Very Low Density Lipoprotein Secretion Research Articles

Fatty acid-binding protein 1 increases steer fat deposition by facilitating the synthesis and secretion of triacylglycerol in liver.

Castration is an important means of improving the beef quality via increasing fat deposition. However, little is known about the molecular mechanism underlying the fat deposition after castration. Here, the intramuscular fat (IMF) content of the steer group was shown to be much higher than the bull group. To understand transcriptional changes in the genes involved in fat deposition following castration, differential expression patterns of mRNAs in liver tissue were investigated in steers and bulls using RNA sequencing. In total, we obtained 58,282,367–54,918,002 uniquely mapped reads, which covered 90.13% of the currently annotated transcripts; 5,864 novel transcripts and optimized 9,088 known genes were determined. These results indicated that castration could change the expression patterns of mRNAs in liver tissue, and 282 differentially expressed genes (DEGs) were detected between steers and bulls. KEGG pathway analysis showed that the DEGs were mostly enriched in PPAR signaling pathway, steroid biosynthesis, steroid hormone biosynthesis, and biosynthesis of fatty acids. Furthermore, eight DEGs were corroborated via quantitative real-time PCR and we found that FABP1 gene knockdown in bovine hepatocytes prominently reduced intracellular triacylglycerol (TAG) synthesis and very low density lipoprotein (VLDL) secretion in culture medium. In summary, these results indicate that FABP1 may promote fat deposition by promoting the production and secretion of TAG and VLDL in steer liver.

Thioesterase Superfamily Member 2 Promotes Hepatic VLDL Secretion by Channeling Fatty Acids Into Triglyceride Biosynthesis.

In nonalcoholic fatty liver disease (NAFLD), triglycerides accumulate within the liver because the rates of fatty acid accrual by uptake from plasma and de novo synthesis exceed elimination by mitochondrial oxidation and secretion as very low-density lipoprotein (VLDL) triglycerides. Thioesterase superfamily member 2 (Them2) is an acyl-coenzyme A (CoA) thioesterase that catalyzes the hydrolysis of fatty acyl-CoAs into free fatty acids plus CoASH. Them2 is highly expressed in the liver, as well as other oxidative tissues. Mice globally lacking Them2 are resistant to diet-induced obesity and hepatic steatosis, and exhibit improved glucose homeostasis. These phenotypes are attributable, at least in part, to roles of Them2 in the suppression of thermogenesis in brown adipose tissue and insulin signaling in skeletal muscle. To elucidate the hepatic function of Them2, we created mice with liver-specific deletion of Them2 (L-Them2-/- ). Although L-Them2-/- mice were not protected against excess weight gain, hepatic steatosis or glucose intolerance, they exhibited marked decreases in plasma triglyceride and apolipoprotein B100 concentrations. These were attributable to reduced rates of VLDL secretion owing to decreased incorporation of plasma-derived fatty acids into triglycerides. The absence of hepatic steatosis in L-Them2-/- mice fed chow was explained by compensatory increases in rates of fatty acid oxidation and by decreased de novo lipogenesis in high fat-fed mice. Consistent with a role for Them2 in hepatic VLDL secretion, THEM2 levels were increased in livers of obese patients with NAFLD characterized by simple steatosis. Conclusion: Them2 functions in the liver to direct fatty acids toward triglyceride synthesis for incorporation into VLDL particles. When taken together with its functions in brown adipose and muscle, these findings suggest that Them2 is a target for the management of NAFLD and dyslipidemia.

GLP-2 Dysregulates Hepatic Lipoprotein Metabolism, Inducing Fatty Liver and VLDL Overproduction in Male Hamsters and Mice.

