Very Low Density Lipoprotein Secretion Research Articles

Silencing of ANGPTL 3 (angiopoietin-like protein 3) in human hepatocytes results in decreased expression of gluconeogenic genes and reduced triacylglycerol-rich VLDL secretion upon insulin stimulation.

Homozygosity of loss-of-function mutations in ANGPTL3 (angiopoietin-like protein 3)-gene results in FHBL2 (familial combined hypolipidaemia, OMIM #605019) characterized by the reduction of all major plasma lipoprotein classes, which includes VLDL (very-low-density lipoprotein), LDL (low-density lipoprotein), HDL (high-density lipoprotein) and low circulating NEFAs (non-esterified fatty acids), glucose and insulin levels. Thus complete lack of ANGPTL3 in humans not only affects lipid metabolism, but also affects whole-body insulin and glucose balance. We used wild-type and ANGPTL3-silenced IHHs (human immortalized hepatocytes) to investigate the effect of ANGPTL3 silencing on hepatocyte-specific VLDL secretion and glucose uptake. We demonstrate that both insulin and PPARγ (peroxisome-proliferator-activated receptor γ) agonist rosiglitazone down-regulate the secretion of ANGPTL3 and TAG (triacylglycerol)-enriched VLDL1-type particles in a dose-dependent manner. Silencing of ANGPTL3 improved glucose uptake in hepatocytes by 20–50% and influenced down-regulation of gluconeogenic genes, suggesting that silencing of ANGPTL3 improves insulin sensitivity. We further show that ANGPTL3-silenced cells display a more pronounced shift from the secretion of TAG-enriched VLDL1-type particles to secretion of lipid poor VLDL2-type particles during insulin stimulation. These data suggest liver-specific mechanisms involved in the reported insulin-sensitive phenotype of ANGPTL3-deficient humans, featuring lower plasma insulin and glucose levels.

Deficiency in Nrf2 transcription factor decreases adipose tissue mass and hepatic lipid accumulation in leptin-deficient mice.

To evaluate whether Nrf2 deficiency impacts insulin resistance and lipid accumulation in liver and white adipose tissue. Lep(ob/ob) mice (OB) with targeted Nrf2 deletion (OB-Nrf2KO) were generated. Pathogenesis of obesity and type 2 diabetes was measured in C57BL/6J, Nrf2KO, OB, and OB-Nrf2KO mice. Hepatic lipid content, lipid clearance, and very low-density lipoprotein (VLDL) secretion were determined between OB and OB-Nrf2KO mice. OB-Nrf2KO mice exhibited decreased white adipose tissue mass and decreased adipogenic and lipogenic gene expression compared with OB mice. Nrf2 deficiency prolonged hyperglycemia in response to glucose challenge, which was paralleled by reduced insulin-stimulated Akt phosphorylation. In OB mice, Nrf2 deficiency decreased hepatic lipid accumulation, decreased peroxisome proliferator-activated receptor γ expression and nicotinamide adenine dinucleotide phosphate (NADPH) content, and enhanced VLDL secretion. However, this observation was opposite in lean mice. Additionally, OB-Nrf2KO mice exhibited increased plasma triglyceride content, decreased HDL-cholesterol content, and enhanced apolipoprotein B expression, suggesting Nrf2 deficiency caused dyslipidemia in these mice. Nrf2 deficiency in Lep(ob/ob) mice reduced white adipose tissue mass and prevented hepatic lipid accumulation but induced insulin resistance and dyslipidemia. This study indicates a dual role of Nrf2 during metabolic dysregulation-increasing lipid accumulation in liver and white adipose tissue but preventing lipid accumulation in obese mice.

