Very Low Density Lipoprotein Synthesis Research Articles

Lipidomic and transcriptomic profiles provide new insights into the triacylglycerol and glucose handling capacities of the Arctic fox.

The Arctic fox (Vulpes lagopus) is a species indigenous to the Arctic and has developed unique lipid metabolism, but the mechanisms remain unclear. Here, the significantly increased body weight of Arctic foxes was consistent with the significantly increased serum very-low-density lipoprotein (VLDL), and the 40% crude fat diet further increased the Arctic fox body weight. The enhanced body weight gain stems primarily from increased subcutaneous adipose tissue accumulation. The adipose triacylglycerol and phosphatidylethanolamine were significantly greater in Arctic foxes. The adipose fatty-acid synthase content was significantly lower in Arctic foxes, highlighting the main role of exogenous fatty-acids in fat accumulation. Considering the same diet, liver-derived fat dominates adipose expansion in Arctic foxes. Liver transcriptome analysis revealed greater fat and VLDL synthesis in Arctic foxes, consistent with the greater VLDL. Glucose homeostasis wasn't impacted in Arctic foxes. And the free fatty-acids in adipose, which promote insulin resistance, also did not differ between groups. However, the hepatic glycogen was greater in Arctic foxes and transcriptome analysis revealed upregulated glycogen synthesis, improving glucose homeostasis. These results suggest that the superior fat accumulation capacity and distinct characteristics of hepatic and adipose lipid and glucose metabolism facilitate glucose homeostasis and massive fat accumulation in Arctic foxes.

α-Tocopherol reduces VLDL secretion through modulation of intracellular ER-to-Golgi transport of VLDL.

Avoiding hepatic steatosis is crucial for preventing liver dysfunction, and one mechanism by which this is accomplished is through synchronization of the rate of very low density lipoprotein (VLDL) synthesis with its secretion. Endoplasmic reticulum (ER)-to-Golgi transport of nascent VLDL is the rate-limiting step in its secretion and is mediated by the VLDL transport vesicle (VTV). Recent in vivo studies have indicated that α-tocopherol (α-T) supplementation can reverse steatosis in nonalcoholic fatty liver disease, but its effects on hepatic lipoprotein metabolism are poorly understood. Here, we investigated the impact of α-T on hepatic VLDL synthesis, secretion, and intracellular ER-to-Golgi VLDL trafficking using an in vitro model. Pulse-chase assays using [3H]-oleic acid and 100 µmol/L α-T demonstrated a disruption of early VLDL synthesis, resulting in enhanced apolipoprotein B-100 expression, decreased expression in markers for VTV budding, ER-to-Golgi VLDL transport, and reduced VLDL secretion. Additionally, an in vitro VTV budding assay indicated a significant decrease in VTV production and VTV-Golgi fusion. Confocal imaging of lipid droplet (LD) localization revealed a decrease in overall LD retention, diminished presence of ER-associated LDs, and an increase in Golgi-level LD retention. We conclude that α-T disrupts ER-to-Golgi VLDL transport by modulating the expression of specific proteins and thus reduces VLDL secretion.

Dietary choline activates the Ampk/Srebp signaling pathway and decreases lipid levels in Pacific white shrimp (Litopenaeus vannamei)