Fundamental complications of insulin resistance and type 2 diabetes include the development of nonalcoholic fatty liver disease and an atherogenic fasting dyslipidemic profile, primarily due to increases in hepatic very-low-density lipoprotein (VLDL) production. Recently, central glucagon-like peptide-2 receptor (GLP2R) signaling has been implicated in regulating hepatic insulin sensitivity; however, its role in hepatic lipid and lipoprotein metabolism is unknown. We investigated the role of glucagon-like peptide-2 (GLP-2) in regulating hepatic lipid and lipoprotein metabolism in Syrian golden hamsters, C57BL/6J mice, and Glp2r-/- mice consuming either a normal chow or high-fat diet (HFD). In the chow-fed hamsters, IP GLP-2 administration significantly increased fasting dyslipidemia, hepatic VLDL production, and the expression of key genes involved in hepatic de novo lipogenesis. In HFD-fed hamsters and chow-fed mice, GLP-2 administration exacerbated or induced hepatic lipid accumulation. HFD-fed Glp2r-/- mice displayed reduced glucose tolerance, VLDL secretion, and microsomal transfer protein lipid transfer activity, as well as exacerbated fatty liver. Thus, we conclude that GLP-2 plays a lipogenic role in the liver by increasing lipogenic gene expression and inducing hepatic steatosis, fasting dyslipidemia, and VLDL overproduction. In contrast, the lack of Glp2r appears to interfere with VLDL secretion, resulting in enhanced hepatic lipid accumulation. These studies have uncovered a role for GLP-2 in maintaining hepatic lipid and lipoprotein homeostasis.

Reticulon 3 regulates very low density lipoprotein secretion by controlling very low density lipoprotein transport vesicle biogenesis.

Secretion of very low density lipoprotein (VLDL) by the liver is an important physiological process; however, the rate of VLDL secretion is determined by its transport from the endoplasmic reticulum (ER) to the Golgi. This transport event is facilitated by a specialized ER-derived vesicle, the VLDL transport vesicle (VTV). We have reported earlier a detailed VTV proteome, which revealed that reticulon 3 (RTN3) is uniquely present in the VTV. Our immunoblotting and electron microscopic data demonstrate that RTN3 is enriched in the VTV; however, other ER-derived vesicles do not contain RTN3. Co-immunoprecipitation data coupled with confocal microscopic analyses strongly suggest that RTN3 interacts with VLDL core protein, apoB100, at the ER level. Our data show that either blocking of RTN3 using specific antibodies or RTN3 knockdown resulted in significant reduction in VTV biogenesis from hepatic ER membranes. Additionally, VLDL secretion from hepatocytes was significantly decreased when RTN3 was silenced by RTN3 siRNA. We conclude that RTN3 regulates VLDL secretion by controlling VTV-mediated ER-to-Golgi transport of nascent VLDL.

Abstract 175: Dynamin-Related Protein 1 Regulates Proteostasis and Proprotein Convertase Subtilisin/Kexin Type 9 Secretion

Objective: Dysfunctional protein homeostasis (proteostasis) contributes to cardiovascular and metabolic disorders. We and others associated the mitochondrial fission protein, Dynamin-related protein 1 (DRP1) with cardiometabolic disease. Liver DRP1-deficiency reduces serum lipids and very-low density lipoprotein secretion in high-fat fed mice; whether DRP1 mediates these effects via proteostasis regulation is unknown. Approach and Results: Using mass spectrometry integrated with network analysis to map the human liver secretome, we found DRP1 associated with cardiovascular disease modules and lipid pathways. Electron microscopy revealed human liver DRP1 at mitochondria, cytosol, vesicles, endoplasmic reticulum (ER), and clustered at membrane tethered to ER exit sites. DRP1 small molecule inhibition (Mdivi-1) or CRISPR/Cas9-mediated DRP1 deletion in human liver cells, and Drp1 -liver deficiency in mice reduced autophagic flux without impairing the amino acid metabolome, or activating the autophagy inhibitor, mammalian target of rapamycin complex 1. DRP1 partially co-localized and co-immunoprecipitated with the ER trafficking and autophagy regulator, Syntaxin 17, in human liver tissue and cells. DRP1 inhibition reduced Proprotein convertase subtilisin/kexin type 9 (PCSK9) secretion in human liver cells and mice (-78.5%), and altered trafficking of the PCSK9-binding and ER maintenance chaperone, Glucose-regulated protein 94. Co-treating human liver cells with Mdivi-1 and proteasome inhibitor (MG132), non-transcriptionally increased intracellular PCSK9, while maintaining Mdivi-1-mediated reduced PCSK9 secretion. Conclusions: We propose a novel function of DRP1 in the regulation of proteostasis, wherein DRP1 may cluster, then tether and/or constrict nascent autophagy-associated membrane at the ER via its interaction with Syntaxin 17. DRP1 inhibition likely reduces lipoprotein and PCSK9 secretion in part by impairing autophagic flux leading to compensatory chaperone-mediated proteasomal degradation for ER maintenance. Proteostasis regulation and the cellular function of DRP1 is more complex than previously thought, potentially providing new avenues to therapeutically target cardiometabolic disease.

