VLDL-TG Secretion Research Articles

246-LB: Stimulation of Hepatic Lipid Secretion by Glucocorticoid Action in the Nucleus of the Solitary Tract

Dysregulated lipid metabolism is a characteristic of diabetes, insulin resistance, and obesity. Increased glucocorticoid (GC) levels and/or action is associated with elevated levels of circulating triglyceride (TG)-rich very low-density lipoproteins (VLDL-TGs). Lipid metabolism is heavily affected by GCs via its direct actions on peripheral organs, e.g. liver, adipose tissue. However, how GCs modulate hepatic VLDL-TG secretion via their actions in the brain is less known. The nucleus of the solitary tract (NTS) in the hindbrain is responsive to various hormones and is an important region involved in whole-body metabolism. Here, we aimed to assess how GC action in the NTS affects hepatic TG secretion and hypothesized that GC receptor (GR) agonism in the NTS stimulates hepatic TG secretion. Sprague-Dawley rats underwent stereotaxic NTS bilateral cannulation and vascular catheterizations to enable simultaneous direct NTS infusions, intravenous injections, and blood sampling. Hepatic TG secretion rates were measured in 10h-fasted rats after intravenous poloxamer injection with concurrent NTS infusions. NTS GC infusion stimulated TG secretion compared to NTS vehicle controls, but this effect was negated with pharmacological and genetic GR antagonism, suggesting that NTS GCs are mediated via its receptor. The NTS GC-induced increase in VLDL-TG was not associated with changes in liver lipogenic gene expression and protein levels or plasma ApoB48/100. However, plasma FFAs were increased in NTS GC infused rats, and this was associated with increased pHSL:HSL protein levels in white adipose tissue. We herein provide evidence that acute GC action in the NTS stimulates hepatic VLDL-TG secretion and affects adipose tissue lipid metabolism. Increased circulating FFA availability may contribute to the hyperlipidemic effects of NTS GCs. Future studies to assess the role of NTS GC action in diabetic and obesity-related metabolic disease dyslipidemic models are warranted. Disclosure B. Vasilev: None. M. S. Grewal: None. B. Lum: None. J. T. Yue: None. Funding Natural Sciences and Engineering Research Council of Canada (RGPIN-2016-06657); Canada Foundation for Innovation (37267); Canada Research Chairs Tier 2 in Brain Regulation of Metabolism

Impaired Glucagon-Mediated Suppression of VLDL-Triglyceride Secretion in Individuals With Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD).

Individuals with metabolic dysfunction-associated fatty liver disease (MAFLD) have elevated plasma lipids as well as glucagon, although glucagon suppresses hepatic VLDL-triglyceride (TG) secretion. We hypothesize that the sensitivity to glucagon in hepatic lipid metabolism is impaired in MAFLD. We recruited 11 subjects with severe MAFLD (MAFLD+), 10 with mild MAFLD (MAFLD-), and 7 overweight control (CON) subjects. We performed a pancreatic clamp with a somatostatin analog (octreotide) to suppress endogenous hormone production, combined with infusion of low-dose glucagon (0.65 ng/kg/min, t = 0-270 min, LowGlucagon), followed by high-dose glucagon (1.5 ng/kg/min, t = 270-450 min, HighGlucagon). VLDL-TG and glucose tracers were used to evaluate VLDL-TG kinetics and endogenous glucose production (EGP). HighGlucagon suppressed VLDL-TG secretion compared with LowGlucagon. This suppression was markedly attenuated in MAFLD subjects compared with CON subjects (MAFLD+: 13% ± [SEM] 5%; MAFLD-: 10% ± 3%; CON: 36% ± 7%, P < 0.01), with no difference between MAFLD groups. VLDL-TG concentration and VLDL-TG oxidation rate increased between LowGlucagon and HighGlucagon in MAFLD+ subjects compared with CON subjects. EGP transiently increased during HighGlucagon without any difference between the three groups. Individuals with MAFLD have a reduced sensitivity to glucagon in the hepatic TG metabolism, which could contribute to the dyslipidemia seen in MAFLD patients. ClinicalTrials.gov: NCT04042142.

Apolipoprotein F is reduced in humans with steatosis and controls plasma triglyceride-rich lipoprotein metabolism.

