatocytes induced and suppressed Mttp mRNA, respectively. However, Mttp induction by ChREBP was much more potent than suppression by SHP. Promoter assays of Mttp and the liver type pyruvate kinase gene revealed that SHP and ChREBP did not affect the transcriptional activity of each other. Mttp mRNA and protein levels of Shp−/− mice were similar to those of wild-types; however, those of Chrebp−/−Shp−/− and Chrebp−/− mice were significantly much lower. Consistent with this, the VLDL particle number and VLDL secretion rates in Shp−/− mice were similar to wild-types but were much lower in Chrebp−/− and Chrebp−/−Shp−/− mice. These findings suggest that ChREBP, rather than SHP, regulates VLDL secretion under normal conditions and that ChREBP and SHP do not affect the transcriptional activities of each other.

MicroRNA-30c reduces plasma cholesterol in homozygous familial hypercholesterolemic and type 2 diabetic mouse models

High plasma cholesterol levels are found in several metabolic disorders and their reductions are advocated to reduce the risk of atherosclerosis. A way to lower plasma lipids is to curtail lipoprotein production; however, this is associated with steatosis. We previously showed that microRNA (miR)-30c lowers diet-induced hypercholesterolemia and atherosclerosis in C57BL/6J and Apoe−/− mice. Here, we tested the effect of miR-30c on plasma lipids, transaminases, and hepatic lipids in different mouse models. Hepatic delivery of miR-30c to chow-fed leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) hypercholesterolemic and hyperglycemic mice reduced cholesterol in total plasma and VLDL/LDL by ∼28% and ∼25%, respectively, without affecting triglyceride and glucose levels. And these mice had lower plasma transaminases and creatine kinase activities than controls. Moreover, miR-30c significantly lowered plasma cholesterol and atherosclerosis in Western diet-fed Ldlr−/− mice with no effect on plasma triglyceride, glucose, and transaminases. In these studies, hepatic lipids were similar in control and miR-30c-injected mice. Mechanistic studies showed that miR-30c reduced hepatic microsomal triglyceride transfer protein activity and lipid synthesis. Thus miR-30c reduced plasma cholesterol in several diet-induced and diabetic hypercholesterolemic mice. We speculate that miR-30c may be beneficial in lowering plasma cholesterol in different metabolic disorders independent of the origin of hypercholesterolemia.

Diverse impacts of the rs58542926 E167K variant in TM6SF2 on viral and metabolic liver disease phenotypes.

A genome-wide exome association study has identified the transmembrane 6 superfamily member 2 (TM6SF2) rs58542926 variant encoding an E167K substitution as a genetic determinant of hepatic steatosis in nonalcoholic fatty liver disease (NAFLD). The roles of this variant across a spectrum of liver diseases and pathologies and on serum lipids comparing viral hepatitis to NAFLD and viral load in chronic viral hepatitis, as well as its intrahepatic molecular signature, have not been well characterized. We undertook detailed analyses in 3260 subjects with viral and nonviral liver diseases and in healthy controls. Serum inflammatory markers and hepatic expression of TM6SF2 and genes regulating lipid metabolism were assessed in a subset with chronic hepatitis C (CHC). The rs58542926 T allele was more prevalent in 502 NAFLD patients than controls (P = 0.02) but not different in cohorts with CHC (n = 2023) and chronic hepatitis B (n = 507). The T allele was associated with alterations in serum lipids and hepatic steatosis in all diseases and with reduced hepatic TM6SF2 and microsomal triglyceride transfer protein expression. Interestingly, the substitution was associated with reduced CHC viral load but increased hepatitis B virus DNA. The rs58542926 T allele had no effect on inflammation, impacted ≥F2 fibrosis in CHC and NAFLD assessed cross-sectionally (odds ratio = 1.39, 95% confidence interval 1.04-1.87, and odds ratio = 1.62, 95% confidence interval 1.03-2.52, respectively; P < 0.03 for both), but had no effect on fibrosis progression in 1174 patients with CHC and a known duration of infection. The TM6SF2 E167K substitution promotes steatosis and lipid abnormalities in part by altering TM6SF2 and microsomal triglyceride transfer protein expression and differentially impacts CHC and chronic hepatitis B viral load, while effects on fibrosis are marginal. (Hepatology 2016;64:34-46).