Defects in TM6SF2 Impairs Lipidation of Nascent VLDL and Leads to Accumulation of Liver Fat

Non‐alcoholic fatty liver disease (NAFLD) is a burgeoning health problem in Western countries. Population‐based surveys indicate that one out of every three adults in the United States has excess liver fat (hepatic steatosis), the first stage of fatty liver disease. Despite the clinical importance of NAFLD, major questions remain regarding its etiology. As a step towards elucidating the pathophysiology of this disorder, we screened individuals in a population‐based study, the Dallas Heart Study (DHS) for DNA sequence variations associated with increased liver fat content. This analysis revealed that a common variant (E167K) in transmembrane 6 superfamily 2 (TM6SF2), a gene of unknown function, was associated with increased hepatic triglyceride (TG) content, reduced plasma lipid levels, and increased levels of circulating liver enzymes. TM6SF2 is expressed at highest levels in the small intestine, liver and kidney. Immunolocalization and biochemical studies localized the protein to the ER and the Golgi. To determine why individuals with the risk variant develop hepatic steatosis, we developed mice expressing no TM6SF2 (Tm6sf2−/−, KO). The Tm6sf2−/− mice have elevated levels of neutral lipids, including TG and cholesteryl esters (CE) in the liver and elevated levels of liver enzymes in the circulation, consistent with liver inflammation. At steady state the Tm6sf2−/− mice have decreased plasma levels of cholesterol and very low density lipoprotein (VLDL)‐TG. Treatment with Triton WR1339 to inhibit peripheral lipases was associated with a non‐specific and dramatic reduction in all of the VLDL associated lipid molecular species, but not apolipoprotein (Apo)‐B100. In addition, it was found that de novo synthesis of radioactive and stable labeled lipids was marginally changed in the liver, but secretion of the newly synthesized labelled lipids was dramatically decreased in the Tm6sf2−/− mouse. As expected, given these observations, VLDL was reduced in size in the KO mice. Thus, TM6SF2 plays a key role in the expansion of the nascent particle, but not in the secretion of nascent VLDL, thereby causes steatosis.Support or Funding InformationFunding was provided by the NIH through research grants R01‐DK090066 and F32‐DK112529.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Phytoceuticals in Fenugreek Ameliorate VLDL Overproduction and Insulin Resistance via the Insig Signaling Pathway.

This study aims to characterize the effect of fenugreek (Trigonella foenum-graecum) seed and its phytoceutical trigonelline in antimetabolic inflammation and ameliorating overproduction of very low density lipoprotein (VLDL) in insulin resistance. Two groups of genetic hyperlipidemic mice generated by depletion of cAMP responsive element binding protein H (CREBH) are fed either a chow containing 2% fenugreek seed or vehicle for 7 weeks. Q-RT-PCR and immunoblotting analysis demonstrated that fenugreek seed containing diet inhibits hepatic SREBP-1c activation and the subsequent de novo lipogenesis by enhancing expression of insulin-inducible gene-1 (Insig-1) and gene-2 (Insig-2). mRNA expression of PPARαand its target genes that are involved in fatty acid β-oxidation are also upregulated in the fenugreek seed fed-mice which is accompanied by significantly reduced hepatic lipid accumulation and VLDL secretion, improved endoplasmic reticulum (ER) stress, and ameliorated metabolic inflammation. These actions enhance insulin sensitivity and improve hyperlipidemia. In vitro, treating a rat hepatoma cell line, McA-RH7777 (McA), with trigonelline is able to recapitulate the results observed in vivo. This study unveils a novel mechanism of fenugreek seed and trigonelline in countering hepatic VLDL overproduction and insulin resistance by enhancing the Insig signaling pathways and ameliorating metabolic inflammatory stress in the liver.