The TM6SF2 Variants, Novel Genetic Predictors for Nonalcoholic Steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of liver disease ranging from benign steatosis (hepatocellular triglyceride [TG] accumulation of >5% of liver weight) through nonalcoholic steatohepatitis (NASH, which is steatosis combined with inflammation and ballooning degeneration) to fibrosis and ultimately cirrhosis and hepatocellular carcinoma (HCC), in the absence of excessive alcohol consumption (Gastroenterology 2007;132:2191–2207; Nat Rev Gastroenterol Hepatol 2013;10:645–655). NAFLD is linked with the features of metabolic syndrome such as obesity, insulin resistance, type 2 diabetes mellitus and dyslipidemia. Recently, 3 independent groups have reported that the single nucleotide polymorphisms (SNPs) related to transmembrane 6 superfamily member 2 (TM6SF2) are associated with NAFLD development. The first paper reported by Kozlitina et al. identified the association between hepatic TG content determined by proton magnetic resonance spectroscopy (1H-MRS) and the TM6SF2 variants (rs58542926) through exome-wide association study in a multiethnic, multi-ancestry, population-based cohort derived from the Dallas Heart Study (DHS; total 2,736 participants; 1,324 African Americans, 882 European Americans, 467 Hispanic, and 63 other ethnicities). This TM6SF2 variant is an adenine-to-guanine transversion at nucleotide position 499, resulting in the replacement of the 167th glutamate by lysine (c.499A>G; p.Glu167Lys). The Glu167 of TM6SF2 is highly conserved among mammals. This variant is more common in people of European-American (7.2%) than of Hispanic (4.7%) or African-American (3.4%) descent. The investigators determined the association of the TM6SF2 variant with elevations in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as a surrogate for NASH through association studies using 3 cohorts (DHS, the Dallas Biobank and the Copenhagen study). The TM6SF2 variant encoding p.Glu167Lys correlated with the increases in serum ALT and AST levels and the decreases in plasma levels of TG and low-density lipoprotein (LDL) cholesterols, but there was no association with the levels of plasma high-density lipoprotein (HDL) cholesterols. The elevated hepatic TG content together with the reduced plasma TG and cholesterol levels suggest TM6SF2 variant to be associated with the secretion of the very-low-density lipoprotein (VLDL). The researchers then performed functional analysis for the TM6SF2 in mouse liver by silencing Tm6sf2 using adeno-associated viral vector that selectively infects the liver. Silencing Tm6sf2 in the liver caused 3-fold increases in hepatic TG levels and decreases in plasma levels of TG, both LDL and HDL cholesterols and TG content of VLDL. Consistently, the VLDL secretion rates were suppressed in Tm6sf2-silenced mice, whereas ALT levels were unchanged. The investigators also tested the effects of a high sucrose diet that enhances hepatic TG synthesis. TG and cholesterols were increased in the liver and decreased in plasma in Tm6sf2-silenced mice fed a high sucrose diet. Their results demonstrated that TM6SF2 regulates hepatic TG secretion; thus, functional impairment of TM6SF2 would increase hepatic TG content, thereby promoting NAFLD. The second study reported by Mahdessian et al used expression quantitative trait locus analysis and showed that the rs10401969 SNP was associated with plasma lipid levels. Using 206 human liver biopsy specimens, the authors reported that the rs10401969 SNP was associated with decreased hepatic mRNA levels of TM6SF2. Furthermore, they found that a positive correlation between hepatic TM6SF2 mRNA and plasma TG levels, but did not find any correlation between hepatic TM6SF2 and plasma LDL and HDL cholesterols. The study then examined the subcellular localization and function of TM6SF2. TM6SF2 is mainly localized in the endoplasmic reticulum and endoplasmic reticulum–Golgi intermediate compartment in human hepatoma cells. The TM6SF2 silencing in hepatoma cell lines reduced the expression of TG synthesis-related genes (ACSS2, DGAT1, DGAT2) and the secretion of TG-rich lipoprotein. The TM6SF2 silencing increased the size and the number of lipid droplets whereas overexpression of full-length TM6SF2 showed a decrease in number and size of lipid droplets. Consistent with the first study (Nat Genet 2014;46:352–356), the current study also demonstrates TM6SF2 to regulate hepatic lipoprotein secretion. Moreover, TM6SF2 was found to influence hepatic TG content through gene regulation of TG synthesis. The third study reported by Liu et al analyzed the relationship between the TM6SF2 rs58542926 SNP and NASH-associated fibrosis/cirrhosis and HCC. Utilizing a (n = 349) “discovery” and (n = 725) “validation” cohort of patients with biopsy-proven NAFLD, the authors found that the TM6SF2 rs58542926 SNP was associated with both the severity of NASH (necroinflammation and ballooning hepatocytes) and advanced liver fibrosis (advanced fibrosis [F2–F4] versus mild fibrosis [F0–F1]) in patients with NAFLD. Although combining the 2 cohorts showed that homozygous individuals have an increased incidence of NAFLD-related HCC, these findings were no longer significant after the adjustment of age, gender, BMI, and type 2 diabetes mellitus to the regression models. Taken together, these 3 studies provide evidence that the TM6SF2 variant is associated with the development of NAFLD/NASH through regulation of lipid metabolism.

