Very Low Density Lipoprotein Secretion 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. Here, we examine 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 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.

Unlocking the mysteries of VLDL: exploring its production, intracellular trafficking, and metabolism as therapeutic targets

Reducing circulating lipid levels is the centerpiece of strategies for preventing and treating atherosclerotic cardiovascular disease (ASCVD). Despite many available lipid-lowering medications, a substantial residual cardiovascular risk remains. Current clinical guidelines focus on plasma levels of low-density lipoprotein (LDL). Recent attention has been given to very low-density lipoprotein (VLDL), the precursor to LDL, and its role in the development of coronary atherosclerosis. Preclinical investigations have revealed that interventions targeting VLDL production or promoting VLDL metabolism, independent of the LDL receptor, can potentially decrease cholesterol levels and provide therapeutic benefits. Currently, methods, such as mipomersen, lomitapide, and ANGPTL3 inhibitors, are used to reduce plasma cholesterol and triglyceride levels by regulating the lipidation, secretion, and metabolism of VLDL. Targeting VLDL represents an avenue for new lipid-lowering strategies. Interventions aimed at reducing VLDL production or enhancing VLDL metabolism, independent of the LDL receptor, hold promise for lowering cholesterol levels and providing therapeutic benefits beyond LDL in the management of ASCVD.

Conditional hepatocyte ablation of PDIA1 uncovers indispensable roles in both APOB and MTTP folding to support VLDL secretion

ObjectivesThe assembly and secretion of hepatic very low-density lipoprotein (VLDL) plays pivotal roles in hepatic and plasma lipid homeostasis. Protein disulfide isomerase A1 (PDIA1/P4HB) is a molecular chaperone whose functions are essential for protein folding in the endoplasmic reticulum. Here we investigated the physiological requirement in vivo for PDIA1 in maintaining VLDL assembly and secretion. MethodsPdia1/P4hb was conditionally deleted in adult mouse hepatocytes and the phenotypes characterized. Mechanistic analyses in primary hepatocytes determined how PDIA1 ablation alters MTTP synthesis and degradation as well as altering synthesis and secretion of Apolipoprotein B (APOB), along with complementary expression of intact PDIA1 vs a catalytically inactivated PDIA1 mutant. ResultsHepatocyte-specific deletion of Pdia1/P4hb inhibited hepatic MTTP expression and dramatically reduced VLDL production, leading to severe hepatic steatosis and hypolipidemia. Pdia1-deletion did not affect mRNA expression or protein stability of MTTP but rather prevented Mttp mRNA translation. We demonstrate an essential role for PDIA1 in MTTP synthesis and function and show that PDIA1 interacts with APOB in an MTTP-independent manner via its molecular chaperone function to support APOB folding and secretion. ConclusionsPDIA1 plays indispensable roles in APOB folding, MTTP synthesis and activity to support VLDL assembly. Thus, like APOB and MTTP, PDIA1 is an obligatory component of hepatic VLDL production.

Current Diagnosis and Management of Familial Hypobetalipoproteinemia 1.

Familial hypobetalipoproteinemia (FHBL) 1 is a rare genetic disorder with an autosomal codominant mode of inheritance and is caused by defects in the apolipoprotein (apo) B (APOB) gene that disable lipoprotein formation. ApoB proteins are required for the formation of very low-density lipoproteins (VLDLs), chylomicrons, and their metabolites. VLDLs transport cholesterol and triglycerides from the liver to the peripheral tissues, whereas chylomicrons transport absorbed lipids and fat-soluble vitamins from the intestine. Homozygous or compound heterozygotes of FHBL1 (HoFHBL1) are extremely rare, and defects in APOB impair VLDL and chylomicron secretion, which result in marked hypolipidemia with malabsorption of fat and fat-soluble vitamins, leading to various complications such as growth disorders, acanthocytosis, retinitis pigmentosa, and neuropathy. Heterozygotes of FHBL1 are relatively common and are generally asymptomatic, except for moderate hypolipidemia and possible hepatic steatosis. If left untreated, HoFHBL1 can cause severe complications and disabilities that are pathologically and phenotypically similar to abetalipoproteinemia (ABL) (an autosomal recessive disorder) caused by mutations in the microsomal triglyceride transfer protein (MTTP) gene. Although HoFHBL1 and ABL cannot be distinguished from the clinical manifestations and laboratory findings of the proband, moderate hypolipidemia in first-degree relatives may help diagnose HoFHBL1. There is currently no specific treatment for HoFHBL1. Palliative therapy including high-dose fat-soluble vitamin supplementation may prevent or delay complications. Registry research on HoFHBL1 is currently ongoing to better understand the disease burden and unmet needs of this life-threatening disease with few therapeutic options.

