Very Low Density Lipoprotein Assembly Research Articles

Biosynthesis and Metabolism of ApoB-Containing Lipoproteins.

Recent advances in human genetics, together with a substantial body of epidemiological, preclinical and clinical trial evidence, strongly support a causal relationship between triglyceride-rich lipoproteins (TRLs) and atherosclerotic cardiovascular disease. Consequently, the secretion and metabolism of TRLs have a significant impact on cardiovascular health. This knowledge underscores the importance of understanding the molecular mechanisms and regulation of very-low-density lipoprotein (VLDL) and chylomicron biogenesis. Fortunately, there has been a resurgence of interest in the intracellular assembly, trafficking, degradation, and secretion of VLDL, leading to many ground-breaking molecular insights. Furthermore, the identification of molecular control mechanisms related to triglyceride metabolism has greatly advanced our understanding of the complex metabolism of TRLs. In this review, we explore recent advances in the assembly, secretion, and metabolism of TRLs. We also discuss available treatment strategies for hypertriglyceridemia.

Betaine Alleviates High-Fat Diet Induced Excessive Lipid Deposition in Gibel Carp Hepatopancreas and L8824 Cells by Enhancing VLDL Secretion through HNF4α/MTTP Pathway.

Betaine, a methyl donor, plays a crucial role in lipid metabolism. Previous studies have shown that appropriate betaine supplementation in a high-fat diet reduces triglycerides (TG) of serum and hepatopancreas in fish. However, the underlying mechanism remains unclear. This study examined whether betaine can enhance the secretion of very low-density lipoprotein (VLDL) and sought to identify the specific mechanisms through which this enhancement occurs. A lipid accumulation model was established in gibel carp and L8824 cells using a high-fat diet and oleic acid, respectively. Different doses of betaine (1, 4, and 16 g/kg in the diet; 400 μmol in cell culture) were administered, and measurements were taken for lipid deposition, gene expression of HNF4α, MTTP, and ApoB, as well as the regulation of Mttp and Apob promoters by HNF4α. The results showed that betaine supplementation mitigated lipid droplet accumulation, TG levels, and VLDL production induced by the high-fat diet in gibel carp hepatopancreas and L8824 cells. Moreover, betaine not only increased VLDL content in the cell culture supernatant but also reversed the inhibitory effects of the high-fat diet on protein expression of MTTP, ApoB, and HNF4α in both gibel carp hepatopancreas and L8824 cells. Additionally, HNF4α exhibits transactivating activity on the promoter of Mttp in gibel carp. These findings suggest that betaine supplementation exerts its effects through the HNF4α/MTTP/ApoB pathway, promoting the assembly and secretion of VLDL and effectively reducing lipid accumulation in the hepatopancreas of farmed gibel carp fed a high-fat diet.

Impaired Hepatic Very Low-Density Lipoprotein Secretion Promotes Tumorigenesis and Is Accelerated with Fabp1 Deletion

Genetic polymorphisms that impair very low-density lipoprotein (VLDL) secretion are linked to hepatic steatosis, fibrosis, and hepatocellular cancer. Liver-specific deletion of microsomal triglyceride transfer protein (Mttp-LKO) impairs VLDL assembly, promoting hepatic steatosis and fibrosis, which are attenuated in Mttp-LKO X Fabp1-null [Fabp1/Mttp double knockout (DKO)] mice. The current study examined the impact of impaired VLDL secretion in Mttp-LKO mice on hepatocellular cancer incidence and progression in comparison to Fabp1/Mttp DKO mice. Diethylnitrosamine-treated Mttp-LKO mice exhibited steatosis with increased tumor burden compared with flox controls, whereas diethylnitrosamine-treated Fabp1/Mttp DKO mice exhibited a paradoxical increase in tumor burden and >50% mortality by 50 weeks. Serum high-density lipoprotein cholesterol was elevated in both Mttp-LKO and Fabp1/Mttp DKO mice, with increased intratumoral expression of apolipoprotein A1 and apolipoprotein E. Lipidomic surveys revealed progressive enrichment in distinct triglyceride species in livers from Mttp-LKO mice with further enrichment in Fabp1/Mttp DKO mice. RNA sequencing revealed mRNA changes suggesting altered monocarboxylic acid use and increased aerobic glycolysis, whereas hepatocytes from Fabp1/Mttp DKO mice exhibited increased capacity to use glucose and glutamine. These metabolic shifts were accompanied by reduced expression of HNF1 homeobox A (HNF1a), which correlated with tumor burden. Taken together, these findings demonstrate that hepatic tumorigenesis is increased in mice with impaired VLDL secretion and further accelerated via pathways including altered fatty acid compartmentalization and shifts in hepatic energy use.