Cathepsin B regulates hepatic lipid metabolism by cleaving liver fatty acid–binding protein

Synthesis and secretion of hepatic triglycerides (TAG) associated with very-low-density lipoprotein (VLDL) play a major role in maintaining overall lipid homeostasis. This study aims to identify factors affecting synthesis and secretion of VLDL-TAG using the growth hormone-deficient Ames dwarf mouse model, which has reduced serum TAG. Proteomic analysis coupled with a bioinformatics-driven approach revealed that these mice express greater amounts of hepatic cathepsin B and lower amounts of liver fatty acid-binding protein (LFABP) than their wildtype littermates. siRNA-mediated knockdown of cathepsin B in McA-RH7777 cells resulted in a 39% increase in [3H]TAG associated with VLDL secretion. Cathepsin B knockdown was accompanied by a 74% increase in cellular LFABP protein levels, but only when cells were exposed to 0.4 mm oleic acid (OA) complexed to BSA. The cathepsin B knockdown and 24-h treatment with OA resulted in increased CD36 expression alone and additively. Co-localization of LFABP and cathepsin B was observed in a distinct Golgi apparatus-like pattern, which required a 1-h OA treatment. Moreover, we observed co-localization of LFABP and apoB, independent of the OA treatment. Overexpression of cathepsin B resulted in decreased OA uptake and VLDL secretion. Co-expression of cathepsin B and cathepsin B-resistant mutant LFABP in McA-RH7777 cells resulted in an increased TAG secretion as compared with cells co-expressing cathepsin B and wildtype LFABP. Together, these data indicate that cathepsin B regulates VLDL secretion and free fatty acid uptake via cleavage of LFABP, which occurs in response to oleic acid exposure.

FADS2 inhibition in essential fatty acid deficiency induces hepatic lipid accumulation via impairment of very low-density lipoprotein (VLDL) secretion

Fatty acid desaturase 2 (FADS2) is responsible for the first desaturation reaction in the synthesis of highly unsaturated fatty acids (HUFAs), such as arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3), and is involved in Mead acid (20:3n-9) production during essential fatty acid deficiency (EFAD). In this study, an obvious hepatic lipid accumulation was observed in EFAD mice treated with a FADS2 inhibitor. FADS2 inhibition in the EFAD state reduced secretion of very low-density lipoprotein (VLDL) and markedly diminished Mead acid in phosphatidylcholine (PC) in the liver and plasma. As the results, the amount of C20 HUFAs in hepatic and plasma PC dramatically reduced in the EFAD mice treated with a FADS2 inhibitor, whereas the decrease of C20 HUFA levels of PC in EFAD mice was not observed because of the increased Mead acid in PC. These results supposed that Mead acid in PC is important as a component of VLDL. It is possible that Mead acid plays the role of a substitute of HUFAs in VLDL secretion during EFAD.