An 8-week feeding trial was conducted in Pacific white shrimp (Litopenaeus vannamei) to evaluate the effects of dietary choline supplementation on choline transport and metabolism, hepatopancreas histological structure and fatty acid profile, and regulation of lipid metabolism. Six isonitrogenous and isolipidic diets were formulated to contain different choline levels of 2.91 (basal diet), 3.85, 4.67, 6.55, 10.70 and 18.90 g/kg, respectively. A total of 960 shrimp (initial weight, 1.38 ± 0.01 g) were distributed randomly into twenty-four 250-L cylindrical fiber-glass tanks, with each diet assigned randomly to 4 replicate tanks. The results indicated that dietary choline significantly promoted the deposition of choline, betaine and carnitine (P < 0.05). The diameters and areas of R cells, total lipid and triglyceride contents in hepatopancreas, and triglyceride and non-esterified fatty acid contents in hemolymph were negatively correlated with dietary choline level. The contents of functional fatty acids in hepatopancreas, the activity of acetyl-CoA carboxylase (Acc), and the mRNA expression of fas, srebp and acc were highest in shrimp fed the diet containing 4.67 g/kg choline, and significantly higher than those fed the diet containing 2.91 g/kg, the lowest level of choline (P < 0.05). The number of R cells, content of very low-density lipoprotein (VLDL), activities of carnitine palmitoyl-transferase (Cpt1), lipoprotein lipase and hepatic lipase, and the mRNA expression levels of cpt1, fabp, fatp, ldlr, and ampk in hepatopancreas increased significantly as dietary choline increased (P < 0.05). In addition, hepatopancreas mRNA expression levels of ctl1, ctl2, oct1, badh, bhmt, ck, cept, and cct were generally up-regulated as dietary choline level increased (P < 0.01). In conclusion, dietary choline promoted the deposition of choline and its metabolites by up-regulating genes related to choline transport and metabolism. Moreover, appropriate dietary choline level promoted the development of hepatopancreas R cells and maintained the normal accumulation of lipids required for development, while high dietary choline not only promoted hepatopancreas lipid export by enhancing VLDL synthesis, but also promoted fatty acid β-oxidation and inhibited de novo fatty acid synthesis by activating the Ampk/Srebp signaling pathway. These findings provided further insight and understanding of the mechanisms by which dietary choline regulated lipid metabolism in L. vannamei.

Dietary inositol improved glucose and lipid metabolism mediated by the Insulin-PI3K-Akt signaling pathway in Pacific white shrimp (Litopenaeus vannamei)

The present study aimed to evaluate the effects of dietary inositol level (0.86, 1.34, 1.40, 2.30, 3.88 and 7.50 g kg−1) on glucose and lipid homeostasis in Litopenaeus vannamei. The results indicated that the contents of inositol, phosphatidyl inositol (PI) and glycogen in hepatopancreas and muscle, very low-density lipoprotein (VLDL) in hepatopancreas and hemolymph, the activity of carnitine palmitoyl-transferase (CPT1) and the expression of hmit and inox were positively related to the dietary inositol levels, while the contents of glucose (GLU), crustacean hyperglycemic hormone (CHH), triglyceride (TG), cholesterol (CHO) and non-esterified fatty acid (NEFA) in hemolymph and the expression of imp, pepck, g6pc and fbp were negatively correlated with the dietary inositol level. TG content, phosphoenolpyruvate carboxykinase (PEPCK) activity and the expression levels of srebp, fas, acc1, g6pd and 6gpd in shrimp fed 3.88 and 7.50 g kg−1 inositol diets were significantly lower compared with shrimp fed the basal diet, while the expression of cpt1, aco, fabp, fatp and idir were showed the reverse change trend (P < 0.05). The activities of hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK) and the expression levels of ir, irs1, pi3k and akt were significantly increased as dietary inositol levels ranged from 1.40 to 7.50 g kg−1, while the expression levels of gsk-3β and foxo1 significantly down-regulated. Hepatopancreas R cells number in shrimp fed 2.30, 3.88 and 7.50 g kg−1 inositol diets were significantly increased compared with shrimp fed 0.86 g kg−1 inositol diet, while the diameters and areas showed an opposite trend. In addition, dietary inositol supplementation significantly increased hemolymph insulin like peptide (ILP) content but had no significant effect on high density lipoprotein cholesterol (HLDC) content in hemolymph, hepatic lipase (HL) activity and the ratio of n-3/ n-6 PUFA and DHA/EPA in hepatopancreas. In conclusion, appropriate dietary inositol could positively regulate the Insulin-PI3K-Akt pathway to promote glucose utilization and maintain the normal accumulation of glycogen and lipids required for development, while high dietary inositol could promote lipid mobilization and prevent hepatopancreas lipids deposition by negatively regulating the Insulin-PI3K-Akt pathway, improving ILP sensitivity and enhancing VLDL synthesis, which may therefore be an important mechanism in the action of inositol on glucose and lipid metabolism. These findings provided further insight and understanding of mechanisms by which dietary inositol regulated glucose and lipid metabolism in L. vannamei.

A three-organelle complex made by wrappER contacts with peroxisomes and mitochondria responds to liver lipid flux changes.