Intracellular tPA-PAI-1 interaction determines VLDL assembly in hepatocytes.

Apolipoprotein B (apoB)-lipoproteins initiate and promote atherosclerotic cardiovascular disease. Plasma tissue plasminogen activator (tPA) activity is negatively associated with atherogenic apoB-lipoprotein cholesterol levels in humans, but the mechanisms are unknown. We found that tPA, partially through the lysine-binding site on its Kringle 2 domain, binds to the N terminus of apoB, blocking the interaction between apoB and microsomal triglyceride transfer protein (MTP) in hepatocytes, thereby reducing very-low-density lipoprotein (VLDL) assembly and plasma apoB-lipoprotein cholesterol levels. Plasminogen activator inhibitor 1 (PAI-1) sequesters tPA away from apoB and increases VLDL assembly. Humans with PAI-1 deficiency have smaller VLDL particles and lower plasma levels of apoB-lipoprotein cholesterol. These results suggest a mechanism that fine-tunes VLDL assembly by intracellular interactions among tPA, PAI-1, and apoB in hepatocytes.

Liver-Specific Bmal1 Depletion Reverses the Beneficial Effects of Nobiletin on Liver Cholesterol Homeostasis in Mice Fed with High-Fat Diet.

Nobiletin (NOB), a naturally occurring small-molecule compound abundant in citrus peels, has displayed potential lipid-lowering and circadian-enhancing properties in preclinical studies. However, the requirement of specific clock genes for the beneficial effects of NOB is not well understood. In the current study, mice with a liver-specific deletion of the core clock component, Bmal1-Bmal1LKO-were fed a high-fat diet (HFD) ad libitum for eight weeks, while NOB (200 mg/kg) was administered by daily oral gavage from the fifth week and throughout the last four weeks. NOB decreased liver triglyceride (TG) alongside the decreasing mRNA levels of de novo lipogenesis (DNL) genes in both Bmal1flox/flox and Bmal1LKO mice. NOB increased serum very low-density lipoprotein (VLDL) levels in Bmal1LKO mice, which was consistent with higher liver Shp and lower Mttp mRNA expression levels, the key genes that facilitate VLDL assembly and secretion. NOB decreased liver and serum cholesterol levels in the Bmal1flox/flox mice, consistent with lower Hmgcr and higher Cyp7a1, Cyp8b1, Gata4 and Abcg5 mRNA levels in the liver. In contrast, in the Bmal1LKO mice, NOB increased Hmgcr mRNA levels and had no effect on the above-mentioned genes related to bile acid synthesis and cholesterol excretion, which might contribute to the elevation of liver and serum cholesterol levels in NOB-treated Bmal1LKO mice. NOB inhibited hepatic DNL and decreased liver TG levels in HFD-fed mice independently of liver Bmal1, whereas liver-specific Bmal1 depletion reversed the beneficial effects of NOB on liver cholesterol homeostasis. The complex interactions between NOB, the circadian clock and lipid metabolism in the liver warrant further research.