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.

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.

Trib1 Deficiency Promotes Hyperlipidemia, Inflammation, and Atherosclerosis in LDL Receptor Knockout Mice.

Genetic variants at the TRIB1 gene locus are strongly associated with plasma lipid traits and the risk of coronary artery disease in humans. Here, we analyzed the consequences of Trib1 deficiency on lipid metabolism and atherosclerotic lesion formation in atherosclerosis-susceptible Ldlr-/- mice. Trib1-/- mice were crossed onto the Ldlr-/- background to generate double-knockout mice (Trib1-/-Ldlr-/-) and fed a semisynthetic, modified AIN76 diet (0.02% cholesterol and 4.3% fat) until 20 weeks of age. Trib1-/-Ldlr-/- mice had profoundly larger (5.8-fold) and more advanced atherosclerotic lesions at the aortic root as compared with Trib1+/+Ldlr-/- controls. Further, we observed significantly elevated plasma total cholesterol and triglyceride levels in Trib1-/-Ldlr-/- mice, resulting from higher VLDL (very-low-density lipoprotein) secretion. Lipidomics analysis revealed that loss of Trib1 altered hepatic lipid composition, including the accumulation of cholesterol and proinflammatory ceramide species, which was accompanied by signs of hepatic inflammation and injury. Concomitantly, we detected higher plasma levels of IL (interleukin)-6 and LCN2 (lipocalin 2), suggesting increased systemic inflammation in Trib1-/-Ldlr-/- mice. Hepatic transcriptome analysis demonstrated significant upregulation of key genes controlling lipid metabolism and inflammation in Trib1-/-Ldlr-/- mice. Further experiments suggested that these effects may be mediated through pathways involving a C/EPB (CCAAT/enhancer binding protein)-PPARγ (peroxisome proliferator-activated receptor γ) axis and JNK (c-Jun N-terminal kinase) signaling. We provide experimental evidence that Trib1 deficiency promotes atherosclerotic lesion formation in a complex manner that includes the modulation of lipid metabolism and inflammation.

Surf4, cargo trafficking, lipid metabolism, and therapeutic implications.

Surfeit 4 is a polytopic transmembrane protein that primarily resides in the endoplasmic reticulum (ER) membrane. It is ubiquitously expressed and functions as a cargo receptor, mediating cargo transport from the ER to the Golgi apparatus via the canonical coat protein complex II (COPII)-coated vesicles or specific vesicles. It also participates in ER-Golgi protein trafficking through a tubular network. Meanwhile, it facilitates retrograde transportation of cargos from the Golgi apparatus to the ER through COPI-coated vesicles. Surf4 can selectively mediate export of diverse cargos, such as PCSK9 very low-density lipoprotein (VLDL), progranulin, α1-antitrypsin, STING, proinsulin, and erythropoietin. It has been implicated in facilitating VLDL secretion, promoting cell proliferation and migration, and increasing replication of positive-strand RNA viruses. Therefore, Surf4 plays a crucial role in various physiological and pathophysiological processes and emerges as a promising therapeutic target. However, the molecular mechanisms by which Surf4 selectively sorts diverse cargos for ER-Golgi protein trafficking remain elusive. Here, we summarize the most recent advances in Surf4, focusing on its role in lipid metabolism.

The role of hepatic Surf4 in lipoprotein metabolism and the development of atherosclerosis in apoE−/− mice

Elevated plasma levels of low-density lipoprotein-C (LDL-C) increase the risk of atherosclerotic cardiovascular disease. Circulating LDL is derived from very low-density lipoprotein (VLDL) metabolism and cleared by LDL receptor (LDLR). We have previously demonstrated that cargo receptor Surfeit 4 (Surf4) mediates VLDL secretion. Inhibition of hepatic Surf4 impairs VLDL secretion, significantly reduces plasma LDL-C levels, and markedly mitigates the development of atherosclerosis in LDLR knockout (Ldlr−/−) mice. Here, we investigated the role of Surf4 in lipoprotein metabolism and the development of atherosclerosis in another commonly used mouse model of atherosclerosis, apolipoprotein E knockout (apoE−/−) mice. Adeno-associated viral shRNA was used to silence Surf4 expression mainly in the liver of apoE−/− mice. In apoE−/− mice fed a regular chow diet, knockdown of Surf4 expression significantly reduced triglyceride secretion and plasma levels of non-HDL cholesterol and triglycerides without causing hepatic lipid accumulation or liver damage. When Surf4 was knocked down in apoE−/− mice fed the Western-type diet, we observed a significant reduction in plasma levels of non-HDL cholesterol, but not triglycerides. Knockdown of Surf4 did not increase hepatic cholesterol and triglyceride levels or cause liver damage, but significantly diminished atherosclerosis lesions. Therefore, our findings indicate the potential of hepatic Surf4 inhibition as a novel therapeutic strategy to reduce the risk of atherosclerotic cardiovascular disease.