Hepatic lipid homeostasis depends on intracellular pathways that respire fatty acid in peroxisomes and mitochondria, and on systemic pathways that secrete fatty acid into the bloodstream, either free or condensed in very-low-density lipoprotein (VLDL) triglycerides. These systemic and intracellular pathways are interdependent, but it is unclear whether and how they integrate into a single cellular circuit. Here, we report that mouse liver wrappER, a distinct endoplasmic reticulum (ER) compartment with apparent fatty acid- and VLDL-secretion functions, connects peroxisomes and mitochondria. Correlative light electron microscopy, quantitative serial section electron tomography and three-dimensional organelle reconstruction analysis show that the number of peroxisome-wrappER-mitochondria complexes changes throughout fasting-to-feeding transitions and doubles when VLDL synthesis stops following acute genetic ablation of Mttp in the liver. Quantitative proteomic analysis of peroxisome-wrappER-mitochondria complex-enriched fractions indicates that the loss of Mttp upregulates global fatty acid β-oxidation, thereby integrating the dynamics of this three-organelle association into hepatic fatty acid flux responses. Therefore, liver lipid homeostasis occurs through the convergence of systemic and intracellular fatty acid-elimination pathways in the peroxisome-wrappER-mitochondria complex.

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.

Low-density lipoprotein receptor is required for cholesteryl ester transfer protein to regulate triglyceride metabolism in both male and female mice.

Elevated triglycerides (TGs) and impaired TG clearance increase the risk of cardiovascular disease in both men and women, but molecular mechanisms remain poorly understood. Cholesteryl ester transfer protein (CETP) is a lipid shuttling protein known for its effects on high‐density lipoprotein cholesterol. Although mice lack CETP, transgenic expression of CETP in mice alters TG metabolism in males and females by sex‐specific mechanisms. A unifying mechanism explaining how CETP alters TG metabolism in both males and females remains unknown. Since low‐density lipoprotein receptor (LDLR) regulates both TG clearance and very low density lipoprotein (VLDL) production, LDLR may be involved in CETP‐mediated alterations in TG metabolism in both males and females. We hypothesize that LDLR is required for CETP to alter TG metabolism in both males and females. We used LDLR null mice with and without CETP to demonstrate that LDLR is required for CETP to raise plasma TGs and to impair TG clearance in males. We also demonstrate that LDLR is required for CETP to increase TG production and to increase the expression and activity of VLDL synthesis targets in response to estrogen. Additionally, we show that LDLR is required for CETP to enhance β‐oxidation. These studies support that LDLR is required for CETP to regulate TG metabolism in both males and females.

The Disruption of Liver Metabolic Circadian Rhythms by a Cafeteria Diet Is Sex-Dependent in Fischer 344 Rats.

Circadian rhythms are ~24 h fluctuations of different biological processes that are regulated by the circadian clock system. They exert a major influence on most of the metabolism, such as the hepatic metabolism. This rhythmicity can be disrupted by obesogenic diets, fact that is considered to be a risk factor for the development of metabolic diseases. Nevertheless, obesogenic diets do not affect both genders in the same manner. We hypothesized that the circadian rhythms disruption of the hepatic metabolism, caused by obesogenic diets, is gender-dependent. Male and female Fischer 344 rats were fed either a standard diet or a cafeteria diet and sacrificed at two different moments, at zeitgeber 3 and 15. Only female rats maintained the circadian variations of the hepatic metabolism under a cafeteria diet. Most of those metabolites were related with the very low density lipoprotein (VLDL) synthesis, such as choline, betaine or phosphatidylcholine. Most of these metabolites were found to be increased at the beginning of the dark period. On the other hand, male animals did not show these time differences. These findings suggest that females might be more protected against the circadian disruption of the hepatic metabolism caused by a cafeteria diet through the increase of the VLDL synthesis at the beginning of the feeding time.

Effects and molecular mechanisms of Ninghong black tea extract in nonalcoholic fatty liver disease of rats.

The aim of this study is to observe the effects of Ninghong black tea extract on fat deposition and high-fat diet-induced nonalcoholic fatty liver disease (NAFLD) and to explore the potential mechanisms of these effect. Under 2% Ninghong black tea extract diet feeding in rat model, the results showed that Ninghong black tea extract decreased the body fat ratio and the number of lipid droplets in the liver and significantly alleviated NAFLD in the rat model. The real-time fluorescence quantitative polymerase chain reaction results showed that Ninghong black tea extract significantly upregulated the expression of peroxisome proliferator-activated receptor α (PPARα), which is important in fatty acid β-oxidation, and microsomal triglyceride transfer protein (MTP), which plays an important role in the synthesis of very low density lipoprotein (VLDL). By promoting the expression of PPARα and MTP in liver tissue and thereby promoting fatty acid β-oxidation and VLDL synthesis, Ninghong black tea extract relieves high-fat diet-induced NAFLD.