A novel gene affecting VLDL assembly/secretion

Background and Aims: Compromised secretion of very low-density lipoprotein (VLDL) can result in hepatic lipid accumulation, a risk factor for non-alcoholic fatty liver disease (NAFLD). Basic knowledge of VLDL assembly and the secretion pathway may help to find solutions for NAFLD. Here, we present a novel gene, SMLR1, identified through contextual co-expression analysis with APOC3 as anchor. SMLR1 expression is strictly confined to liver and small intestine. Here, we present a first study on the role of SMLR1 in hepatic and plasma lipid metabolism.

Sirtuin 3 Restores Synthesis and Secretion of Very Low-Density Lipoproteins in Cow Hepatocytes Challenged with Nonesterified Fatty Acids In Vitro.

Fatty liver is closely associated with elevated concentrations of nonesterified fatty acids (NEFA) and a low level of very low-density lipoproteins (VLDL) in blood of dairy cows. High NEFA inhibit the VLDL synthesis and assembly, and cause hepatic triacylglycerol (TAG) deposition. Sirtuin 3 (SIRT3), a mitochondrial deacetylase, antagonizes NEFA-induced TAG accumulation through modulating expressions of fatty acid synthesis and oxidation genes in cow hepatocytes. However, the role of SIRT3 in the VLDL synthesis and assembly was largely unknown. Here we aimed to test whether SIRT3 would recover the synthesis and assembly of VLDL in cow hepatocytes induced by high NEFA. Primary cow hepatocytes were isolated from 3 Holstein cows. Hepatocytes were infected with SIRT3 overexpression adenovirus (Ad-SIRT3), SIRT3-short interfering (si) RNA, or first infected with Ad-SIRT3 and then incubated with 1.0 mM NEFA (Ad-SIRT3 + NEFA). Expressions of key genes in VLDL synthesis and the VLDL contents in cell culture supernatants were measured. SIRT3 overexpression significantly increased the mRNA abundance of microsomal triglyceride transfer protein (MTP), apolipoprotein B100 (ApoB100) and ApoE (p < 0.01), and raised VLDL contents in the supernatants (p < 0.01). However, SIRT3 silencing displayed a reverse effect in comparison to SIRT3 overexpression. Compared with NEFA treatment alone, the Ad-SIRT3 + NEFA significantly upregulated the mRNA abundance of MTP, ApoB100 and ApoE (p < 0.01), and increased VLDL contents in the supernatants (p < 0.01). Our data demonstrated that SIRT3 restored the synthesis and assembly of VLDL in cow hepatocytes challenged with NEFA, providing an in vitro basis for further investigations testing its feasibility against hepatic TAG accumulation in dairy cows during the perinatal period.

Elucidation of the mechanism of NEFA-induced PERK-eIF2α signaling pathway regulation of lipid metabolism in bovine hepatocytes

During the periparturient transition period, negative energy balance (NEB) characterized by high concentrations of non-esterified fatty acids (NEFA) may cause fatty liver and ketosis in dairy cows. Previous studies have shown that the protein kinase R-like endoplasmic reticulum kinase (PERK) branch of the endoplasmic reticulum stress (ERS) response plays an important role in lipid metabolism in hepatocytes. This study, therefore, investigated the role of the PERK-branch in NEFA-induced fatty liver. Different concentrations of NEFA or GSK2656157 (a novel catalytic inhibitor of PERK) were used to treat hepatocytes isolated from calves. The NEFA treatment significantly increased the triacylglycerol (TG) content, the phosphorylation level of PERK and eukaryotic initiation factor 2α (eIF2α), and the abundance of glucose-regulated protein 78 (Grp78), C/EBP homologous protein (CHOP), sterol regulatory element-binding protein 1c (SREBP-1c), fatty acid synthase (FASN), peroxisome proliferator-activated receptor-α (PPARα), carnitine palmitoyltransferase 1A (CPT1A), apolipoprotein B (APOB), and the low-density lipoprotein receptor (LDLR). Compared with the 1.2 mM NEFA group, inhibition of PERK activity further increased the TG content in hepatocytes, the very-low-density lipoprotein (VLDL) content in the supernatant and the protein abundance of APOB while reducing the expression and nuclear levels of SREBP-1c and PPARα, as well as the expression of CPT1A and CPT2. In conclusion, the results showed that the NEFA-induced PERK-eIF2α signaling pathway promotes lipid synthesis, lipid oxidation, but inhibits the assembly and secretion of VLDL. Therefore, during the transition period, the activation of the PERK-eIF2α signaling pathway in the liver of dairy cows could defeat the acid-induced lipotoxicity and provide energy to alleviate NEB.