Role of sortilin 1 (SORT1) on lipid metabolism in bovine liver

High circulating concentrations of fatty acids cause triacylglycerol (TAG) accumulation in hepatocytes of dairy cows, a common metabolic disorder after calving. Low secretion of apolipoprotein B (APOB) and very low density lipoprotein (VLDL) are thought to be the major factors for TAG accumulation in hepatocytes. Recent data in nonruminant models revealed that sortilin 1 (SORT1) is a key regulator of VLDL secretion in part due to its ability to bind APOB. Thus, SORT1 could play a role in the susceptibility of dairy cows to develop fatty liver. To gain mechanistic insights in vivo and in vitro, we performed experiments using liver biopsies or isolated primary hepatocytes. For the in vivo study, blood and liver samples were collected from healthy multiparous dairy cows (n = 6; 9.0 ± 2.1 d in milk) and cows with fatty liver (n = 6; 9.7 ± 2.2 d in milk). In vitro, hepatocytes isolated from 4 healthy female calves (1 d old, 42-51 kg) were challenged with (fatty acids) or without (control) a 1.2 mM mixture of fatty acids in an attempt to induce metabolic stress. Furthermore, hepatocytes were treated with empty adenovirus vectors (Ad-GFP) or SORT1 overexpressing adenovirus (Ad-SORT1) for 6 h, or SORT1 inhibitor for 2 h followed by a challenge with (Ad-GFP + fatty acids, Ad-SORT1 + fatty acids, or SORT1 inhibitor + fatty acids) or without (Ad-GFP, Ad-SORT1, or SORT1 inhibitor) the 1.2 mM mixture of fatty acids for 12 h. Data from liver biopsies were compared using a 2-tailed unpaired Student's t-test. Data from calf hepatocytes were analyzed by one-way ANOVA. Data revealed that both fatty liver and in vitro challenge with fatty acids were associated with greater concentrations of TAG and mRNA and protein abundance of SORT1, SREBF1, FASN, and ACACA. In contrast, mRNA and protein abundance of CPT1A and APOB, and mRNA abundance of MTTP were markedly lower. Compared with fatty acid challenge alone, SORT1 overexpression led to greater concentration of TAG and mRNA abundance of SREBF1, FASN, ACACA, DGAT1, and DGAT2, and protein abundance of SREBF1, FASN, and ACACA. In contrast, concentration of secreted VLDL-APOB and mRNA abundance of APOB and MTTP, and protein abundance of CPT1A, APOB, and MTTP were lower. Compared with fatty acid challenge alone, SORT1 inhibitor + fatty acids led to lower concentrations of TAG and mRNA abundance of SREBF1, FASN, and DGAT2, and protein abundance of FASN, ACACA, and DGAT1. Concentrations of secreted VLDL-APOB and mRNA abundance of CPT1A and protein abundance of CPT1A and APOB were greater. Overall, in vitro data suggested that greater SORT1 abundance induced by exogenous fatty acids caused a reduction in VLDL-APOB secretion and increased hepatocyte TAG synthesis. Such mechanism was also apparent in tissue from cows with fatty liver. Thus, targeted downregulation of hepatic SORT1 could represent a viable mechanism to unload lipid during conditions where the influx of fatty acids increases markedly.

Emerging Evidence of Pathological Roles of Very-Low-Density Lipoprotein (VLDL).