S-Adenosylmethionine increases circulating very-low density lipoprotein clearance in non-alcoholic fatty liver disease

Very-low-density lipoproteins (VLDLs) export lipids from the liver to peripheral tissues and are the precursors of low-density-lipoproteins. Low levels of hepatic S-adenosylmethionine (SAMe) decrease triglyceride (TG) secretion in VLDLs, contributing to hepatosteatosis in methionine adenosyltransferase 1A knockout mice but nothing is known about the effect of SAMe on the circulating VLDL metabolism. We wanted to investigate whether excess SAMe could disrupt VLDL plasma metabolism and unravel the mechanisms involved. Glycine N-methyltransferase (GNMT) knockout (KO) mice, GNMT and perilipin-2 (PLIN2) double KO (GNMT-PLIN2-KO) and their respective wild type (WT) controls were used. A high fat diet (HFD) or a methionine deficient diet (MDD) was administrated to exacerbate or recover VLDL metabolism, respectively. Finally, 33 patients with non-alcoholic fatty-liver disease (NAFLD); 11 with hypertriglyceridemia and 22 with normal lipidemia were used in this study. We found that excess SAMe increases the turnover of hepatic TG stores for secretion in VLDL in GNMT-KO mice, a model of NAFLD with high SAMe levels. The disrupted VLDL assembly resulted in the secretion of enlarged, phosphatidylethanolamine-poor, TG- and apoE-enriched VLDL-particles; special features that lead to increased VLDL clearance and decreased serum TG levels. Re-establishing normal SAMe levels restored VLDL secretion, features and metabolism. In NAFLD patients, serum TG levels were lower when hepatic GNMT-protein expression was decreased. Excess hepatic SAMe levels disrupt VLDL assembly and features and increase circulating VLDL clearance, which will cause increased VLDL-lipid supply to tissues and might contribute to the extrahepatic complications of NAFLD.

Selective regulation of hepatic lipid metabolism by the AMP-activated protein kinase pathway in late-pregnant rats.

The liver plays an essential role in maternal metabolic adaptation during late pregnancy. With regard to lipid metabolism, increased secretion of very low-density lipoprotein (VLDL) is characteristic of late pregnancy. Despite this well-described metabolic plasticity, the molecular changes underlying the hepatic adaptation to pregnancy remain unclear. As AMPK is a key intracellular energy sensor, we investigated whether this protein assumes a causal role in the hepatic adaptation to pregnancy. Pregnant Wistar rats were treated with vehicle or AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) for 5 days starting at gestational day 14. At the end of treatment, the rats were subjected to an intraperitoneal pyruvate tolerance test and in situ liver perfusion with pyruvate. The livers were processed for Western blot analysis, quantitative PCR, thin-layer chromatography, enzymatic activity, and glycogen content measurements. Blood biochemical profiles were also assessed. We found that AMPK and ACC phosphorylation were reduced in the livers of pregnant rats in parallel with a reduced level of hepatic gluconeogenesis of pyruvate. This effect was accompanied by both a reduction in the levels of hepatic triglycerides (TG) and an increase in circulating levels of TG. Treatment with AICAR restored hepatic levels of TG to those observed in nonpregnant rats. Additionally, AMPK activation reduced the upregulation of genes related to VLDL synthesis and secretion observed in the livers of pregnant rats. We conclude that the increased secretion of hepatic TG in late pregnancy is concurrent with a transcriptional profile that favors VLDL production. This transcriptional profile results from the reduction in hepatic AMPK activity.

Modulation of Triglyceride and Cholesterol Ester Synthesis Impairs Assembly of Infectious Hepatitis C Virus

In hepatitis C virus infection, replication of the viral genome and virion assembly are linked to cellular metabolic processes. In particular, lipid droplets, which store principally triacylglycerides (TAGs) and cholesterol esters (CEs), have been implicated in production of infectious virus. Here, we examine the effect on productive infection of triacsin C and YIC-C8-434, which inhibit synthesis of TAGs and CEs by targeting long-chain acyl-CoA synthetase and acyl-CoA:cholesterol acyltransferase, respectively. Our results present high resolution data on the acylglycerol and cholesterol ester species that were affected by the compounds. Moreover, triacsin C, which blocks both triglyceride and cholesterol ester synthesis, cleared most of the lipid droplets in cells. By contrast, YIC-C8-434, which only abrogates production of cholesterol esters, induced an increase in size of droplets. Although both compounds slightly reduced viral RNA synthesis, they significantly impaired assembly of infectious virions in infected cells. In the case of triacsin C, reduced stability of the viral core protein, which forms the virion nucleocapsid and is targeted to the surface of lipid droplets, correlated with lower virion assembly. In addition, the virus particles that were released from cells had reduced specific infectivity. YIC-C8-434 did not alter the association of core with lipid droplets but appeared to decrease production of infectious virus particles, suggesting a block in virion assembly. Thus, the compounds have antiviral properties, indicating that targeting synthesis of lipids stored in lipid droplets might be an option for therapeutic intervention in treating chronic hepatitis C virus infection.