Protective Effect of Patchouli Alcohol Against High-Fat Diet Induced Hepatic Steatosis by Alleviating Endoplasmic Reticulum Stress and Regulating VLDL Metabolism in Rats.

Nonalcoholic fatty liver disease (NAFLD) is currently the most common chronic hepatic disorder worldwide. The earliest stage of NAFLD is simple steatosis, which is characterized by the accumulation of triglycerides in hepatocytes. Inhibition of steatosis is a potential treatment for NAFLD. Patchouli alcohol (PA) is an active component of Pogostemon cablin (Blanco) Benth. (Labiatae), which is a medicinal food in Asia countries and proved to possess hepatoprotective effect. This research aimed to investigate the effectiveness of PA against high fat diet (HFD)-induced hepatic steatosis in rats. In this study, male Sprague Dawley rats were fed a HFD for 4 weeks to induce NAFLD. Oral administration with PA significantly reduced pathological severity of steatosis in HFD-fed rats. It was associated with suppressing endoplasmic reticulum (ER) stress and regulating very low-density lipoprotein (VLDL) metabolism. Our data showed that PA treatment effectively attenuated ER stress by inhibiting the activation of protein kinase-like ER kinase (PERK), inositol-requiring transmembrane kinase/endoribonuclease 1 (IRE1), and activating transcription factor 6 (ATF6). Moreover, PA decreased hepatic VLDL uptake by suppressing very low-density lipoprotein receptor (VLDLR) expression. It also restored VLDL synthesis and export by increasing apolipoprotein B100 (apoB 100) secretion and microsomal triglyceride-transfer protein (MTP) activity. Taken together, PA exerted a protective effect on the treatment of NAFLD in HFD-fed rats and may be potential therapeutic agent acting on hepatic steatosis.

Unravelling the multiple roles of apolipoprotein E in the hepatitis C virus life cycle.

Worldwide, about 3% of the population is infected with HCV, and chronic infection is associated with a risk of serious progressive liver disease. The recent introduction of direct-acting antiviral agents (DAAs) has considerably improved the treatment of chronic HCV infection, and most patients are now cured without major side effects.1 However, subgroups of patients difficult-to-treat and requiring additional therapeutic approaches will probably emerge. Furthermore, DAAs are very expensive, limiting their use in some countries.1 Thus, in the absence of prophylactic vaccines, improvements to our understanding of the mechanisms underlying virus assembly, maturation and secretion from the cell are required, to facilitate the development of new treatment options. In patients, HCV particles circulate as hybrid particles, known as lipoviroparticles (LVPs), combining components of both HCV and very low-density lipoprotein (VLDL).2 LVPs have a low density (<1.06 g/mL) and are highly infectious. Their maturation and release are tightly associated with VLDL synthesis, leading to cholesterol and triglyceride export from hepatocytes. Similar characteristics have been described for viruses produced in cell culture (HCVcc) by human Huh7.5 hepatoma cells.2 LVPs are rich in triacylglycerol and total cholesterol. They contain the viral RNA, capsid protein, envelope glycoproteins and apolipoproteins. They carry highly variable amounts of lipids and are therefore highly heterogeneous in size and density. LVPs have been shown to associate with apolipoprotein B and apolipoprotein E (ApoE).3 ApoE is a soluble and exchangeable apolipoprotein. It plays a critical role in VLDL assembly and is also involved in cellular lipid transport.2 The importance of ApoE for the life cycle of HCV has been confirmed by studies showing ApoE to be required for LVP production4 ,5 HCV infectivity being associated with the presence of ApoE in …

Abstract 397: Transgenic Expression of Cholesteryl Ester Transfer Protein in Female Mice Disrupts Liver and Plasma Triglyceride Metabolism With Estrogen Treatment in a Liver Network Involving Estrogen Receptor Alpha and Small Heterodimer Partner