NAFLD affects nearly 25% of the global population. Cardiovascular disease (CVD) is the most common cause of death among patients with NAFLD, in line with highly prevalent dyslipidemia in this population. Increased plasma triglyceride (TG)-rich lipoprotein (TRL) concentrations, an important risk factor for CVD, are closely linked with hepatic TG content. Therefore, it is of great interest to identify regulatory mechanisms of hepatic TRL production and remnant uptake in the setting of hepatic steatosis. To identify liver-regulated pathways linking intrahepatic and plasma TG metabolism, we performed transcriptomic analysis of liver biopsies from two independent cohorts of obese patients. Hepatic encoding apolipoprotein F ( APOF ) expression showed the fourth-strongest negatively correlation with hepatic steatosis and the strongest negative correlation with plasma TG levels. The effects of adenoviral-mediated human ApoF (hApoF) overexpression on plasma and hepatic TG were assessed in C57BL6/J mice. Surprisingly, hApoF overexpression increased both hepatic very low density lipoprotein (VLDL)-TG secretion and hepatic lipoprotein remnant clearance, associated a ~25% reduction in plasma TG levels. Conversely, reducing endogenous ApoF expression reduced VLDL secretion in vivo , and reduced hepatocyte VLDL uptake by ~15% in vitro . Transcriptomic analysis of APOF -overexpressing mouse livers revealed a gene signature related to enhanced ApoB-lipoprotein clearance, including increased expression of Ldlr and Lrp1 , among others. These data reveal a previously undescribed role for ApoF in the control of plasma and hepatic lipoprotein metabolism by favoring VLDL-TG secretion and hepatic lipoprotein remnant particle clearance.

Metabolic subtypes of patients with NAFLD exhibit distinctive cardiovascular risk profiles.

We previously identified subsets of patients with NAFLD with different metabolic phenotypes. Here we align metabolomic signatures with cardiovascular disease (CVD) and genetic risk factors. We analyzed serum metabolome from 1154 individuals with biopsy-proven NAFLD, and from four mouse models of NAFLD with impaired VLDL-triglyceride (TG) secretion, and one with normal VLDL-TG secretion. We identified three metabolic subtypes: A (47%), B (27%), and C (26%). Subtype A phenocopied the metabolome of mice with impaired VLDL-TG secretion; subtype C phenocopied the metabolome of mice with normal VLDL-TG; and subtype B showed an intermediate signature. The percent of patients with NASH and fibrosis was comparable among subtypes, although subtypes B and C exhibited higher liver enzymes. Serum VLDL-TG levels and secretion rate were lower among subtype A compared with subtypes B and C. Subtype A VLDL-TG and VLDL-apolipoprotein B concentrations were independent of steatosis, whereas subtypes B and C showed an association with these parameters. Serum TG, cholesterol, VLDL, small dense LDL5,6 , and remnant lipoprotein cholesterol were lower among subtype A compared with subtypes B and C. The 10-year high risk of CVD, measured with the Framingham risk score, and the frequency of patatin-like phospholipase domain-containing protein 3 NAFLD risk allele were lower in subtype A. Metabolomic signatures identify three NAFLD subgroups, independent of histological disease severity. These signatures align with known CVD and genetic risk factors, with subtype A exhibiting a lower CVD risk profile. This may account for the variation in hepatic versus cardiovascular outcomes, offering clinically relevant risk stratification.

Hepatic TM6SF2 Is Required for Lipidation of VLDL in a Pre-Golgi Compartment in Mice and Rats.

Background & AimsSubstitution of lysine for glutamic acid at residu 167 in Transmembrane 6 superfamily member 2 (TM6SF2) is associated with fatty liver disease and reduced plasma lipid levels. Tm6sf2-/- mice replicate the human phenotype but were not suitable for detailed mechanistic studies. As an alternative model, we generated Tm6sf2-/- rats to determine the subcellular location and function of TM6SF2.MethodsTwo lines of Tm6sf2-/- rats were established using gene editing. Lipids from tissues and from newly secreted very low density lipoproteins (VLDLs) were quantified using enzymatic assays and mass spectrometry. Neutral lipids were visualized in tissue sections using Oil Red O staining. The rate of dietary triglyceride (TG) absorption and hepatic VLDL-TG secretion were compared in Tm6sf2-/- mice and in their wild-type littermates. The intracellular location of TM6SF2 was determined by cell fractionation. Finally, TM6SF2 was immunoprecipitated from liver and enterocytes to identify interacting proteins.ResultsTm6sf2-/- rats had a 6-fold higher mean hepatic TG content (56.1 ± 28.9 9 vs 9.8 ± 3.9 mg/g; P < .0001) and lower plasma cholesterol levels (99.0 ± 10.5 vs 110.6 ± 14.0 mg/dL; P = .0294) than their wild-type littermates. Rates of appearance of dietary and hepatic TG into blood were reduced significantly in Tm6sf2-/- rats (P < .001 and P < .01, respectively). Lipid content of newly secreted VLDLs isolated from perfused livers was reduced by 53% (TG) and 62% (cholesterol) (P = .005 and P = .01, respectively) in Tm6sf2-/- mice. TM6SF2 was present predominantly in the smooth endoplasmic reticulum and endoplasmic reticulum–Golgi intermediate compartments, but not in Golgi. Both apolipoprotein B-48 and acyl-CoA synthetase long chain family member 5 physically interacted with TM6SF2.ConclusionsTM6SF2 acts in the smooth endoplasmic reticulum to promote bulk lipidation of apolipoprotein B–containing lipoproteins, thus preventing fatty liver disease.