Abstract 634: Treatment with Mipomersen Reduces Levels of ApoB-Containing Lipoproteins by Increasing Fractional Removal of VLDL and LDL-apoB Without Reducing VLDL-apob Secretion

Objectives: Mipomersen (MIPO), a second generation antisense oligonucleotide, targets apoB mRNA, thereby inhibiting apolipoprotein B (apoB) synthesis. In humans, MIPO reduces plasma levels of low density lipoprotein-cholesterol (LDL-C), and plasma triglycerides (TG). We hypothesized that these changes are due to reduced assembly and secretion of very low density lipoproteins (VLDL) and lower production of LDL. Methods: Healthy volunteers (HVs) (9M, 8F), mean age 43.5 ± 14.2 yr, completed a single-blind, fixed-sequence, phase I study. They received sc-placebo injections once weekly for 3-wks followed by 200mg sc-MIPO injections once weekly for 7-9 wks. Stable isotope turnover studies were performed after each treatment. Blood samples were collected over 48-hrs to determine fractional catabolic rates (FCRs) and production rates (PRs) of apoB in VLDL, IDL, and LDL, and of TG in VLDL. Rates of de novo lipogenesis (DNL) were also measured. Results: MIPO treatment resulted in significant reductions in plasma LDL-C (45%), TG (29%), and apoB (40%). VLDL, IDL, and LDL apoB levels fell by 29%, 25%, and 42%, respectively. These changes were associated with increases in FCRs of VLDL apoB (42%) and LDL apoB (30%), and by reductions in PRs of IDL apoB (15%) and LDL apoB (27%). The PR of VLDL apoB was unaffected. The FCR of VLDL-TG increased 46% without change in PR. DNL did not change. Conclusion: In summary, 7 wks of MIPO significantly reduced levels of all apoB-lipoproteins in HVs by increasing the FCRs of VLDL and LDL apoB. The absence of a reduction in VLDL apoB secretion is consistent with many studies in isolated hepatocytes demonstrating both intracellular degradation and secretion of newly synthesized apoB. Thus, if MIPO submaximally inhibited apoB synthesis in this study, the liver could have compensated by increasing the efficiency of VLDL assembly and secretion. The basis of increases in VLDL and LDL FCRs requires further investigation.

Role of ACOT1 in hepatic lipid trafficking (821.6)

Peroxisome proliferator‐activated regulator alpha (PPARα) is a major regulator of fasting lipid metabolism. PPARα induces transcription of cytosolic Acyl‐CoA Thioesterase 1 (ACOT1), which hydrolyzes acyl‐CoAs into long‐chain free fatty acids and Coenzyme A. However, the physiological role of ACOT1 in lipid metabolism is not known. Thus, the objective of this research was to determine the contribution of hepatic ACOT1 to lipid metabolism and signaling. To test our objectives, we injected C57/Bl6 mice with an adenovirus harboring shRNA targeted to ACOT1 or a scrambled control. One week post‐injection, mice were fasted overnight and sacrificed. Compared to control mice, ACOT1 knockdown resulted in an increase in serum β‐hydroxybutyrate, a marker of fatty acid oxidation, and an increase in serum triglyceride, indicating a possible increase in very‐low density lipoprotein secretion. These changes were not accompanied by a significant change in liver triglyceride. In addition, genes involved in mitochondrial biogenesis were decreased in response to ACOT1 knockdown suggesting that ACOT1 alters cell signaling to influence gene expression. In summary, these results show that ACOT1 plays an important role in regulating hepatic lipid metabolism and may serve an important novel role in trafficking of lipids to influence cell signaling.