Background: Triglycerides are an important risk factor for cardiovascular disease, especially in women. Hormone replacement therapy raises triglycerides (TG) in postmenopausal women, but mechanisms responsible remain poorly understood. Cholesteryl Ester Transfer Protein (CETP) is a serum protein that shuttles TG and cholesteryl ester between lipoproteins in humans and plays an important role in lipoprotein metabolism. The role of CETP in regulating whole body triglyceride homeostasis remains unclear. Mice (which naturally lack CETP) expressing a transgenic copy of CETP or their wild-type (WT) littermates were used to determine the role of CETP in regulating plasma and liver TG metabolism in response to estrogen. Methods and Results: Female CETP and WT mice were ovariectomized and given vehicle or estrogen treatment. Estrogen raised plasma TG in CETP females, but not in WT females. CETP expression impaired TG clearance independent of estrogen. Estrogen treatment raised very-low density lipoprotein (VLDL) TG production in CETP females, but not in WT females. Estrogen treatment augmented mRNA expression and protein activity of VLDL assembly and secretion targets (Apob, Mttp, PDI) in CETP females, but not in WT females. Additionally, CETP expression lowered liver TG content and increased mRNA expression of TG oxidation targets (Ppara, Acox1, Acadm, Cpt2). Deletion of liver estrogen receptor alpha prevented CETP-mediated decreases in liver TG content and increases in mRNA expression of TG oxidation targets. Liver-specific deletion of estrogen receptor alpha did not attenuate estrogen-mediated increases in plasma TG and VLDL-TG production in female mice expressing CETP. Deletion of liver small heterodimer partner attenuated CETP-mediated increases in plasma TG and VLDL-TG production with estrogen treatment. Deletion of small heterodimer partner also attenuated CETP-mediated increases in mRNA expression and protein activity of VLDL synthesis and assembly targets with estrogen treatment. Conclusion: These studies indicate several novel functions of CETP in regulating plasma and liver TG homeostasis. We also discovered that CETP expression in female mice also augments several aspects of estrogen action in liver that impact liver TG biology.

ABHD5/CGI-58, the Chanarin-Dorfman Syndrome Protein, Mobilises Lipid Stores for Hepatitis C Virus Production.

Hepatitis C virus (HCV) particles closely mimic human very-low-density lipoproteins (VLDL) to evade humoral immunity and to facilitate cell entry. However, the principles that govern HCV association with VLDL components are poorly defined. Using an siRNA screen, we identified ABHD5 (α/β hydrolase domain containing protein 5, also known as CGI-58) as a new host factor promoting both virus assembly and release. ABHD5 associated with lipid droplets and triggered their hydrolysis. Importantly, ABHD5 Chanarin-Dorfman syndrome mutants responsible for a rare lipid storage disorder in humans were mislocalised, and unable to consume lipid droplets or support HCV production. Additional ABHD5 mutagenesis revealed a novel tribasic motif that does not influence subcellular localization but determines both ABHD5 lipolytic and proviral properties. These results indicate that HCV taps into the lipid droplet triglyceride reservoir usurping ABHD5 lipase cofactor function. They also suggest that the resulting lipid flux, normally devoted to VLDL synthesis, also participates in the assembly and release of the HCV lipo-viro-particle. Altogether, our study provides the first association between the Chanarin-Dorfman syndrome protein and an infectious disease and sheds light on the hepatic manifestations of this rare genetic disorder as well as on HCV morphogenesis.

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

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

Effects of β-hydroxybutyricacid on the synthesis and assembly of very low-density lipoprotein in bovine hepatocytes invitro.

β-Hydroxybutyricacid (BHBA) is an important metabolite that involved in the development of ketosis and fatty liver in dairy cows. Dairy cows with fatty liver displayed high blood concentration of BHBA and very low-density lipoprotein (VLDL) assembly. The effects of BHBA on VLDL synthesis and assembly in hepatocytes of cows were unclear. In this study, bovine hepatocytes were cultured and treated with different concentrations of BHBA. We found that BHBA treatment upregulated the mRNA and protein levels of apolipoprotein B100 (ApoB 100), apolipoprotein E (ApoE) and microsomal triglyceride transfer protein (MTTP) and showed in a firstly increased and then decreased trend. Meanwhile, the mRNA and protein levels of LDLR showed in a reverse trend. Consequently, VLDL content was significantly increased in medium-dose BHBA treatment group, while decreased in high-dose group. These results indicate that the effects of BHBA on the VLDL synthesis showed in a dose-dependent manner that low levels of BHBA increase VLDL synthesis and high levels of BHBA decrease VLDL synthesis.

Identification of protein disulfide isomerase 1 as a key isomerase for disulfide bond formation in apolipoprotein B100.