The effects of PCB126 on intra-hepatic mechanisms associated with non alcoholic fatty liver disease.

BackgroundNon alcoholic fatty liver disease (NAFLD) results from alteration in lipid synthesis and elimination mechanisms such as very-low density lipoprotein (VLDL) production and de novo lipogenesis. Persistent organic pollutants (POPs) are chemicals that were mostly used historically as pesticides, solvents, flame retardant, and other applications. Among POPs, polychlorinated biphenyls (PCB) have been recognized to be of environmental and potential toxicologic concerns. Specifically, PCB126 could act as endocrine disruptors and has recently been associated with hepatic fat accumulation. The purpose of the study was to investigate the effects of PCB126 on the molecular development of NAFLD using hepatocyte and rat models.MethodsHepatocytes were exposed to PCB 126 for 72 h and lipid accumulation in cells was quantified by Oil-Red-O. Rats were injected with a single dose of PCB126 or vehicle. Seven days later, liver triglycerides (TAG) content was measured along with protein quantification of hepatic microsomal triglyceride transfer protein (MTP), sterol regulatory element-binding protein 1c (SREBP1c) and diacylglycerol O-acyltransferase 2 (DGAT-2).ResultsExposure to PCB126 resulted in significant increases of lipid accumulation in hepatocytes (38 %, P <0.05) and hepatic TAG concentrations (64 %, P <0.001) in rats compared to respective control groups. Rats with fatty livers depicted lower MTP (40 %, P <0.02), higher SREBP1c (27 %, P < 0.05) and DGAT-2 (120 %, P < 0.02) protein content levels compared to Placebo group in rats.ConclusionsIt seems that exposure to PCB126 has an important emerging role in the pathophysiology of NAFLD by 1) altering elimination mechanisms such as VLDL synthesis and secretion, through MTP; and 2) increasing hepatic TAG synthesis mechanisms through DGAT 2 and SREBP1c.

Central effects of humanin on hepatic triglyceride secretion.

Humanin (HN) is an endogenous mitochondria-associated peptide that has been shown to protect against various Alzheimer's disease-associated insults, myocardial ischemia-reperfusion injury, and reactive oxygen species-induced cell death. We have shown previously that HN improves whole body glucose homeostasis by improving insulin sensitivity and increasing glucose-stimulated insulin secretion (GSIS) from the β-cells. Here, we report that intraperitoneal treatment with one of HN analogs, HNG, decreases body weight gain, visceral fat, and hepatic triglyceride (TG) accumulation in high-fat diet-fed mice. The decrease in hepatic TG accumulation is due to increased activity of hepatic microsomal triglyceride transfer protein (MTTP) and increased hepatic TG secretion. Both intravenous (iv) and intracerebroventricular (icv) infusion of HNG acutely increase TG secretion from the liver. Vagotomy blocks the effect on both iv and icv HNG on TG secretion, suggesting that the effects of HNG on hepatic TG flux are centrally mediated. Our data suggest that HN is a new player in central regulation of peripheral lipid metabolism.

Abstract 115: Microsomal Triglyceride Transfer Protein Is a Major Determinant of Plasma Ceramide And Sphingomyelin but Not of Hexosylceramide and Lactosylceramide

Sphingolipids are present in cell membranes and in plasma lipoproteins. Mechanisms of sphingolipid transport to plasma lipoproteins are unknown. We found that plasma levels of ceramide and sphingomyelin were significantly lower in homozygous abetalipoproteinemia subjects defective in microsomal triglyceride transfer protein (MTP) function compared with heterozygotes, and in mice deficient in both intestinal and hepatic MTP compared with control mice. In contrast, plasma concentrations of hexosylceramide, lactosylceramide and different sphingosines were not affected by MTP deficiency. MTP deficiency had no effect on ceramide and sphingomyelin synthesis, but reduced their secretion. MTP was found to transfer ceramide and sphingomyelin. These studies suggest that MTP is a critical determinant of plasma ceramide and sphingomyelins, but not of glycosylceramide and sphingosine. The MTP-dependent transport processes to carry ceramide and sphingomyelin might have evolved to protect against toxicity associated with cellular ceramide accretions and to supply sphingomyelin to other tissues to maintain membrane integrity and to control cellular differentiation and proliferation.