Severe Hypertriglyceridemia With Pancreatitis

Recurrent pancreatitis is a potentially fatal complication of severe hypertriglyceridemia. Genetic defects and lifestyle risk factors may render this condition unresponsive to current treatments. We report this first case of long-term management of intractable near-fatal recurrent pancreatitis secondary to severe hypertriglyceridemia by a novel use of lomitapide, an inhibitor of microsomal triglyceride transfer protein, recently approved for treatment of familial homozygous hypercholesterolemia. The patient had been hospitalized many times for pancreatitis since age 15 years. Her serum triglyceride level averaged 3900 mg/dL while she received therapy with approved lipid drugs. She is homozygous for a coding mutation (P234L) in lipoprotein lipase, leaving her unable to metabolize triglycerides in chylomicrons and very low density lipoproteins (VLDL). Lomitapide reduces the secretion of chylomicrons and VLDL. Lomitapide, which was started when she was 44 years old after near-fatal pancreatitis, lowered her fasting triglyceride level from greater than 3000 mg/dL to a mean (SD) of 903 (870) mg/dL while she received 30 mg/d and to 524 (265) mg/dL while she received 40 mg/d; eliminated chronic abdominal pain; and prevented pancreatitis. However, fatty liver, present before treatment, progressed to steatohepatitis and fibrosis after 12 to 13 years. Lomitapide prevented pancreatitis in severe intractable hypertriglyceridemia but at a potential long-term cost of hepatotoxicity.

Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease. To elucidate the molecular basis of NAFLD we performed an exome-wide association study of liver fat content. Three variants were associated with increased liver fat at the exome-wide significance level: two in PNPLA3, an established locus for NAFLD, and one (Glu167Lys) in TM6SF2, a gene of unknown function. The Glu167LysTM6SF2 variant was also associated with higher circulating levels of alanine transaminase, a marker of liver injury, and lower levels of LDL-cholesterol, triglycerides and alkaline phosphatase in 3 independent populations (n>80,000). Recombinant Glu167LysTM6SF2 produced 50% less protein than wild-type TM6SF2 when expressed in cultured hepatocytes. Adeno-associated virus-mediated shRNA knockdown of Tm6sf2 in mice increased liver triglyceride content 3-fold and decreased VLDL secretion by 50%. Taken together, these data indicate that TM6SF2 activity is required for normal VLDL secretion, and that impaired TM6SF2 function causally contributes to NAFLD.

Reduction of VLDL secretion decreases cholesterol excretion in niemann-pick C1-like 1 hepatic transgenic mice.

An effective way to reduce LDL cholesterol, the primary risk factor of atherosclerotic cardiovascular disease, is to increase cholesterol excretion from the body. Our group and others have recently found that cholesterol excretion can be facilitated by both hepatobiliary and transintestinal pathways. However, the lipoprotein that moves cholesterol through the plasma to the small intestine for transintestinal cholesterol efflux (TICE) is unknown. To test the hypothesis that hepatic very low-density lipoproteins (VLDL) support TICE, antisense oligonucleotides (ASO) were used to knockdown hepatic expression of microsomal triglyceride transfer protein (MTP), which is necessary for VLDL assembly. While maintained on a high cholesterol diet, Niemann-Pick C1-like 1 hepatic transgenic (L1Tg) mice, which predominantly excrete cholesterol via TICE, and wild type (WT) littermates were treated with control ASO or MTP ASO. In both WT and L1Tg mice, MTP ASO decreased VLDL triglyceride (TG) and cholesterol secretion. Regardless of treatment, L1Tg mice had reduced biliary cholesterol compared to WT mice. However, only L1Tg mice treated with MTP ASO had reduced fecal cholesterol excretion. Based upon these findings, we conclude that VLDL or a byproduct such as LDL can move cholesterol from the liver to the small intestine for TICE.

Apolipoprotein B100 quality control and the regulation of hepatic very low density lipoprotein secretion

Apolipoprotein B (apoB) is the main protein component of very low density lipoprotein (VLDL) and is necessary for the assembly and secretion of these triglyceride (TG)-rich particles. Following release from the liver, VLDL is converted to low density lipoprotein (LDL) in the plasma and increased production of VLDL can therefore play a detrimental role in cardiovascular disease. Increasing evidence has helped to establish VLDL assembly as a target for the treatment of dyslipidemias. Multiple factors are involved in the folding of the apoB protein and the formation of a secretion-competent VLDL particle. Failed VLDL assembly can initiate quality control mechanisms in the hepatocyte that target apoB for degradation. ApoB is a substrate for endoplasmic reticulum associated degradation (ERAD) by the ubiquitin proteasome system and for autophagy. Efficient targeting and disposal of apoB is a regulated process that modulates VLDL secretion and partitioning of TG. Emerging evidence suggests that significant overlap exists between these degradative pathways. For example, the insulin-mediated targeting of apoB to autophagy and postprandial activation of the unfolded protein response (UPR) may employ the same cellular machinery and regulatory cues. Changes in the quality control mechanisms for apoB impact hepatic physiology and pathology states, including insulin resistance and fatty liver. Insulin signaling, lipid metabolism and the hepatic UPR may impact VLDL production, particularly during the postprandial state. In this review we summarize our current understanding of VLDL assembly, apoB degradation, quality control mechanisms and the role of these processes in liver physiology and in pathologic states.