Hypolipidemia associated with inactivation of TM6SF2 is due to decreased VLDL-lipids secretion

Abstract Background A missense variant in Transmembrane 6 Superfamily Member 2 [TM6SF2 (E167K)] is associated with reduced plasma lipid levels and protection from coronary atherosclerosis. The substitution of lysine for glutamate at residue 167 is associated with a marked decrease in TM6SF2 protein expression, consistent with a loss-of-function mutation. However the biological role of TM6SF2 is not known, and the mechanism(s) responsible for the hypolipidemia associated with mutation gene has not been fully defined. To elucidate the pathological mechanism for the hypolipidemia associated with TM6SF2 deficiency, we inactivated Tm6sf2 in mice and rats. Methods Tm6sf2−/− mice were generated as described previously. Two lines of Tm6sf2−/− rats with different frameshift mutations in exon 1 were generated using CRISPR/Cas9 technology. Primary hepatocytes were isolated from WT and Tm6sf2−/− mice for microscopy. Rats were fasted 16 or 4 hours and tissues were collected on ice for cell fractionation, and in liquid nitrogen for biochemical analyses. Frozen samples were stored at −80°C for subsequent analyses. Result In both mice and rats, inactivation of Tm6sf2 recapitulated the phenotype of humans with the E167K substitution: steatosis, reduced plasma lipid levels, and transaminitis. The phenotype was readily apparent in animals fed chow diets. Both species had reduced secretion of VLDL-TG, as determined by TRITON WR1399 injection, with no decrease in secretion of ApoB. Experiments in isolated perfused livers from WT and Tm6sf2−/− mice confirmed that the decreased TG secretion observed in intact animals reflected reduced TG secretion from the liver. Lipidomic analysis of the liver perfusates by by LC-MS indicated that secretion of cholesteryl esters, and phospholipids was also decreased in the KO animals. Taken together, these findings are consistent with a role for TM6SF2 in lipidation of ApoB-containing lipoproteins. To further elucidate the function of TM6SF2, we used fluorescence microscopy and cell fractionation to determine the subcellular localization of the protein. Microscopic analysis showed that TM6SF2 co-localized with ER and Golgi markers, but cell fractionation studies indicated that the protein is located primarily in the smooth ER. The ratio of TG to ApoB was lower in Golgi fractions from TM6sf2−/− rats than in corresponding fractions from WT animals. Conclusions Since the sequela of TM6SF2 inactivation are already apparent in the Golgi, we speculate that TM6SF2 promotes lipidation of VLDL in a pre-Golgi compartment. We are currently performing additional studies to further define the specific mechanism whereby TM6SF2 promotes lipidation of ApoB-containing lipoproteins. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): National Institutes of Health

Gut inflammation exacerbates high-fat diet induced steatosis by suppressing VLDL-TG secretion through HNF4α pathway

Nonalcoholic fatty liver disease (NAFLD) is increasingly identified in inflammatory bowel disease (IBD) patients with unclear etiology. In the current study we assessed the contribution of colonic inflammation to NAFLD development and the underlying mechanism in a mouse model for IBD. Our results showed that dextran sulfate sodium (DSS)-induced gut colitis directly led to hepatic inflammation, injury and further exacerbated hepatic steatosis caused by high fat diet (HF) feeding. The essential genes assessment, hepatic metabolic analysis and triglyceride-rich very low-density lipoprotein (VLDL-TG) secretion assays revealed a higher β-oxidation of fatty acids (FAs) but impaired VLDL-TG secretion in liver of DSS-treated mice. Disruption of the intestinal barrier by DSS promoted liver inflammation, which strongly suppressed hepatic VLDL-TG secretion and further aggravated HF-induced VLDL-TG secretion impairment through down-regulation of apolipoprotein B (APOB), hence promoting the storage of triglycerides (TG) in the liver. Inflammation induced by mixed proinflammatory cytokines or LPS obviously inhibited the expression of microsomal triglyceride transfer protein (MTP) and APOB expression and subsequently increased TG content via the suppression of HNF4α in mouse primary hepatocytes. In addition, the downregulation of MTP and APOB by proinflammatory cytokines was also rescued through activating Hnf4α by cortisol. Altogether, our results demonstrated that chronic inflammation exacerbated hepatic steatosis by inhibiting the secreting of hepatic VLDL-TG through HNF4α pathway, suggesting that restoring hepatic VLDL-TG secretion may be a novel strategy for treatment of NAFLD in IBD.