High fat diet-induced oxidative stress blocks hepatocyte nuclear factor 4α and leads to hepatic steatosis in mice.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease with manifestation of over-accumulation of fat in liver. Increasing evidences indicate that NAFLD may be in part caused by malfunction of very low density lipoprotein (VLDL) secretion. Hepatocyte nuclear factor 4α (HNF4α), a nuclear receptor protein, plays an important role in sustain hepatic lipid homeostasis via transcriptional regulation of genes involved in secretion of VLDL, such as apolipoprotein B (ApoB). However, the exact functional change of HNF4α in NAFLD remains to be elucidated. In the present study, we found that high fat diet (HFD) induced cytoplasmic retention of HNF4α in hepatocytes, which led to down-regulation of hepatic ApoB expression and its protein level in serum, as well as reduced secretion of VLDL. We further revealed that oxidative stress, elevated in fatty liver, was the key factor inducing the cytoplasmic retention of HNF4α in hepatocytes by activating protein kinase C (PKC)-mediated phosphorylation in HNF4α. Thus, our findings reveal a novel mechanism underlying HFD-induced fatty liver that oxidative stress impairs function of HNF4α on ApoB expression and VLDL secretion via PKC activation, eventually promoting fat accumulation in the liver. Therefore, oxidative stress/PKC/HNF4α pathway may be a novel target to treat diet-induced fatty liver.

Autophagy Is Required for Sortilin-Mediated Degradation of Apolipoprotein B100.

Genome-wide association studies identified single-nucleotide polymorphisms near the SORT1 locus strongly associated with decreased plasma LDL-C (low-density lipoprotein cholesterol) levels and protection from atherosclerotic cardiovascular disease and myocardial infarction. The minor allele of the causal SORT1 single-nucleotide polymorphism locus creates a putative C/EBPα (CCAAT/enhancer-binding protein α)-binding site in the SORT1 promoter, thereby increasing in homozygotes sortilin expression by 12-fold in liver, which is rich in this transcription factor. Our previous studies in mice have showed reductions in plasma LDL-C and its principal protein component, apoB (apolipoprotein B) with increased SORT1 expression, and in vitro studies suggested that sortilin promoted the presecretory lysosomal degradation of apoB associated with the LDL precursor, VLDL (very-low-density lipoprotein). To determine directly that SORT1 overexpression results in apoB degradation and to identify the mechanisms by which this reduces apoB and VLDL secretion by the liver, thereby contributing to understanding the clinical phenotype of lower LDL-C levels. Pulse-chase studies directly established that SORT1 overexpression results in apoB degradation. As noted above, previous work implicated a role for lysosomes in this degradation. Through in vitro and in vivo studies, we now demonstrate that the sortilin-mediated route of apoB to lysosomes is unconventional and intersects with autophagy. Increased expression of sortilin diverts more apoB away from secretion, with both proteins trafficking to the endosomal compartment in vesicles that fuse with autophagosomes to form amphisomes. The amphisomes then merge with lysosomes. Furthermore, we show that sortilin itself is a regulator of autophagy and that its activity is scaled to the level of apoB synthesis. These results strongly suggest that an unconventional lysosomal targeting process dependent on autophagy degrades apoB that was diverted from the secretory pathway by sortilin and provides a mechanism contributing to the reduced LDL-C found in individuals with SORT1 overexpression.

GLP-1 Elicits an Intrinsic Gut–Liver Metabolic Signal to Ameliorate Diet-Induced VLDL Overproduction and Insulin Resistance

Perturbations in hepatic lipid and very-low-density lipoprotein (VLDL) metabolism are involved in the pathogenesis of obesity and hepatic insulin resistance. The objective of this study is to delineate the mechanism of subdiaphragmatic vagotomy in preventing obesity, hyperlipidemia, and insulin resistance. By subjecting the complete subdiaphragmatic vagotomized mice to various nutritional conditions and investigating hepatic de novo lipogenesis pathway, we found that complete disruption of subdiaphragmatic vagal signaling resulted in a significant decrease of circulating VLDL-triglyceride compared with the mice obtained sham procedure. Vagotomy further prevented overproduction of VLDL-triglyceride induced by an acute fat load and a high-fat diet-induced obesity, hyperlipidemia, hepatic steatosis, and glucose intolerance. Mechanistic studies revealed that plasma glucagon-like peptide-1 was significantly raised in the vagotomized mice, which was associated with significant reductions in mRNA and protein expression of SREBP-1c (sterol regulatory element-binding protein 1c), SCD-1 (stearoyl-CoA desaturase-1), and FASN (fatty acid synthase), as well as enhanced hepatic insulin sensitivity. In vitro, treating mouse primary hepatocytes with a glucagon-like peptide-1 receptor agonist, exendin-4, for 48 hours inhibited free fatty acid, palmitic acid treatment induced de novo lipid synthesis, and VLDL secretion from hepatocytes. Elevation of glucagon-like peptide-1 in vagotomized mice may prevent VLDL overproduction and insulin resistance induced by high-fat diet. These novel findings, for the first time, delineate an intrinsic gut-liver regulatory circuit that is mediated by glucagon-like peptide-1 in regulating hepatic energy metabolism.