Hepatic SH2B1 and SH2B2 Regulate Liver Lipid Metabolism and VLDL Secretion in Mice

SH2B1 is an SH2 and PH domain-containing adaptor protein. Genetic deletion of SH2B1 results in obesity, type 2 diabetes, and fatty liver diseases in mice. Mutations in SH2B1 are linked to obesity in humans. SH2B1 in the brain controls energy balance and body weight at least in part by enhancing leptin sensitivity in the hypothalamus. SH2B1 in peripheral tissues also regulates glucose and lipid metabolism, presumably by enhancing insulin sensitivity in peripheral metabolically-active tissues. However, the function of SH2B1 in individual peripheral tissues is unknown. Here we generated and metabolically characterized hepatocyte-specific SH2B1 knockout (HKO) mice. Blood glucose and plasma insulin levels, glucose tolerance, and insulin tolerance were similar between HKO, albumin-Cre, and SH2B1f/f mice fed either a normal chow diet or a high fat diet (HFD). Adult-onset deletion of SH2B1 in the liver either alone or in combination with whole body SH2B2 knockout also did not exacerbate HFD-induced insulin resistance and glucose intolerance. Adult-onset, but not embryonic, deletion of SH2B1 in the liver attenuated HFD-induced hepatic steatosis. In agreement, adult-onset deletion of hepatic SH2B1 decreased the expression of diacylglycerol acyltransferase-2 (DGAT2) and increased the expression of adipose triglyceride lipase (ATGL). Furthermore, deletion of liver SH2B1 in SH2B2 null mice attenuated very low-density lipoprotein (VLDL) secretion. These data indicate that hepatic SH2B1 is not required for the maintenance of normal insulin sensitivity and glucose metabolism; however, it regulates liver triacylglycerol synthesis, lipolysis, and VLDL secretion.

A Regulator of Secretory Vesicle Size, Kelch-Like Protein 12, Facilitates the Secretion of Apolipoprotein B100 and Very-Low-Density Lipoproteins—Brief Report

One of the major risk factors for atherosclerosis is the plasma level of low-density lipoprotein (LDL), which is a product of very-low-density lipoprotein (VLDL). Hepatic apolipoprotein B100 (apoB100) is the essential component that provides structural stability to VLDL particles. Newly translated apoB100 is partially lipidated in the endoplasmic reticulum (ER), forming nascent apoB100-VLDL particles. These particles are further modified to form fully mature VLDLs in the Golgi apparatus. Therefore, the transport of nascent VLDL from the ER to the Golgi represents a critical step during VLDL maturation and secretion and in regulating serum LDL cholesterol levels. Our previous studies showed that apoB100 exits the ER in coat complex II vesicles (COPII), but the cohort of related factors that control trafficking is poorly defined. Expression levels of Kelch-like protein 12 (KLHL12), an adaptor protein known to assist COPII-dependent transport of procollagen, were manipulated by using a KLHL12-specific small interfering RNA and a KLHL12 expression plasmid in the rat hepatoma cell line, McArdle RH7777. KLHL12 knockdown decreased the secreted and intracellular pools of apoB100, an effect that was attenuated in the presence of an autophagy inhibitor. KLHL12 knockdown also significantly reduced secretion of the most lipidated apoB100-VLDL species and led to the accumulation of apoB100 in the ER. Consistent with these data, KLHL12 overexpression increased apoB100 recovery and apoB100-VLDL secretion. Images obtained from confocal microscopy revealed colocalization of apoB100 and KLHL12, further supporting a direct link between KLHL12 function and VLDL trafficking from the ER. KLHL12 plays a critical role in facilitating the ER exit and secretion of apoB100-VLDL particles, suggesting that KLHL12 modulation would influence plasma lipid levels.