Nutrient infusion in the dorsal vagal complex controls hepatic lipid and glucose metabolism in rats

SummaryHypothalamic regulation of lipid and glucose homeostasis is emerging, but whether the dorsal vagal complex (DVC) senses nutrients and regulates hepatic nutrient metabolism remains unclear. Here, we found in rats DVC oleic acid infusion suppressed hepatic secretion of triglyceride-rich very-low-density lipoprotein (VLDL-TG), which was disrupted by inhibiting DVC long-chain fatty acyl-CoA synthetase that in parallel disturbed lipid homeostasis during intravenous lipid infusion. DVC glucose infusion elevated local glucose levels similarly as intravenous glucose infusion and suppressed hepatic glucose production. This was independent of lactate metabolism as inhibiting lactate dehydrogenase failed to disrupt glucose sensing and neither could DVC lactate infusion recapitulate glucose effect. DVC oleic acid and glucose infusion failed to lower VLDL-TG secretion and glucose production in high-fat fed rats, while inhibiting DVC farnesoid X receptor enhanced oleic acid but not glucose sensing. Thus, an impairment of DVC nutrient sensing may lead to the disruption of lipid and glucose homeostasis in metabolic syndrome.

Abstract 13164: Tm6sf2 is a Regulator of Liver Fat Metabolism in Smooth Er Influencing Vldl Lipidation

Backgrounds: A variant in Transmembrane 6 Superfamily Member 2 [ TM6SF2 (E167K)] that is associated with a loss of function is strongly associated with both alcoholic and nonalcoholic fatty liver disease. TM6SF2 is a polytopic protein that is expressed predominantly in the liver and small intestines. Immunolocalization studies are consistent with the protein being present in the endoplasmic reticulum (ER) and the Golgi complex. Tm6sf2 -/- mice replicate the phenotype of individuals with TM6SF2 -167K variant: hepatic steatosis accompanied by hypocholesterolemia, and transaminitis. These mice have a reduced rate of secretion of VLDL-TG without any change in the rate of secretion of ApoB. Thus, TM6SF2 is required for normal lipidation of VLDL. To determine where in the secretory pathway TM6SF2 promotes lipid addition to nascent VLDL, we have generated Tm6sf2 -/- rats. Methods: Two lines of Tm6sf2 -/- rats with different frameshift mutations in exon 1 were generated using CRISPR/Cas9 technology. Rats were fasted 4 hours and tissues were collected and frozen at -80°C. Cell fractionation studies were performed to isolate the smooth and rough ER along with trans - and cis -Golgi membranes. Result: Both lines of rats were confirmed by immunoblotting to produce no TM6SF2 in the liver or intestines. The phenotype of the Tm6sf2 -/- rats resembles that of mice and humans. Cell fractionation studies revealed that TM6SF2 mainly localized to the smooth ER. Unlike what was observed previously by immunocytochemistry, no TM6SF2 was found in the Golgi fraction. Analysis of the lipid profile of the Golgi apparatus in the KO rat revealed that the majority triglyceride (TG) and fatty acids (FA) subclasses were decreased by one to two folds compared to WT in terms of TG or FA to ApoB48 ratios. Conclusions: TM6SF2 is localized predominantly in the smooth ER and regulates the lipidation of VLDL.

Hepatic Carbohydrate Response Element Binding Protein Activation Limits Nonalcoholic Fatty Liver Disease Development in a Mouse Model for Glycogen Storage Disease Type 1a.

Background and AimsGlycogen storage disease (GSD) type 1a is an inborn error of metabolism caused by defective glucose‐6‐phosphatase catalytic subunit (G6PC) activity. Patients with GSD 1a exhibit severe hepatomegaly due to glycogen and triglyceride (TG) accumulation in the liver. We have shown that the activity of carbohydrate response element binding protein (ChREBP), a key regulator of glycolysis and de novo lipogenesis, is increased in GSD 1a. In the current study, we assessed the contribution of ChREBP to nonalcoholic fatty liver disease (NAFLD) development in a mouse model for hepatic GSD 1a.Approach and ResultsLiver‐specific G6pc–knockout (L‐G6pc −/−) mice were treated with adeno‐associated viruses (AAVs) 2 or 8 directed against short hairpin ChREBP to normalize hepatic ChREBP activity to levels observed in wild‐type mice receiving AAV8–scrambled short hairpin RNA (shSCR). Hepatic ChREBP knockdown markedly increased liver weight and hepatocyte size in L‐G6pc −/− mice. This was associated with hepatic accumulation of G6P, glycogen, and lipids, whereas the expression of glycolytic and lipogenic genes was reduced. Enzyme activities, flux measurements, hepatic metabolite analysis and very low density lipoprotein (VLDL)‐TG secretion assays revealed that hepatic ChREBP knockdown reduced downstream glycolysis and de novo lipogenesis but also strongly suppressed hepatic VLDL lipidation, hence promoting the storage of “old fat.” Interestingly, enhanced VLDL‐TG secretion in shSCR‐treated L‐G6pc −/− mice associated with a ChREBP‐dependent induction of the VLDL lipidation proteins microsomal TG transfer protein and transmembrane 6 superfamily member 2 (TM6SF2), the latter being confirmed by ChIP‐qPCR.ConclusionsAttenuation of hepatic ChREBP induction in GSD 1a liver aggravates hepatomegaly because of further accumulation of glycogen and lipids as a result of reduced glycolysis and suppressed VLDL‐TG secretion. TM6SF2, critical for VLDL formation, was identified as a ChREBP target in mouse liver. Altogether, our data show that enhanced ChREBP activity limits NAFLD development in GSD 1a by balancing hepatic TG production and secretion.