Knockdown of phosphatase and tensin homolog (PTEN) inhibits fatty acid oxidation and reduces very low density lipoprotein assembly and secretion in calf hepatocytes

Dairy cows with fatty liver exhibit hepatic lipid accumulation and disturbances in fatty acid oxidation and lipid transport. Phosphatase and tensin homolog (PTEN), a lipid phosphatase, regulates intrahepatic fatty acid oxidation and lipid transport in mice. Whether PTEN play a role in fatty acid oxidation and very low density lipoprotein (VLDL) assembly in calf hepatocytes are unknown. Hepatocytes isolated from 3 healthy female Holstein calves (1 d old, 30-40 kg) were infected with empty adenovirus with green fluorescent protein for 48 h (Ad-GFP group) or infected with PTEN knockdown adenovirus for 48 h (Ad-shPTEN group), or cultured in RPMI-1640 without Ad-shPTEN or Ad-GFP (control group). Compared with the Ad-GFP group, PTEN knockdown decreased mRNA and protein abundance and the activity of fatty acid oxidation-related molecules, including acyl-coA synthetase long-chain 1, carnitine palmitoyltransferase 1, carnitine palmitoyltransferase 2, and 3-hydroxy acyl-coA dehydrogenase. Furthermore, PTEN knockdown decreased mRNA and protein abundance of VLDL assembly-related molecules, including apolipoprotein B100, apolipoprotein E, microsomal triglyceride transfer protein, and low density lipoprotein receptor. Importantly, PTEN knockdown promoted triglyceride accumulation in hepatocytes and reduced the VLDL content in culture medium. A subsequent study was conducted on the following 4 groups: cells infected with Ad-GFP for 48 h and then treated with 2% BSA for another 24 h (Ad-GFP + BSA); cells infected with Ad-GFP for 48 h and then treated with 1.2 mM free fatty acids (FFA) and 2% BSA for another 24 h (Ad-GFP + 1.2 mM FFA); cells infected with Ad-shPTEN for 48 h and then treated with 2% BSA for another 24 h (Ad-shPTEN + BSA); cells infected with Ad-shPTEN for 48 h and then treated with 1.2 mM FFA and 2% BSA for another 24 h (Ad-shPTEN + 1.2 mM FFA). Compared with Ad-GFP + BSA, the abundances of PTEN and of fatty acid oxidation- and VLDL assembly-related proteins were lower in the Ad-GFP + 1.2 mM FFA group. Importantly, PTEN knockdown heightened the increase in triglyceride accumulation of hepatocytes and the decrease in VLDL content in culture medium induced by FFA. Overall, these in vitro data indicate that FFA inhibits PTEN expression, leading to triglyceride accumulation and the inhibition of VLDL assembly in calf hepatocytes. These findings suggest that PTEN may be a potential therapeutic target for FFA-induced hepatic steatosis in dairy cows.