Intestine-specific MTP and global ACAT2 deficiency lowers acute cholesterol absorption with chylomicrons and HDLs

Intestinal cholesterol absorption involves the chylomicron and HDL pathways and is dependent on microsomal triglyceride transfer protein (MTP) and ABCA1, respectively. Chylomicrons transport free and esterified cholesterol, whereas HDLs transport free cholesterol. ACAT2 esterifies cholesterol for secretion with chylomicrons. We hypothesized that free cholesterol accumulated during ACAT2 deficiency may be secreted with HDLs when chylomicron assembly is blocked. To test this, we studied cholesterol absorption in mice deficient in intestinal MTP, global ACAT2, and both intestinal MTP and global ACAT2. Intestinal MTP ablation significantly increased intestinal triglyceride and cholesterol levels and reduced their transport with chylomicrons. In contrast, global ACAT2 deficiency had no effect on triglyceride absorption but significantly reduced cholesterol absorption with chylomicrons and increased cellular free cholesterol. Their combined deficiency reduced cholesterol secretion with both chylomicrons and HDLs. Thus, contrary to our hypothesis, free cholesterol accumulated in the absence of MTP and ACAT2 is unavailable for secretion with HDLs. Global ACAT2 deficiency causes mild hypertriglyceridemia and reduces hepatosteatosis in mice fed high cholesterol diets by increasing hepatic lipoprotein production by unknown mechanisms. We show that this phenotype is preserved in the absence of intestinal MTP in global ACAT2-deficient mice fed a Western diet. Further, we observed increases in hepatic MTP activity in these mice. Thus, ACAT2 deficiency might increase MTP expression to avoid hepatosteatosis in cholesterol-fed animals. Therefore, ACAT2 inhibition might avert hepatosteatosis associated with high cholesterol diets by increasing hepatic MTP expression and lipoprotein production.

Inhibition of hepatic microsomal triglyceride transfer protein &amp;ndash; a novel therapeutic option for treatment of homozygous familial hypercholesterolemia

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the low-density lipoprotein (LDL)-receptor gene (LDLR). Patients with homozygous FH (hoFH) have inherited a mutated LDLR gene from both parents, and therefore all their LDL-receptors are incapable of functioning normally. In hoFH, serum LDL levels often exceed 13 mmol/L and tendon and cutaneous xanthomata appear early (under 10 years of age). If untreated, this extremely severe form of hypercholesterolemia may cause death in childhood or in early adulthood. Based on recent data, it can be estimated that the prevalence of hoFH is about 1:500,000 or even 1:400,000. Until now, the treatment of hoFH has been based on high-dose statin treatment combined with LDL apheresis. Since the LDL cholesterol-lowering effect of statins is weak in this disease, and apheresis is a cumbersome treatment and not available at all centers, alternative novel pharmaceutical therapies are needed. Lomitapide is a newly introduced drug, capable of effectively decreasing serum LDL cholesterol concentration in hoFH. It inhibits the microsomal triglyceride transfer protein (MTTP). By inhibiting in hepatocytes the transfer of triglycerides into very low density lipoprotein particles, the drug blocks their assembly and secretion into the circulating blood. Since the very low density lipoprotein particles are precursors of LDL particles in the circulation, the reduced secretion of the former results in lower plasma concentration of the latter. The greatest concern in lomitapide treatment has been the increase in liver fat, which can be, however, counteracted by strictly adhering to a low-fat diet. Lomitapide is a welcome addition to the meager selection of drugs currently available for the treatment of refractory hypercholesterolemia in hoFH patients.

Differential impact of hepatic deficiency and total body inhibition of MTP on cholesterol metabolism and RCT in mice