Spontaneous liver disease in wild-type C57BL/6JOlaHsd mice fed semisynthetic diet.

Mouse models are frequently used to study mechanisms of human diseases. Recently, we observed a spontaneous bimodal variation in liver weight in C57BL/6JOlaHsd mice fed a semisynthetic diet. We now characterized the spontaneous variation in liver weight and its relationship with parameters of hepatic lipid and bile acid (BA) metabolism. In male C57BL/6JOlaHsd mice fed AIN-93G from birth to postnatal day (PN)70, we measured plasma BA, lipids, Very low-density lipoprotein (VLDL)-triglyceride (TG) secretion, and hepatic mRNA expression patterns. Mice were sacrificed at PN21, PN42, PN63 and PN70. Liver weight distribution was bimodal at PN70. Mice could be subdivided into two nonoverlapping groups based on liver weight: 0.6 SD 0.1 g (approximately one-third of mice, small liver; SL), and 1.0 SD 0.1 g (normal liver; NL; p<0.05). Liver histology showed a higher steatosis grade, inflammation score, more mitotic figures and more fibrosis in the SL versus the NL group. Plasma BA concentration was 14-fold higher in SL (p<0.001). VLDL-TG secretion rate was lower in SL mice, both absolutely (-66%, p<0.001) and upon correction for liver weight (-44%, p<0.001). Mice that would later have the SL-phenotype showed lower food efficiency ratios during PN21-28, suggesting the cause of the SL phenotype is present at weaning (PN21). Our data show that approximately one-third of C57BL/6JOlaHsd mice fed semisynthetic diet develop spontaneous liver disease with aberrant histology and parameters of hepatic lipid, bile acid and lipoprotein metabolism. Study designs involving this mouse strain on semisynthetic diets need to take the SL phenotype into account. Plasma lipids may serve as markers for the identification of the SL phenotype.

Chronic Stress Potentiates High Fructose-Induced Lipogenesis in Rat Liver and Kidney.

Intake of fructose-sweetened beverages and chronic stress (CS) both increase risk of cardiometabolic diseases. The aim is to investigate whether these factors synergistically perturb lipid metabolism in rat liver and kidney. Fractional de novo lipogenesis (fDNL), intrahepatic- and intrarenal-triglycerides (IHTG and IRTG), de novo palmitate (DNPalm) content, FA composition, VLDL-TGs kinetics, and key metabolic gene expression at the end of the feeding and non-feeding phases in rats exposed to standard chow diet, chow diet + CS, 20% liquid high-fructose supplementation (HFr), or HFr+CS are measured. HFr induces hypertriglyceridemia, up-regulates fructose-metabolism and gluconeogenic enzymes, increases IHTG and DNPalm content in IHTG and IRTG, and augments fDNL at the end of the feeding phase. These changes are diminished after the non-feeding phase. CS does not exert such effects, but when combined with HFr, it reduces IHTG and visceral adiposity, enhances lipogenic gene expression and fDNL, and increases VLDL-DNPalm secretion. Liquid high-fructose supplementation increases IHTG and VLDL-TG secretion after the feeding phase, the latter being the result of stimulated hepatic and renal DNL. Chronic stress potentiates the effects of high fructose on fDNL and export of newly synthesized VLDL-TGs, and decreases fructose-induced intrahepatic TG accumulation after the feeding phase.

Niacin increases diet-induced hepatic steatosis in B6129 mice

Nonalcoholic fatty liver disease (NAFLD) is a very common disorder affecting between 20 and 30% of adults in the United States. However, there is no effective pharmacotherapy for treating NAFLD. Niacin, a water-soluble vitamin (B3), at pharmacological doses, decreases hepatic triglyceride (TG) content in NAFLD through inhibition of diacylglycerol acyltransferase 2, a key enzyme that catalyzes the final step in TG synthesis. Alternatively, some studies indicate that niacin induces fatty liver in high-fat diet (HFD)-fed rats. Therefore, in this study we investigated whether niacin is beneficial in treating NAFLD in two strains of mice, C57BL/6J (B6) and B6129SF2/J (B6129) mice, with 20 weeks of HFD feeding. Niacin treatment was started from week 5 until the end of the study. Niacin treatment increased normalized liver weight, hepatic TG content and NAFLD score in HFD-fed B6129 mice but had no impact on B6 mice. Metabolomics analysis revealed that in B6129 mice, 4-hydroxyphenylpyruvic acid (4-HPP), which is associated with fatty acid oxidation, did not change with HFD feeding but significantly decreased with niacin treatment. Lipidomics analysis discovered that the abundance of phosphocholine (PC), which is critical for very low-density lipoprotein (VLDL)–TG production and secretion, was decreased in HFD-fed B6129 with niacin treatment. In conclusion, niacin had no impact on diet-induced NAFLD development in B6 mice but potentiated hepatic steatosis in HFD-fed B6129 mice due to impaired fatty acid oxidation and decreased VLDL-TG production and secretion.