Drug-induced hepatic steatosis in absence of severe mitochondrial dysfunction in HepaRG cells: proof of multiple mechanism-based toxicity

Steatosis is a liver lesion reported with numerous pharmaceuticals. Prior studies showed that severe impairment of mitochondrial fatty acid oxidation (mtFAO) constantly leads to lipid accretion in liver. However, much less is known about the mechanism(s) of drug-induced steatosis in the absence of severe mitochondrial dysfunction, although previous studies suggested the involvement of mild-to-moderate inhibition of mtFAO, increased de novo lipogenesis (DNL), and impairment of very low-density lipoprotein (VLDL) secretion. The objective of our study, mainly carried out in human hepatoma HepaRG cells, was to investigate these 3 mechanisms with 12 drugs able to induce steatosis in human: amiodarone (AMIO, used as positive control), allopurinol (ALLO), d-penicillamine (DPEN), 5-fluorouracil (5FU), indinavir (INDI), indomethacin (INDO), methimazole (METHI), methotrexate (METHO), nifedipine (NIF), rifampicin (RIF), sulindac (SUL), and troglitazone (TRO). Hepatic cells were exposed to drugs for 4 days with concentrations decreasing ATP level by less than 30% as compared to control and not exceeding 100 × Cmax. Among the 12 drugs, AMIO, ALLO, 5FU, INDI, INDO, METHO, RIF, SUL, and TRO induced steatosis in HepaRG cells. AMIO, INDO, and RIF decreased mtFAO. AMIO, INDO, and SUL enhanced DNL. ALLO, 5FU, INDI, INDO, SUL, RIF, and TRO impaired VLDL secretion. These seven drugs reduced the mRNA level of genes playing a major role in VLDL assembly and also induced endoplasmic reticulum (ER) stress. Thus, in the absence of severe mitochondrial dysfunction, drug-induced steatosis can be triggered by different mechanisms, although impairment of VLDL secretion seems more frequently involved, possibly as a consequence of ER stress.

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.

Molecular Insights into the Mechanisms Underlying the Cholesterol- Lowering Effects of Phytosterols.

Dietary phytosterols, which comprise plant sterols and stanols, reduce plasma Low-Density Lipoprotein-Cholesterol (LDL-C) levels when given 2 g/day. Since this dose has not been reported to cause health-related side effects in long-term human studies, food products containing these plant compounds are used as potential therapeutic dietary options to reduce LDL-C and cardiovascular disease risk. Several mechanisms have been proposed to explain the cholesterol-lowering action of phytosterols. They may compete with dietary and biliary cholesterol for micellar solubilization in the intestinal lumen, impairing intestinal cholesterol absorption. Recent evidence indicates that phytosterols may also regulate other pathways. Impaired intestinal cholesterol absorption is usually associated with reduced cholesterol transport to the liver, which may reduce the incorporation of cholesterol into Very-Low- Density Lipoprotein (VLDL) particles, thereby lowering the rate of VLDL assembly and secretion. Impaired liver VLDL production may reduce the rate of LDL production. On the other hand, significant evidence supports a role for plant sterols in the Transintestinal Cholesterol Excretion (TICE) pathway, although the exact mechanisms by which they promote the flow of cholesterol from the blood to enterocytes and the intestinal lumen remains unknown. Dietary phytosterols may also alter the conversion of bile acids into secondary bile acids, and may lower the bile acid hydrophobic/hydrophilic ratio, thereby reducing intestinal cholesterol absorption. This article reviews the progress to date in research on the molecular mechanisms underlying the cholesterol-lowering effects of phytosterols.