Because apoB-containing lipoproteins are pro-atherogenic and their secretion by liver and intestine largely depends on microsomal triglyceride transfer protein (MTP) activity, MTP inhibition strategies are actively pursued. How decreasing the secretion of apoB-containing lipoproteins affects intracellular rerouting of cholesterol is unclear. Therefore, the aim of the present study was to determine the effects of reducing either systemic or liver-specific MTP activity on cholesterol metabolism and reverse cholesterol transport (RCT) using a pharmacological MTP inhibitor or a genetic model, respectively. Plasma total cholesterol and triglyceride levels were decreased in both MTP inhibitor-treated and liver-specific MTP knockout (L-Mttp(-/-)) mice (each P < 0.001). With both inhibition approaches, hepatic cholesterol as well as triglyceride content was consistently increased (each P < 0.001), while biliary cholesterol and bile acid secretion remained unchanged. A small but significant decrease in fecal bile acid excretion was observed in inhibitor-treated mice (P < 0.05), whereas fecal neutral sterol excretion was substantially increased by 75% (P < 0.001), conceivably due to decreased intestinal absorption. In contrast, in L-Mttp(-/-) mice both fecal neutral sterol and bile acid excretion remained unchanged. However, while total RCT increased in inhibitor-treated mice (P < 0.01), it surprisingly decreased in L-Mttp(-/-) mice (P < 0.05). These data demonstrate that: i) pharmacological MTP inhibition increases RCT, an effect that might provide additional clinical benefit of MTP inhibitors; and ii) decreasing hepatic MTP decreases RCT, pointing toward a potential contribution of hepatocyte-derived VLDLs to RCT.

Abstract 617: Intestine-Specific MTP Deficiency with ACAT2 Gene Ablation Lowers Acute Cholesterol Absorption With Chylomicrons and High-Density Lipoproteins

Intestinal cholesterol absorption involves chylomicron and high density lipoprotein (HDL) pathways. Microsomal triglyceride transfer protein (MTP) and ATP binding cassette family A protein 1 (ABCA1) are critical for cholesterol transport by these pathways, respectively. Additionally, acyl Co-A:cholesterol acyltransferase 2 (ACAT2) plays an important role in cholesterol absorption. Intestinal MTP ablation significantly increased intestinal triglyceride and cholesterol levels and reduced their acute absorption. In contrast, ACAT2 deficiency had no effect on triglyceride absorption but significantly reduced cholesterol absorption. Individual deficiencies of ACAT2 and MTP reduced cholesterol absorption with chylomicrons. We hypothesized that their combined deficiency would increase cholesterol secretion with HDL; unexpectedly, their deficiency reduced secretion with both chylomicrons and HDL. Further, we observed significant reductions in intestinal ABCA1 expression in combined deficient mice. Thus, free cholesterol is unavailable for secretion by the HDL pathway in these mice. We speculate that reductions in ABCA1 expression and HDL secretion might be secondary to massive triglyceride accumulation associated with intestinal MTP deficiency. Besides its role in cholesterol absorption, ACAT2 deficiency causes mild hypertriglyceridemia and reduces steatosis in mice fed high cholesterol diets by increasing hepatic lipoprotein production by unknown mechanisms. We show that this phenotype is preserved in the absence of intestinal MTP in ACAT2 deficient mice fed a Western diet. Further, we observed increases in hepatic MTP activity in these mice. Thus, ACAT2 deficiency might increase MTP expression to avoid steatosis. Therefore, ACAT2 inhibition might avert steatosis associated with high cholesterol diets by increasing MTP expression.

Alpha-Lipoic Acid Reduces LDL-Particle Number and PCSK9 Concentrations in High-Fat Fed Obese Zucker Rats

We characterized the hypolipidemic effects of alpha-lipoic acid (LA, R-form) and examined the associated molecular mechanisms in a high fat fed Zucker rat model. Rats (n = 8) were assigned to a high fat (HF) diet or the HF diet with 0.25% LA (HF-LA) for 30 days and pair fed to remove confounding effects associated with the anorectic properties of LA. Compared with the HF controls, the HF-LA group was protected against diet-induced obesity (102.5±3.1 vs. 121.5±3.6,% change BW) and hypercholesterolemia with a reduction in total-C (−21%), non-HDL-C (−25%), LDL-C (−16%), and total LDL particle number (−46%) and an increase in total HDL particles (∼22%). This cholesterol-lowering response was associated with a reduction in plasma PCSK9 concentration (−70%) and an increase in hepatic LDLr receptor protein abundance (2 fold of HF). Compared with the HF-fed animals, livers of LA-supplemented animals were protected against TG accumulation (−46%), likely through multiple mechanisms including: a suppressed lipogenic response (down-regulation of hepatic acetyl-CoA carboxylase and fatty acid synthase expression); enhanced hepatic fat oxidation (increased carnitine palmitoyltransferase Iα expression); and enhanced VLDL export (increased hepatic diacylglycerol acyltransferase and microsomal triglyceride transfer protein expression and elevated plasma VLDL particle number). Study results also support an enhanced fatty acid uptake (2.8 fold increase in total lipase activity) and oxidation (increased CPT1β protein abundance) in muscle tissue in LA-supplemented animals compared with the HF group. In summary, in the absence of a change in caloric intake, LA was effective in protecting against hypercholesterolemia and hepatic fat accumulation under conditions of strong genetic and dietary predisposition toward obesity and dyslipidemia.