Dysfunction of estrogen-related receptor alpha-dependent hepatic VLDL secretion contributes to sex disparity in NAFLD/NASH development.

Rationale: Men and postmenopausal women are more prone to developing non-alcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) than premenopausal women. However, the pathological links and underlying mechanisms of this disparity are still elusive. The sex-difference in hepatic very low-density lipoprotein (VLDL) assembly and secretion may contribute to NAFLD development. Estrogen-related receptor alpha (ERRα) is a key regulator of several metabolic processes. We hypothesized that ERRα plays a role contributing to the sex-difference in hepatic VLDL assembly and secretion.Methods: VLDL secretion and essential genes governing said process were assessed in male and female mice. Liver-specific ERRα-deficient (ERRαLKO) mice were generated to assess the rate of hepatic VLDL secretion and alteration in target gene expression. Overexpression of either microsomal triglyceride transfer protein (Mttp) or phospholipase A2 G12B (Pla2g12b) by adenovirus was performed to test if the fatty liver phenotype in male ERRαLKO mice was due to defects in hepatic VLDL secretion. Female ERRαLKO mice were put on a diet high in saturated fat, fructose and cholesterol (HFHC) to promote NASH development. Wild type female mice were either ovariectomized or treated with tamoxifen to induce a state of estrogen deficiency or disruption in estrogen signaling. Adenovirus was used to overexpress ERRα in these mice to test if ERRα was sufficient to rescue the suppressed VLDL secretion due to estrogen dysfunction. Finally, wild type male mice on a high-fat diet (HFD) were treated with an ERRα inverse agonist to assess if suppressing ERRα activity pharmacologically would lead to fatty liver development.Results: ERRα is an indispensable mediator modulating hepatic triglyceride-rich very low-density lipoprotein (VLDL-TG) assembly and secretion through coordinately controlling target genes apolipoprotein B (Apob), Mttp and Pla2g12b in a sex-different manner. Hepatic VLDL-TG secretion is blunted in ERRαLKO mice, leading to hepatosteatosis which exacerbates endoplasmic reticulum stress and inflammation paving ways for NASH development. Importantly, ERRα acts downstream of estrogen/ERα signaling in contributing to the sex-difference in hepatic VLDL secretion effecting hepatic lipid homeostasis.Conclusions: Our results highlight ERRα as a key mediator which contributes to the sex disparity in NAFLD development, suggesting that selectively restoring ERRα activity in the liver may be a novel strategy for treating NAFLD/NASH.

Pathophysiological importance of bile cholesterol reabsorption: hepatic NPC1L1-exacerbated steatosis and decreasing VLDL-TG secretion in mice fed a high-fat diet

BackgroundNon-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide, although its pathogenesis remains to be elucidated. A recent study revealed that hepatic Niemann-Pick C1-Like 1 (NPC1L1), a cholesterol re-absorber from bile to the liver expressed on the bile canalicular membrane, is an exacerbation factor of NAFLD. Indeed, transgenic mice with hepatic expression of human NPC1L1 under a liver-specific promoter (L1-Tg mice) developed steatosis with a high-fat diet (HFD) containing cholesterol within a few weeks. However, the mechanism underlying diet-induced hepatic NPC1L1-mediated lipid accumulation is poorly defined.MethodsTo achieve a deeper understanding of steatosis development in L1-Tg mice, the biochemical features of hepatic NPC1L1-mediated steatosis were investigated. Hemizygous L1-Tg mice and wild-type littermate controls fed a HFD or control-fat diet were used. At the indicated time points, the livers were evaluated for cholesterol and triglyceride (TG) contents as well as mRNA levels of hepatic genes involved in the maintenance of lipid homeostasis. The hepatic ability to secrete very low-density lipoprotein (VLDL)-TG was also investigated.ResultsUnlike the livers of wild-type mice that have little expression of hepatic Npc1l1, the livers of L1-Tg mice displayed time-dependent changes that indicated steatosis formation. In steatosis, there were three different stages of development: mild accumulation of hepatic cholesterol and TG (early stage), acceleration of hepatic TG accumulation (middle stage), and further accumulation of hepatic cholesterol (late stage). In the early stage, between WT and L1-Tg mice fed a HFD for 2 weeks, there were no significant differences in the hepatic expression of Pparα, Acox1, Fat/Cd36, Srebf1, and Srebf2; however, the hepatic ability to secrete VLDL-TG decreased in L1-Tg mice (P < 0.05). Furthermore, this decrease was completely prevented by administration of ezetimibe, an NPC1L1-selective inhibitor.ConclusionHepatic NPC1L1 exacerbates diet-induced steatosis, which was accompanied by decreased hepatic ability of VLDL-TG secretion. The obtained results provide a deeper understanding of L1-Tg mice as a promising NAFLD animal model that is able to re-absorb biliary-secreted cholesterol similar to humans. Furthermore, this work supports further studies of the pathophysiological impact of re-absorbed biliary cholesterol on the regulation of hepatic lipid homeostasis.