Embraced with apolipoproteins (Apo) B and Apo E, triglyceride-enriched very-low-density lipoprotein (VLDL) is secreted by the liver into circulation, mainly during post-meal hours. Here, we present a brief review of the physiological role of VLDL and a systemic review of the emerging evidence supporting its pathological roles. VLDL promotes atherosclerosis in metabolic syndrome (MetS). VLDL isolated from subjects with MetS exhibits cytotoxicity to atrial myocytes, induces atrial myopathy, and promotes vulnerability to atrial fibrillation. VLDL levels are affected by a number of endocrinological disorders and can be increased by therapeutic supplementation with cortisol, growth hormone, progesterone, and estrogen. VLDL promotes aldosterone secretion, which contributes to hypertension. VLDL induces neuroinflammation, leading to cognitive dysfunction. VLDL levels are also correlated with chronic kidney disease, autoimmune disorders, and some dermatological diseases. The extra-hepatic secretion of VLDL derived from intestinal dysbiosis is suggested to be harmful. Emerging evidence suggests disturbed VLDL metabolism in sleep disorders and in cancer development and progression. In addition to VLDL, the VLDL receptor (VLDLR) may affect both VLDL metabolism and carcinogenesis. Overall, emerging evidence supports the pathological roles of VLDL in multi-organ diseases. To better understand the fundamental mechanisms of how VLDL promotes disease development, elucidation of the quality control of VLDL and of the regulation and signaling of VLDLR should be indispensable. With this, successful VLDL-targeted therapies can be discovered in the future.

FGF21 Reduces Lipid Accumulation in Bovine Hepatocytes by Enhancing Lipid Oxidation and Reducing Lipogenesis via AMPK Signaling.

Simple SummaryThe negative energy balance (NEB) caused by decreased energy intake and increased demand during the perinatal period of dairy cows causes lipid mobilization, which leads to the accumulation of non-esterified fatty acids (NEFAs) in the liver. NEFAs are substrates that reduce triglyceride (TG) esterification synthesis, leading to energy metabolic disorders such as ketosis and fatty liver. Fibroblast growth factor 21 (FGF21) treatment can reduce TG content in the liver of dairy cows by 50%. This study further found that FGF21 regulated lipid metabolism in bovine liver cells through the AMPK signaling pathway. The expressions of SREBF1, the peroxisome proliferator-activated receptor α (PPARA) and their target genes were changed. This had a negative effect on lipid formation, enhanced lipid oxidation, and increased lipid transport. The present data suggest the possibility that FGF21 has potential value in alleviating perinatal metabolic diseases in dairy cows, and specific research in vivo should be studied in more detail.During the periparturient period, dairy cows suffer drastic metabolic stress because of plasma increased non-esterified fatty acids (NEFAs) that stem from a negative energy balance. Fibroblast growth factor 21 (FGF21) is a hepatokine that activates the AMP-activated protein kinase (AMPK) signaling pathway to maintain intracellular energy balance and tissue integrity via the promotion of catabolism and the inhibition of anabolic regulation. FGF21 treatment caused a 50% reduction in triglyceride (TG) content in liver in dairy cows. However, it is not clear whether FGF21 regulates lipid metabolism in bovine liver. The purpose of this study was to evaluate the influence of FGF21 on lipid metabolism via AMPK signaling in bovine hepatocytes. The hepatocytes isolated from calves were treated with different concentrations of FGF21 or co-treated with AMPK inhibitor (BML-275). Herein, the study showed that FGF21 significantly reduced TG content in a dose–response manner and promoted very-low-density lipoprotein (VLDL) secretion via an up-regulation of the proteins (ApoB 100, ApoE and MTTP) involved in VLDL secretion. Otherwise, the genes associated with lipid transport (LDLR and CD36) and lipid oxidation (PPARGC1A, ACOX1 and CPT1A), were up-regulated following FGF21 treatment. Moreover, FGF21 treatment inhibited lipogenesis via SREBF1, ACACA, FASN and ACLY inhibition. After being co-treated with the AMPK inhibitor, FGF21-induced changes were reversed in some genes. In conclusion, these results indicate that FGF21 adaptively regulates energy metabolism for a negative impact on lipogenesis, strengthens lipid oxidation, and inhibited lipid transportation via AMPK signaling in bovine hepatocytes. The present data suggest the possibility that FGF21 has potential value in alleviating perinatal metabolic diseases in dairy cows, and specific research in vivo should be studied in more detail.

Measurement of In Vivo VLDL and Chylomicron Secretion.