MiR-130b is a potent stimulator of hepatic very-low-density lipoprotein assembly and secretion via marked induction of microsomal triglyceride transfer protein

miR-130b is a microRNA whose expression is particularly elevated within adipose tissue and in the circulation in diabetic states. Hepatic miR-130b expression has been linked to hepatocellular carcinoma and changes in lipid metabolism. Here, we investigated the role of miR-130b in hepatic lipid homeostasis and lipoprotein export. We observed that overexpression of miR-130b-3p or -5p in HepG2 cells markedly enhanced the secretion of very-low-density lipoprotein (VLDL) particles, enhanced the secretion of [3H]glycerol metabolically labeled triglyceride (TG), and significantly increased the number or the average size of lipid droplets (LDs), respectively. Overexpression of miR-130b also altered the expression of key genes involved in lipid metabolism and in particular markedly increased both mRNA and protein expression levels of microsomal triglyceride transfer protein (MTP). Conversely, the miR-130b inhibitor decreased mRNA levels of MTP and fatty acid synthase (FAS) in HepG2 cells. However, dual-luciferase reporter assays indicated that MTP is not a direct target of miR-130b-3p. miR-130b overexpression did not alter de novo synthesized TG or the stability and secretion of apolipoprotein B 100. Interestingly, knockdown of phosphatase and tensin homolog (PTEN) blocked the upregulation of MTP mRNA induced by miR-130b. Finally, miR-130b-induced stimulation of VLDL secretion was also observed in a second hepatocyte cell culture model, immortalized human hepatocytes, confirming the effects observed in HepG2 cells. Overall, these data suggest a potential role for miR-130b in promoting hepatic VLDL assembly and secretion mediated by marked stimulation of MTP expression and TG mobilization. Thus miR-130b overexpression corrects the defect in VLDL production in HepG2 cells.

Regulation of triglyceride synthesis by estradiol in the livers of hybrid tilapia (Oreochromis niloticus ♀ × O. aureus ♂).

Estradiol (E2) is a sex steroid hormone that modulates multiple physiological processes in teleosts. The aim of this study was to explore the role of E2 in the hepatic lipid metabolism of hybrid tilapia. The hybrid tilapias were injected with different concentrations of E2 (0 mg/kg, 10 mg/kg, 25 mg/kg and 50 mg/kg) and ICI 182,780 (ICI) (35 mg/kg) (an E2 receptor antagonist). Subsequently, the liver lipid depositions were analyzed by tissue sections with oil red O staining. Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and nonesterified fatty acids (NEFAs) were assayed from the fish in different groups. Genes related to very low-density lipoprotein (VLDL) assembly, lipoprotein lipase and lipoprotein receptors, fatty acid uptake and triacylglycerol metabolism were determined by quantitative RT-PCR. The results showed that 50 mg/kg E2 injections enlarged the lipid droplets significantly. Simultaneously, the E2 injections tended to upregulate TC, TG, LDL, and HDL in the serum. The 50 mg/kg E2 group showed a significantly higher expression of the VLDL assembly genes but depressed levels of LDLR and LRP1. In addition, FABP3, FABP11a and DGAT2 were significantly elevated, while CD36 and ACO1 decreased in the 50 mg/kg E2 injection. The ICI injection inhibited the expression of MTP, LPL, LRP1, CD36, FABP11a, ACO1 and FAS in tilapia livers. These results demonstrated that by stimulating the expression of genes associated with the VLDL assembly, inhibiting lipoprotein lipase and lipoprotein receptor-related genes and promoting the rate-limiting enzyme in the synthesis of the TG, E2 induced deposition of lipids in the livers of hybrid tilapia. Overall, the results suggest a role for E2 in fish lipid metabolisms that provide new clues to illustrate the sex steroid function in energy metabolism in livers.

Glucagon attenuates lipid accumulation in cow hepatocytes through AMPK signaling pathway activation.