Targeting hepatic heparin-binding EGF-like growth factor (HB-EGF) induces anti-hyperlipidemia leading to reduction of angiotensin II-induced aneurysm development.

ObjectiveThe upregulated expression of heparin binding EGF-like growth factor (HB-EGF) in the vessel and circulation is associated with risk of cardiovascular disease. In this study, we tested the effects of HB-EGF targeting using HB-EGF-specific antisense oligonucleotide (ASO) on the development of aortic aneurysm in a mouse aneurysm model.Approach and resultsLow-density lipoprotein receptor (LDLR) deficient mice (male, 16 weeks of age) were injected with control and HB-EGF ASOs for 10 weeks. To induce aneurysm, the mice were fed a high fat diet (22% fat, 0.2% cholesterol; w/w) at 5 week point of ASO administration and infused with angiotensin II (AngII, 1,000ng/kg/min) for the last 4 weeks of ASO administration. We confirmed that the HB-EGF ASO administration significantly downregulated HB-EGF expression in multiple tissues including the liver. Importantly, the HB-EGF ASO administration significantly suppressed development of aortic aneurysms including thoracic and abdominal types. Interestingly, the HB-EGF ASO administration induced a remarkable anti-hyperlipidemic effect by suppressing very low density lipoprotein (VLDL) level in the blood. Mechanistically, the HB-EGF targeting suppressed hepatic VLDL secretion rate without changing heparin-releasable plasma triglyceride (TG) hydrolytic activity or fecal neutral cholesterol excretion rate.ConclusionThis result suggested that the HB-EGF targeting induced protection against aneurysm development through anti-hyperlipidemic effects. Suppression of hepatic VLDL production process appears to be a key mechanism for the anti-hyperlipidemic effects by the HB-EGF targeting.

Hepatitis C Virus Lipoviroparticles Assemble in the Endoplasmic Reticulum (ER) and Bud off from the ER to the Golgi Compartment in COPII Vesicles.

Hepatitis C virus (HCV) exists as a lipoprotein-virus hybrid lipoviroparticle (LVP). In vitro studies have demonstrated the importance of apolipoproteins in HCV secretion and infectivity, leading to the notion that HCV coopts the secretion of very-low-density lipoprotein (VLDL) for its egress. However, the mechanisms involved in virus particle assembly and egress are still elusive. The biogenesis of VLDL particles occurs in the endoplasmic reticulum (ER), followed by subsequent lipidation in the ER and Golgi compartment. The secretion of mature VLDL particles occurs through the Golgi secretory pathway. HCV virions are believed to latch onto or fuse with the nascent VLDL particle in either the ER or the Golgi compartment, resulting in the generation of LVPs. In our attempt to unravel the collaboration between HCV and VLDL secretion, we studied HCV particles budding from the ER en route to the Golgi compartment in COPII vesicles. Biophysical characterization of COPII vesicles fractionated on an iodixanol gradient revealed that HCV RNA is enriched in the highly buoyant COPII vesicle fractions and cofractionates with apolipoprotein B (ApoB), ApoE, and the HCV core and envelope proteins. Electron microscopy of immunogold-labeled microsections revealed that the HCV envelope and core proteins colocalize with apolipoproteins and HCV RNA in Sec31-coated COPII vesicles. Ultrastructural analysis also revealed the presence of HCV structural proteins, RNA, and apolipoproteins in the Golgi stacks. These findings support the hypothesis that HCV LVPs assemble in the ER and are transported to the Golgi compartment in COPII vesicles to embark on the Golgi secretory route.IMPORTANCE HCV assembly and release accompany the formation of LVPs that circulate in the sera of HCV patients and are also produced in an in vitro culture system. The pathway of HCV morphogenesis and secretion has not been fully understood. This study investigates the exact site where the association of HCV virions with host lipoproteins occurs. Using immunoprecipitation of COPII vesicles and immunogold electron microscopy (EM), we characterize the existence of LVPs that cofractionate with lipoproteins, viral proteins, RNA, and vesicular components. Our results show that this assembly occurs in the ER, and LVPs thus formed are carried through the Golgi network by vesicular transport. This work provides a unique insight into the HCV LVP assembly process within infected cells and offers opportunities for designing antiviral therapeutic cellular targets.