Central Sfrp5 regulates hepatic glucose flux and VLDL-triglyceride secretion

ObjectiveSecreted frizzled-related protein 5 (Sfrp5) has been shown to be associated with energy homeostasis and insulin resistance in mouse models of obesity and diabetes. However, its central role in glucose and lipid metabolism is unknown. MethodsHFD-fed rats received ICV infusions of vehicle or Sfrp5 during a pancreatic euglycemic clamp procedure. To delineate the pathway(s) by which ICV Sfrp5 modulates HGP and VLDL-TG secretion, we inhibited the hypothalamic KATP channel using glibenclamide, the DVC NMDA receptor with MK801, and selectively transected the hepatic branch of the vagal nerve while centrally infusing Sfrp5. ResultsICV Sfrp5 in HFD-fed rats significantly increased the glucose infusion required to maintain euglycemia due to HGP inhibition during the clamp procedure; moreover, hepatic PEPCK and G6Pase expression was decreased, and InsR and Akt phosphorylation was increased in the liver. ICV Sfrp5 also decreased circulating triglyceride levels via inhibiting hepatic VLDL-TG secretion. These changes were accompanied by the inhibition of enzymes related to lipogenesis in the liver. ICV Sfrp5 significantly increased insulin-stimulated phosphorylation of InsR and Akt in the hypothalamus of HFD-fed rats, and insulin-stimulated immunodetectable PIP3 levels were higher in Sfrp5 group than in control group both in vitro and vivo. The glucose- and lipid-lowering effects of ICV Sfrp5 were eliminated by NMDA receptor or DVC KATP channel inhibition or HVAG. ConclusionsThe present study demonstrates that central Sfrp5 signaling activates a previously unappreciated InsR-Akt-PI3k-KATP channel pathway in the hypothalamus and brain-hepatic vagus neurocircuitry to decrease HGP and VLDL-TG secretion.

44-LB: Hypothalamic Glucocorticoid Action Regulates Lipid Metabolism

Aberrant lipid homeostasis is a feature of diabetes and obesity. This metabolic imbalance is associated with excessive levels and/or actions of glucocorticoids (GCs) and contributes to dyslipidemia including elevated hepatic triglyceride (TG)-rich very low-density lipoprotein (VLDL-TG) secretion. The mediobasal hypothalamus (MBH) is a nutrient and hormone sensing brain region that regulates whole-body lipid metabolism and VLDL-TG secretion. We aimed to assess GC action in the MBH in regulating VLDL-TG secretion in health and diet-induced hyperlipidemia. Sprague Dawley rats received stereotaxic MBH cannulation and vascular catheterizations to enable direct MBH infusions, intravenous injections, and blood sampling. Plasma TG levels and the rate VLDL-TG secretion were measured in 10h-fasted rats following intravenous poloxamer injection with concomitant MBH infusions. In healthy rats, direct MBH GC infusion increased VLDL-TG secretion compared to controls. MBH GC action is mediated via its receptor (GR) since mifepristone (GR antagonist) co-infusion, chronic inhibition of GRs in the MBH with GR shRNA, or inhibition of heat shock protein 90 (required for in vivo GR function) negated the MBH GC-induced increases in VLDL-TG secretion rate. High-fat diet-fed rats had both elevated basal plasma GC levels and hepatic TG secretion compared to healthy rats. Remarkably, chronic MBH GR loss-of-function lowered VLDL-TG secretion in high-fat diet-induced hyperlipidemic rats, independent of changes in body weight. Here, we provide novel in vivo evidence that MBH GC action modulates VLDL-TG secretion and suggest that blocking excessive GC action in the brain improves lipid homeostasis in diet-induced hyperlipidemic rats. These findings may provide insight on lowering lipid levels in diabetes and obesity. Disclosure M. Cardoso: None. M. Wang: None. E. Ling: None. H. Lee: None. E. Beaulieu-Bayne: None. J.T. Yue: None. Funding Diabetes Canada; Natural Sciences and Engineering Research Council of Canada; Canadian Lipoprotein Conference

174-OR: Dorsal Vagal Complex Is a Site for Brain Lipid Sensing to Regulate Lipid Homeostasis