The ketotic cows displayed hepatic lipid metabolic disorder and high blood concentration of glucagon. Importantly, adenosine monophosphate-activated protein kinase (AMPK) signaling pathway plays an important role in the hepatic lipid homeostasis. Therefore, the aim of this study was to investigate the effect of glucagon on AMPK pathway and its underlying mechanism on lipid metabolism in cow hepatocytes. Cow hepatocytes were cultured and treated with glucagon and AMPK inhibitor (BML-275). The results showed that glucagon significantly promoted the expression of glucagon receptor and increased the phosphorylation level and activity of AMPKα. Activated AMPKα increased the expression level and transcriptional activity of peroxisome proliferator-activated receptor α, which further increased the expression of fatty acid oxidation genes and lipid oxidation. Furthermore, activated AMPKα inhibited the expression level and transcriptional activity of sterol regulatory element binding protein-1c and carbohydrate response element binding protein, which decreased the expression of lipogenic genes, thereby decreasing lipid synthesis. In addition, glucagon also increased the expression of very-low-density lipoprotein (VLDL) assembly to export intracellular triglycerides (TG). Consequently, the content of intracellular TG was significantly decreased in cow hepatocytes. These results indicate that glucagon activates the AMPK signaling pathway to increase lipid oxidation and VLDL assembly and decrease lipid synthesis in cow hepatocytes, thereby reducing liver fat accumulation.

Insulin suppresses the AMPK signaling pathway to regulate lipid metabolism in primary cultured hepatocytes of dairy cows.

Dairy cows with type II ketosis display hepatic fat accumulation and hyperinsulinemia, but the underlying mechanism is not completely clear. This study aimed to clarify the regulation of lipid metabolism by insulin in cow hepatocytes. In vitro, cow hepatocytes were treated with 0, 1, 10, or 100 nm insulin in the presence or absence of AICAR (an AMP-activated protein kinase alpha (AMPKα) activator). The results showed that insulin decreased AMPKα phosphorylation. This inactivation of AMPKα increased the gene and protein expression levels of carbohydrate responsive element-binding protein (ChREBP) and sterol regulatory element-binding protein-1c (SREBP-1c), which downregulated the expression of lipogenic genes, thereby decreasing lipid biosynthesis. Furthermore, AMPKα inactivation decreased the gene and protein expression levels of peroxisome proliferator-activated receptor-α (PPARα), which upregulated the expression of lipid oxidation genes, thereby increasing lipid oxidation. In addition, insulin decreased the very low density lipoprotein (VLDL) assembly. Consequently, triglyceride content was significantly increased in insulin treated hepatocytes. Activation of AMPKα induced by AICAR could reverse the effect of insulin on PPARα, SREBP-1c, and ChREBP, thereby decreasing triglyceride content. These results indicate that insulin inhibits the AMPKα signaling pathway to increase lipid synthesis and decrease lipid oxidation and VLDL assembly in cow hepatocytes, thereby inducing TG accumulation. This mechanism could partly explain the causal relationship between hepatic fat accumulation and hyperinsulinemia in dairy cows with type II ketosis.

Quercetin ameliorates HFD-induced NAFLD by promoting hepatic VLDL assembly and lipophagy via the IRE1a/XBP1s pathway

The consumption of a quercetin-rich diet has been well-established as a feasible method against non-alcoholic fatty liver disease (NAFLD); however, the molecular mechanisms underlying the progression of NAFLD and its intervention by quercetin remain largely obscure. Male Sprague-Dawley rats fed high-fat diet (HFD), and HepG2 cells stimulated with free fatty acid, were treated with quercetin and various pharmacological reagents to explore the effect of signaling pathways involved in endoplasmic reticulum stress on very low-density lipoprotein (VLDL) assembly and lipophagy. Quercetin intake decreased hepatic TG content by 39%, with a 1.5-fold increase in VLDL, and up-regulated spliced X-box binding protein 1 (XBP1s) expression compared with the HFD group. Thapsigargin or STF-083010 (an IRE1α endonuclease inhibitor) decreased VLDL content in a dose-dependent manner, partially counteracting the protective effects of quercetin, 4-PBA or APY-29 (an IRE1α endonuclease activator). Additionally, microsomal TG-transfer protein complex expression was increased with quercetin-treated and down-regulated by STF-083010. Moreover, quercetin increased co-localization of lysosomes with lipid droplets (LDs) accompanied by decreased p62 accumulation. STF-083010 partially abolished the effect of quercetin on LDs autophagy in an mTOR-independent manner. Collectively, these findings demonstrate that hepatic VLDL assembly and lipophagy are the main targets of quercetin against NAFLD via the IRE1a/XBP1s pathway.