Apolipoprotein (apo) B is an obligatory component of very low density lipoprotein (VLDL), and its cotranslational and posttranslational modifications are important in VLDL synthesis, secretion, and hepatic lipid homeostasis. ApoB100 contains 25 cysteine residues and eight disulfide bonds. Although these disulfide bonds were suggested to be important in maintaining apoB100 function, neither the specific oxidoreductase involved nor the direct role of these disulfide bonds in apoB100-lipidation is known. Here we used RNA knockdown to evaluate both MTP-dependent and -independent roles of PDI1 in apoB100 synthesis and lipidation in McA-RH7777 cells. Pdi1 knockdown did not elicit any discernible detrimental effect under normal, unstressed conditions. However, it decreased apoB100 synthesis with attenuated MTP activity, delayed apoB100 oxidative folding, and reduced apoB100 lipidation, leading to defective VLDL secretion. The oxidative folding-impaired apoB100 was secreted mainly associated with LDL instead of VLDL particles from PDI1-deficient cells, a phenotype that was fully rescued by overexpression of wild-type but not a catalytically inactive PDI1 that fully restored MTP activity. Further, we demonstrate that PDI1 directly interacts with apoB100 via its redox-active CXXC motifs and assists in the oxidative folding of apoB100. Taken together, these findings reveal an unsuspected, yet key role for PDI1 in oxidative folding of apoB100 and VLDL assembly.

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

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

SREBP-1c overexpression induces triglycerides accumulation through increasing lipid synthesis and decreasing lipid oxidation and VLDL assembly in bovine hepatocytes

The natural incidence of fatty liver in ruminants is significantly higher than in monogastric animals. Fatty liver is associated with sterol regulatory element-binding protein 1c (SREBP-1c). The aim of this study was to investigate the regulatory network effects of SREBP-1c on the lipid metabolic genes involved in fatty acid uptake, activation, oxidation, synthesis, and very low-density lipoprotein (VLDL) assembly in bovine hepatocytes. In vitro, bovine hepatocytes were transfected with an adenovirus-mediated SREBP-1c overexpression vector. SREBP-1c overexpression significantly up-regulated the expression and activity of the fatty acid uptake, activation, and synthesis enzymes: liver fatty acid binding protein, fatty acid translocase, acyl-CoA synthetase long-chain 1, acetyl-CoA carboxylase 1, and fatty acid synthase, increasing triglyceride (TG) synthesis and accumulation. SREBP-1c overexpression down-regulated the expression and activity of the lipid oxidation enzymes: carnitine palmitoyltransferase 1 and carnitine palmitoyltransferase 2. Furthermore, the apolipoprotein B100 expression and microsomal triglyceride transfer protein activity were significantly decreased. SREBP-1c overexpression reduced lipid oxidation and VLDL synthesis, thereby decreasing TG disposal and export. Therefore, large amounts of TG accumulated in the bovine hepatocytes. Taken together, these results indicate that SREBP-1c overexpression increases lipid synthesis and decreases lipid oxidation and VLDL export, thereby inducing TG accumulation in bovine hepatocytes.

Effects of nonesterified fatty acids on the synthesis and assembly of very low density lipoprotein in bovine hepatocytes in vitro

High serum concentrations of nonesterified fatty acids (NEFA), which may affect the synthesis and assembly of very low density lipoproteins (VLDL), are associated with fatty liver during the early lactation period. Therefore, the objective of this study was to investigate the effects of NEFA on the synthesis and assembly of VLDL in bovine hepatocytes. Bovine hepatocytes were cultured and treated with different concentrations of NEFA. The mRNA expression of apolipoprotein B100 (ApoB100) and apolipoprotein E (ApoE) was significantly lower in the NEFA treatment groups than in the control group (0mM NEFA). The abundance of mRNA from microsomal triglyceride transfer protein (MTP) and the low density lipoprotein receptor (LDLR) was significantly lower in the medium- and high-dose NEFA treatment groups. The protein expression of ApoB100, ApoE, MTP, and LDLR was found to be significantly and dose-dependently decreased in groups of NEFA-treated hepatocytes. The VLDL content was also significantly decreased in the NEFA-treated hepatocytes. Large amounts of triglycerides accumulated in the hepatocytes. These results indicate that NEFA significantly inhibits the expression of ApoB100, ApoE, MTP, and LDLR, thereby decreasing the synthesis and assembly of VLDL and inducing TG accumulation in bovine hepatocytes.

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

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