Tamoxifen reduces hepatic VLDL production and GH secretion in women: a possible mechanism for steatosis development.

Growth hormone (GH) stimulates hepatic synthesis of very-low-density lipoproteins (VLDL), whereas hepatic steatosis develops as a result of GH deficiency. Steatosis is also a complication of tamoxifen treatment, the cause of which is not known. As tamoxifen inhibits the secretion and action of GH, we hypothesize that it induces steatosis by inhibiting hepatic VLDL export. To investigate whether tamoxifen reduces hepatic VLDL secretion. Eight healthy, normolipidemic women (age: 64.4 ± 2.1 years) were studied in random sequence at baseline, after 2 weeks of tamoxifen (20 mg/day) and after 2 weeks of estradiol valerate (EV; 2 mg/day) treatments, separated by a 4-week washout period. The kinetics of apolipoprotein B (apoB), the structural protein of VLDL particles, were measured using a stable isotope 2H3-leucine turnover technique. VLDL-apoB fractional catabolic rate (FCR) was determined using a multicompartment model. VLDL-apoB secretion was estimated as the product of FCR and VLDL-apoB concentration. GH response to arginine stimulation, circulating levels of IGF-1, FFA, and TG, along with TG content in VLDL were measured. Tamoxifen significantly (P < 0.05) reduced VLDL-apoB concentration and secretion by 27.3 ± 7.8% and 29.8 ± 10.2%, respectively. In contrast, EV did not significantly change VLDL-apoB concentration or secretion. Tamoxifen but not EV significantly reduced (P < 0.05) GH response to arginine stimulation. Both treatments significantly lowered (P < 0.05) circulating IGF-1. Inhibition of VLDL secretion may contribute to the development of fatty liver during tamoxifen therapy. As GH stimulates VLDL secretion, the development of steatosis may arise secondarily from GH insufficiency induced by tamoxifen.

Abstract 196: Acute Phase Response Transcription Factor CREBH Mediates Metabolic Inflammation to Hepatic VLDL Overproduction and Hyperlipidemia