FoxO6 Integrates Insulin Signaling With MTP for Regulating VLDL Production in the Liver

Excessive production of triglyceride-rich very low-density lipoproteins (VLDL-TG) contributes to hypertriglyceridemia in obesity and type 2 diabetes. To understand the underlying mechanism, we studied hepatic regulation of VLDL-TG production by (forkhead box O6) FoxO6, a forkhead transcription factor that integrates insulin signaling to hepatic metabolism. We showed that transgenic mice expressing a constitutively active FoxO6 allele developed hypertriglyceridemia, culminating in elevated VLDL-TG levels and impaired postprandial TG clearance. This effect resulted in part from increased hepatic VLDL-TG production. We recapitulated these findings in cultured HepG2 cells and human primary hepatocytes, demonstrating that FoxO6 promoted hepatic VLDL-TG secretion. This action correlated with the ability of FoxO6 to stimulate hepatic production of microsomal triglyceride transfer protein (MTP), a molecular chaperone that catalyzes the rate-limiting step in VLDL-TG assembly and secretion. FoxO6 was shown to bind to the MTP promoter and stimulate MTP promoter activity in HepG2 cells. This effect was inhibited by insulin, consistent with the ability of insulin to promote FoxO6 phosphorylation and disable FoxO6 DNA-binding activity. Mutations of the FoxO6 target site within the MTP promoter abrogated FoxO6-mediated induction of MTP promoter activity. Hepatic FoxO6 expression became deregulated in insulin-resistant mice with obesity and type 2 diabetes. FoxO6 inhibition in insulin-resistant liver suppressed hepatic MTP expression and curbed VLDL-TG overproduction, contributing to the amelioration of hypertriglyceridemia in obese and diabetic db/db mice. These results characterize FoxO6 as an important signaling molecule upstream of MTP for regulating hepatic VLDL-TG production.

ω‐3 Fatty Acids Prevent Hepatic Steatosis, Independent of PPAR‐α Activity, in a Murine Model of Parenteral Nutrition–Associated Liver Disease

ω-3 Fatty acids (FAs), natural ligands for the peroxisome proliferator-activated receptor-α (PPAR-α), attenuate parenteral nutrition-associated liver disease (PNALD). However, the mechanisms underlying the protective role of ω-3 FAs are still unknown. The aim of this study was to determine the effects of ω-3 FAs on hepatic triglyceride (TG) accumulation in a murine model of PNALD and to investigate the role of PPAR-α and microsomal triglyceride transfer protein (MTP) in this experimental setting. 129S1/SvImJ wild-type or 129S4/SvJaePparatm/Gonz/J PPAR-α knockout mice were fed chow and water (controls); oral, fat-free PN solution only (PN-O); PN-O plus intraperitoneal (IP) ω-6 FA-predominant supplements (PN-ω-6); or PN-O plus IP ω-3 FA (PN-ω-3). Control and PN-O groups received sham IP injections of 0.9% NaCl. Hepatic histology, TG and cholesterol, MTP activity, and PPAR-α messenger RNA were assessed after 19 days. In all experimental groups, PN feeding increased hepatic TG and MTP activity compared with controls. Both PN-O and PN-ω-6 groups accumulated significantly greater amounts of TG when compared with PN-ω-3 mice. Studies in PPAR-α null animals showed that PN feeding increases hepatic TG as in wild-type mice. PPAR-α null mice in the PN-O and PN-ω-6 groups demonstrated variable degrees of hepatic steatosis, whereas no evidence of hepatic fat accumulation was found after 19 days of oral PN plus IP ω-3 FAs. PN induces TG accumulation (steatosis) in wild-type and PPAR-α null mice. In PN-fed wild-type and PPAR-α null mice given IP ω-3 FAs, reduced hepatic TG accumulation and absent steatosis are found. Prevention of steatosis by ω-3 FAs results from PPAR-α-independent pathways.