Leukocytosis and Enhanced Susceptibility to Endotoxemia but Not Atherosclerosis in Adrenalectomized APOE Knockout Mice

Hyperlipidemic apolipoprotein E (APOE) knockout mice show an enhanced level of adrenal-derived anti-inflammatory glucocorticoids. Here we determined in APOE knockout mice the impact of total removal of adrenal function through adrenalectomy (ADX) on two inflammation-associated pathologies, endotoxemia and atherosclerosis. ADX mice exhibited 91% decreased corticosterone levels (P<0.001), leukocytosis (WBC count: 10.0 ± 0.4 x 10E9/L vs 6.5 ± 0.5 x 10E9/L; P<0.001) and an increased spleen weight (P<0.01). FACS analysis on blood leukocytes revealed increased B-lymphocyte numbers (55 ± 2% vs 46 ± 1%; P<0.01). T-cell populations in blood appeared to be more immature (CD62L+: 26 ± 2% vs 19 ± 1% for CD4+ T-cells, P<0.001 and 58 ± 7% vs 47 ± 4% for CD8+ T-cells, P<0.05), which coincided with immature CD4/CD8 double positive thymocyte enrichment. Exposure to lipopolysaccharide failed to increase corticosterone levels in ADX mice and was associated with a 3-fold higher (P<0.05) TNF-alpha response. In contrast, the development of initial fatty streak lesions and progression to advanced collagen-containing atherosclerotic lesions was unaffected. Plasma cholesterol levels were decreased by 35% (P<0.001) in ADX mice. This could be attributed to a decrease in pro-atherogenic very-low-density lipoproteins (VLDL) as a result of a diminished hepatic VLDL secretion rate (-24%; P<0.05). In conclusion, our studies show that adrenalectomy induces leukocytosis and enhances the susceptibility for endotoxemia in APOE knockout mice. The adrenalectomy-associated rise in white blood cells, however, does not alter atherosclerotic lesion development probably due to the parallel decrease in plasma levels of pro-atherogenic lipoproteins.

Perfusion Defatting at Subnormothermic Temperatures in Steatotic Rat Livers

Hepatic steatosis is a major risk factor in liver transplantation. Use of machine perfusion to reduce steatosis has been reported previously at normothermic (37°C) temperatures, with minimal media as well as specialized defatting cocktails. In this work, we tested if subnormothermic (room temperature) machine perfusion, a more practical version of machine perfusion approach that does not require temperature control or oxygen carriers, could also be used to reduce fat content in steatotic livers. Steatotic livers recovered from obese Zucker rats were perfused for 6 hours. A significant increase of very low density lipoprotein (VLDL) and triglyceride (TG) content in perfusate, with or without a defatting cocktail, was observed although the changes in histology were minimal and changes in intracellular TG content were not statistically significant. The oxygen uptake rate, VLDL secretion, TG secretion, and venous resistance were similar in both groups. This study confirms lipid export during subnormothermic machine perfusion; however, the duration of perfusion necessary appears much higher than required in normothermic perfusion.

Hepatocyte Nuclear Factor 4α and Downstream Secreted Phospholipase A 2 GXIIB Regulate Production of Infectious Hepatitis C Virus

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans. The life cycle of HCV is closely associated with the metabolism of lipids, especially very-low-density lipoprotein (VLDL) in hepatocytes. Hepatocyte nuclear factor 4α (HNF4α), the most abundant transcription factor in the liver, regulates the VLDL secretory pathway. However, the effects of HNF4α on the HCV life cycle are unclear. In this study, we investigated the regulatory effects of HNF4α on HCV assembly and secretion. HCV in HNF4α-deficient hepatocytes showed reduced assembly and secretion but unchanged entry and RNA replication. Bezafibrate, a chemical inhibitor of HNF4α, suppressed HCV assembly and secretion. HNF4α downregulation resulted in rearrangement of cytosolic lipid droplets (LDs), as evidenced by the aggregation of large LDs and distorted cytosolic distribution. Phospholipase A2 GXIIB (PLA2GXIIB), an HNF4α-regulated factor involved in VLDL secretion, was found to be crucial in HCV secretion. PLA2GXIIB expression was upregulated in hepatocytes harboring HCV subgenomic replicons or in HCV-infected hepatocytes. This upregulation was transcriptionally controlled in an HNF4α-dependent manner after HCV infection. Furthermore, PLA2GXIIB combined with microsomal triglyceride transfer protein was found to be responsible for the regulation of HNF4α-induced HCV infectivity. These results suggest that HNF4α and its downstream PLA2GXIIB are important factors affecting the late stage of the HCV life cycle and may serve as potential drug targets for the treatment of HCV infection.