Hyperlipidemia is a major complication of insulin-resistant states, such as obesity and type 2 diabetes, and contributes to an increased risk of cardiovascular disease. One key mechanism underlying the hyperlipidemia in metabolic syndrome is the overproduction of apolipoprotein B (apoB), the essential structural protein of the triglyceride-rich lipoproteins, such as very-low-density lipoproteins (VLDL) and chylomicrons. Currently, the underlying mechanisms for the overproduction of VLDL in metabolic syndrome are not fully understood. In this study, we demonstrated that the cAMP responsive element binding protein H (CREBH), an acute phase transcription factor, enhanced VLDL assembly and secretion by upregulating apoB expression, which contributed to hyperlipidemia associated metabolic inflammation. Specifically, we showed that over-expression of CREBH significantly induced mRNA and protein expression of apoB in McA-7777 cells. A luciferase assay further revealed that the presence of CREBH positively enhanced the activity of the apoB gene promoter. Genetic depletion of CREBH in mice resulted in significant reduction in expression of hepatic apoB mRNA. Moreover, challenging mice with a fat load via oral gavage upregulated VLDL secretion in wild type but not in the CREBH-null mice, suggesting the essential role of CREBH in apoB expression. Inflammatory cytokine TNFα treatment activated hepatic CREBH expression in wild type mice which subsequently enhanced hepatic apoB biosynthesis and VLDL secretion. This phenotype was not observed in CREBH-null mice. Metabolic inflammation induced by LPS or HFD also resulted in overproduction of apoB and hyperlipoproteinemia in wild type but not in CREBH-null mice. Conclusion: This study demonstrated that, for the first time, CREBH could be a mediator between metabolic inflammation and hepatic VLDL overproduction in chronic metabolic disorders. This novel finding establishes CREBH as the first transcription factor that regulates apoB expression on the transcription level and the subsequent VLDL biosynthesis in response to metabolic inflammation, and further provides novel mechanistic insight into the pathogenesis of hyperlipidemia in metabolic syndrome.

CREBH mediates metabolic inflammation to hepatic VLDL overproduction and hyperlipoproteinemia.

CREBH mediates inflammatory signaling to VLDL overproduction in metabolic stress. Activation of CREBH in inflammation enhances mRNA and protein expression of apoB. CREBH presents a potential novel therapeutic target for hyperlipoproteinemia.

GLP‐1 Mediates the Intrinsic Gut‐Liver Metabolic Signaling in Anti‐VLDL Overproduction and Insulin Resistance in Vagotomized Mice

Perturbations in hepatic lipid and very low density lipoprotein (VLDL) metabolism are involved in the pathogenesis of obesity and hepatic insulin resistance. In this study, our objective was to investigate the intrinsic peripheral gut‐liver vagal nerve circuit in the regulation of hepatic VLDL secretion and insulin sensitivity in sub‐diaphragmatic vagotomized mice. Specifically, by disrupting both hepatic and gastric divisions of the vagal nerve and using fast protein liquid chromatography (FPLC) to subclassify the lipoprotein particles, we discovered that vagotomy significantly decreased triglyceride (TG) contents in VLDL particles compared to that in the control mice with sham‐surgery. Vagotomy further prevented overproduction of VLDL‐TG and VLDL‐apoB induced by an acute fat load. Espousing both sham control and vagotomized mice to a long term high fat diet (HFD) for 22 weeks, we found that vagotomy protected mice from HFD induced hyperlipidemia, obesity, hepatic steatosis and insulin resistance. In an attempt to delineate the molecular mechanism for the anti‐hyperlipidemia and anti‐insulin resistant effect of vagotomy, we investigated the gut hormones and discovered that plasma glucagon‐like peptide‐1(GLP‐1) was significantly higher in the vagotomised mice compared to sham controls, which was associated with significant reduction on mRNA and protein expression of hepatic stearoyl CoA desaturase‐1 (SCD‐1), an enzyme that catalyzes the synthesis of oleyl CoA from stearolyl CoA. In vitro, pre‐treating a rat hepatoma cell line, McA‐7777 cells, with GLP‐1 inhibited SCD‐1 and Fasn mRNA expression which resulted in significant reduction on hepatic de novo lipid synthesis and VLDL secretion. Vagotomy further sensitized insulin sensitivity by upregulating expression of insulin receptors and enhanced activation of downstream signaling molecule, AKT. In conclusion, we demonstrated that increased secretion of GLP‐1 in vagotomized mice mediates the intrinsic gut‐liver metabolic signaling in preventing VLDL overproduction and insulin resistance induced by HFD. GLP‐1 inhibits SCD‐1 activity which in turn inhibits hepatic de novo lipogenesis and VLDL biogenesis. These novel findings, for the first time, provide mechanistic rationale for the application of vagotomy in clinical treatment in patients with obesity and diabetes.Support or Funding InformationHatch Funds from USDA/NIFA to Q. Su