Referee Report: Methyl donor deficient diets cause distinct alterations in lipid metabolism but are poorly representative of human NAFLD. Wellcome Open Res 2017, 2:67

Background: Non-alcoholic fatty liver disease (NAFLD) is a global health issue. Dietary methyl donor restriction is used to induce a NAFLD/non-alcoholic steatohepatitis (NASH) phenotype in rodents, however the extent to which this model reflects human NAFLD remains incompletely understood. To address this, we undertook hepatic transcriptional profiling of methyl donor restricted rodents and compared these to published human NAFLD datasets. Methods: Adult C57BL/6J mice were maintained on control, choline deficient (CDD) or methionine/choline deficient (MCDD) diets for four weeks; the effects on methyl donor and lipid biology were investigated by bioinformatic analysis of hepatic gene expression profiles followed by a cross-species comparison with human expression data of all stages of NAFLD. Results: Compared to controls, expression of the very low density lipoprotein (VLDL) packaging carboxylesterases ( Ces1d, Ces1f, Ces3b) and the NAFLD risk allele Pnpla3 were suppressed in MCDD; with Pnpla3 and the liver predominant Ces isoform, Ces3b, also suppressed in CDD. With respect to 1-carbon metabolism, down-regulation of Chka, Chkb, Pcty1a, Gnmt and Ahcy with concurrent upregulation of Mat2a suggests a drive to maintain S-adenosylmethionine levels. There was minimal similarity between global gene expression patterns in either dietary intervention and any stage of human NAFLD, however some common transcriptomic changes in inflammatory, fibrotic and proliferative mediators were identified in MCDD, NASH and HCC. Conclusions: This study suggests suppression of VLDL assembly machinery may contribute to hepatic lipid accumulation in these models, but that CDD and MCDD rodent diets are minimally representative of human NAFLD at the transcriptional level.

Methyl donor deficient diets cause distinct alterations in lipid metabolism but are poorly representative of human NAFLD.

Background: Non-alcoholic fatty liver disease (NAFLD) is a global health issue. Dietary methyl donor restriction is used to induce a NAFLD/non-alcoholic steatohepatitis (NASH) phenotype in rodents, however the extent to which this model reflects human NAFLD remains incompletely understood. To address this, we undertook hepatic transcriptional profiling of methyl donor restricted rodents and compared these to published human NAFLD datasets. Methods: Adult C57BL/6J mice were maintained on control, choline deficient (CDD) or methionine/choline deficient (MCDD) diets for four weeks; the effects on methyl donor and lipid biology were investigated by bioinformatic analysis of hepatic gene expression profiles followed by a cross-species comparison with human expression data of all stages of NAFLD. Results: Compared to controls, expression of the very low density lipoprotein (VLDL) packaging carboxylesterases ( Ces1d, Ces1f, Ces3b) and the NAFLD risk allele Pnpla3 were suppressed in MCDD; with Pnpla3 and the liver predominant Ces isoform, Ces3b, also suppressed in CDD. With respect to 1-carbon metabolism, down-regulation of Chka, Chkb, Pcty1a, Gnmt and Ahcy with concurrent upregulation of Mat2a suggests a drive to maintain S-adenosylmethionine levels. There was minimal similarity between global gene expression patterns in either dietary intervention and any stage of human NAFLD, however some common transcriptomic changes in inflammatory, fibrotic and proliferative mediators were identified in MCDD, NASH and HCC. Conclusions: This study suggests suppression of VLDL assembly machinery may contribute to hepatic lipid accumulation in these models, but that CDD and MCDD rodent diets are minimally representative of human NAFLD at the transcriptional level.

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.