Glycosyltransferase GLT8D2 positively regulates ApoB100 protein expression in hepatocytes.

Non-alcoholic fatty liver disease (NAFLD) is characterized by triglyceride (TG) accumulation in hepatocytes. Very low density lipoprotein (VLDL) is a major secretory product of the liver that transports endogenously synthesized TG. Disrupted VLDL secretion may contribute to the accumulation of TG in hepatocytes. ApoB100 (apolipoprotein B100) is a glycoprotein and an essential protein component of VLDL. Its glycosylation may affect VLDL assembly and secretion. However, which glycosyltransferase catalyzes apoB100 glycosylation is unknown. In this study, we cloned the GLT8D2 (glycosyltransferase 8 domain containing 2) gene from HepG2 cells and generated a series of plasmids for in vitro studies of its molecular functions. We discovered that GLT8D2 was localized in the ER, interacted with apoB100, and positively regulated the levels of apoB100 protein in HepG2 cells. Based on these results, we propose that GLT8D2 is a glycosyltransferase of apoB100 that regulates apoB100 levels in hepatocytes.

Saturated fatty acids activate ERK signaling to downregulate hepatic sortilin 1 in obese and diabetic mice

Hepatic VLDL overproduction is a characteristic feature of diabetes and an important contributor to diabetic dyslipidemia. Hepatic sortilin 1 (Sort1), a cellular trafficking receptor, is a novel regulator of plasma lipid metabolism and reduces plasma cholesterol and triglycerides by inhibiting hepatic apolipoprotein B production. Elevated circulating free fatty acids play key roles in hepatic VLDL overproduction and the development of dyslipidemia. This study investigated the regulation of hepatic Sort1 in obesity and diabetes and the potential implications in diabetic dyslipidemia. Results showed that hepatic Sort1 protein was markedly decreased in mouse models of type I and type II diabetes and in human individuals with obesity and liver steatosis, whereas increasing hepatic Sort1 expression reduced plasma cholesterol and triglycerides in mice. Mechanistic studies showed that the saturated fatty acid palmitate activated extracellular signal-regulated kinase (ERK) and inhibited Sort1 protein by mechanisms involving Sort1 protein ubiquitination and degradation. Consistently, hepatic ERK signaling was activated in diabetic mice, whereas blocking ERK signaling by an ERK inhibitor increased hepatic Sort1 protein in mice. These results suggest that increased saturated fatty acids downregulate liver Sort1 protein, which may contribute to the development of dyslipidemia in obesity and diabetes.

Methionine restriction prevents the progression of hepatic steatosis in leptin-deficient obese mice

ObjectiveThis study investigated the effects of dietary methionine restriction (MR) on the progression of established hepatic steatosis in the leptin-deficient ob/ob mouse. Material/MethodsTen-week-old ob/ob mice were fed diets containing 0.86% (control-fed; CF) or 0.12% methionine (MR) for 14weeks. At 14weeks, liver and fat were excised and blood was collected for analysis. In another study, blood was collected to determine in vivo triglyceride (TG) and very-low-density lipoprotein (VLDL) secretion rates. Liver histology was conducted to determine the severity of steatosis. Hepatic TG, free fatty acid levels, and fatty acid oxidation (FAO) were also measured. Gene expression was analyzed by quantitative PCR. ResultsMR reversed the severity of steatosis in the ob/ob mouse. This was accompanied by reduced body weight despite similar weight-specific food intake. Compared with the CF group, hepatic TG levels were significantly reduced in response to MR, but adipose tissue weight was not decreased. MR reduced insulin and HOMA ratios but increased total and high-molecular-weight adiponectin levels. Scd1 gene expression was significantly downregulated, while Acadvl, Hadha, and Hadhb were upregulated in MR, corresponding with increased β-hydroxybutyrate levels and a trend toward increased FAO. The VLDL secretion rate was also significantly increased in the MR mice, as were the mRNA levels of ApoB and Mttp. The expression of inflammatory markers, such as Tnf-α and Ccr2, was also downregulated by MR. ConclusionsOur data indicate that MR reverses steatosis in the ob/ob mouse liver by promoting FAO, increasing the export of lipids, and reducing obesity-related inflammatory responses.