Obesity and diabetes are characterized by dyslipidemia due partly to an overproduction of hepatic triglyceride-rich very-low-density lipoproteins (VLDL-TG). The hypothalamus regulates whole-body lipid homeostasis in rodents but whether extra-hypothalamic brain regions can impact lipid homeostasis remain unclear. We here assessed whether the dorsal vagal complex (DVC), a region in the brainstem which contains a circumventricular organ termed the area postrema, as well as the nucleus of the solitary tract and the dorsal motor nucleus of the vagus, senses oleic acids to regulate whole-body lipid homeostasis. We first surgically implanted a guide cannula into the DVC of male 280-300g rats for vehicle (3% cyclodextrin in saline) or oleic acid (1 mM dissolved in vehicle) infusion, followed by catheter placements (7 days after) in the left carotid artery and right jugular vein for sampling and infusion. After an additional 5 days of recovery, 10h-fasted rats received either vehicle or oleic acid pre-infusion to the DVC for 10 min, and DVC infusion was maintained for an additional 150 min after an i.v. injection of poloxamer (600 mg/kg; a lipoprotein lipase inhibitor), while plasma samples were collected every 15 min for TG measurement. The rate of plasma TG appearance as a function of time (slope) reflects the rate of VLDL-TG secretion in the current experimental context. DVC oleic acid vs. vehicle infusion (0.33 ul/h) lowered VLDL-TG secretion (mM/min) in healthy rats (0.032±0.003, n=7 vs. 0.049±0.006, n=5; p&amp;lt;0.001), but interestingly failed to do so in high-fat fed rats. Taken together, our findings indicate that: (i) the DVC is a novel site of fatty acid sensing that impacts whole-body lipid homeostasis by regulating hepatic VLDL-TG secretion, and (ii) a disruption in DVC fatty acid sensing may contribute to the overproduction of hepatic VLDL-TG and a disruption in lipid homeostasis in obesity and diabetes. Disclosure B. Batchuluun: None. T. Waise: None. J.T. Yue: None. T.K. Lam: None. Funding Canadian Institutes of Health Research

Estrogen-related receptor γ regulates hepatic triglyceride metabolism through phospholipase A2 G12B.

Hypersecretion of hepatic very LDL (VLDL)-associated triglyceride (TG) is the hallmark of hypertriglyceridemia. The estrogen-related receptor γ (ERRγ), an orphan nuclear receptor, plays crucial roles in the regulation of metabolic homeostasis, including TG formation in the liver. It remains unclear whether ERRγ regulates hepatic VLDL-TG secretion. We demonstrated that knockdown of ERRγ impairs hepatic VLDL-TG secretion in mice, whereas overexpression of ERRγ favors the secretion, indicating a novel role of ERRγ in hepatic TG metabolism. We found that ERRγ transcriptionally regulates the expression of PLA2G12B by binding to the promoter region of the Pla2g12b gene. In Pla2g12b-null mice, ERRγ fails to regulate hepatic VLDL-TG secretion. There is an apparent accumulation of large lipid droplets in the liver of Pla2g12b-null mice. These data suggest that ERRγ is a novel regulator of hepatic VLDL-TG secretion, which is mediated through the action on PLA2G12B.-Chen, L., Wu, M., Zhang, S., Tan, W., Guan, M., Feng, L., Chen, C., Tao, J., Chen, L., Qu, L. Estrogen-related receptor γ regulates hepatic triglyceride metabolism through phospholipase A2 G12B.

Vitamin E alleviates non-alcoholic fatty liver disease in phosphatidylethanolamine N-methyltransferase deficient mice

Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC), mainly in the liver. Pemt−/− mice are protected from high-fat diet (HFD)-induced obesity and insulin resistance, but develop severe non-alcoholic fatty liver disease (NAFLD) when fed a HFD, mostly due to impaired VLDL secretion. Oxidative stress is thought to be an essential factor in the progression from simple steatosis to steatohepatitis. Vitamin E is an antioxidant that has been clinically used to improve NAFLD pathology. Our aim was to determine whether supplementation of the diet with vitamin E could attenuate HFD-induced hepatic steatosis and its progression to NASH in Pemt−/− mice. Treatment with vitamin E (0.5 g/kg) for 3 weeks improved VLDL-TG secretion and normalized cholesterol metabolism, but failed to reduce hepatic TG content. Moreover, vitamin E treatment was able to reduce hepatic oxidative stress, inflammation and fibrosis. We also observed abnormal ceramide metabolism in Pemt−/− mice fed a HFD, with elevation of ceramides and other sphingolipids and higher expression of mRNAs for acid ceramidase (Asah1) and ceramide kinase (Cerk). Interestingly, vitamin E supplementation restored Asah1 and Cerk mRNA and sphingolipid levels. Together this study shows that vitamin E treatment efficiently prevented the progression from simple steatosis to steatohepatitis in mice lacking PEMT.