Intestinal lipid absorption as well as secretion of absorbed lipids as chylomicrons by the enterocytes is a direct measure of the availability of dietary lipids. Measurement of this parameter is central to the understanding of the influence of diet on plasma lipids, specifically when modulation of intestinal lipid absorption by targeted interventions is being examined. In the post-prandial state, very low-density lipoprotein (VLDL) secreted from the liver represent the major source of plasma lipids and rate of VLDL secretion reports on hepatic lipid homeostasis. Here, we describe the methods to specifically measure secretion of chylomicron and VLDL in vivo. Tight regulation of dietary lipid absorption (chylomicron secretion) and hepatic secretion of VLDL underlies the development of dyslipidemia preceding hepatic lipid accumulation seen in non-alcoholic fatty liver disease (NAFLD) and subsequent progression to non-alcoholic steatohepatitis (NASH) underscoring the importance of measurement of lipoprotein secretion in vivo.

Intracellular C3 prevents hepatic steatosis by promoting autophagy and very-low-density lipoprotein secretion.

Complement component C3, mainly synthesized by hepatocytes, acts as the convergence point of three different pathways in activating the complement cascade. Besides its well-established roles in the extracellular milieu, C3 performs various intracellular functions such as immunomodulation and pathogen recognition. Although C3 is present at extremely high concentrations in hepatocytes, little is known about its intrahepatic function. In this study, we found that C3knockout (C3-/- ) mice displayed accelerated hepatic triglyceride (TG) accumulation compared with C57BL/6J wild type mice. Mechanistically, C3 deficiency impaired lipophagy in hepatocytes, owing to the disrupted interaction between C3 and autophagy-related 16like 1, which is essential for autolysosome assembly. Furthermore, lipophagy deficiency affected the function of the endoplasmic reticulum in C3-/- mice, subsequently affecting the expression of protein disulfide isomerase and activity of microsomal TG transfer protein, and ultimately impairing the production of hepatic very-low-density lipoproteins (VLDLs). Rapamycin and thapsigargin treatment accelerated VLDL secretion and alleviated hepatic lipid accumulation in C3-/- mice. Our study demonstrates that C3 promotes lipophagy to facilitate VLDL secretion in hepatocytes, thus playing an essential role in balancing TG levels in the liver.

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.

Lipid overload impairs hepatic VLDL secretion via oxidative stress-mediated PKCδ-HNF4α-MTP pathway in large yellow croaker (Larimichthys crocea)

Lipid overload-induced hepatic steatosis is a major public health problem worldwide. However, the potential molecular mechanism is not completely understood. Herein, we found that high-fat diet (HFD) or oleic acid (OA) treatment induced oxidative stress which prevented the entry of hepatocyte nuclear factor 4 alpha (HNF4α) into the nucleus by activating protein kinase C delta (PKCδ) in vivo and in vitro in large yellow croaker (Larimichthys crocea). This reduced the level of microsomal triglyceride transfer protein (MTP) transcription, resulting in the impaired secretion of very-low-density lipoprotein (VLDL) and the abnormal accumulation of triglyceride (TG) in hepatocytes. Meanwhile, the detrimental effects induced by lipid overload could be partly alleviated by pretreating hepatocytes with Go6983 (PKCδ inhibitor) or N-acetylcysteine (NAC, reactive oxygen species (ROS) scavenger). In conclusion, for the first time, we revealed that lipid overload impaired hepatic VLDL secretion via oxidative stress-mediated PKCδ-HNF4α-MTP pathway in fish. This study may provide critical insights into potential intervention strategies against lipid overload-induced hepatic steatosis of fish and human beings.

Liver-Specific Deletion of Mouse Tm6sf2 Promotes Steatosis, Fibrosis, and Hepatocellular Cancer.

Human transmembrane 6 superfamily 2 (TM6SF2) variant rs58542926 is associated with NAFLD and HCC. However, conflicting reports in germline Tm6sf2 knockout mice suggest no change or decreased very low density lipoprotein (VLDL) secretion and either unchanged or increased hepatic steatosis, with no increased fibrosis. We generated liver-specific Tm6Sf2 knockout mice (Tm6 LKO) to study VLDL secretion and the impact on development and progression of NAFLD. Two independent lines of Tm6 LKO mice exhibited spontaneous hepatic steatosis. Targeted lipidomic analyses showed increased triglyceride species whose distribution and abundance phenocopied findings in mice with liver-specific deletion of microsomal triglyceride transfer protein. The VLDL triglyceride secretion was reduced with small, underlipidated particles and unchanged or increased apolipoprotein B. Liver-specific adeno-associated viral, serotype 8 (AAV8) rescue using either wild-type or mutant E167K-Tm6 reduced hepatic steatosis and improved VLDL secretion. The Tm6 LKO mice fed a high milk-fat diet for 3weeks exhibited increased steatosis and fibrosis, and those phenotypes were further exacerbated when mice were fed fibrogenic, high fat/fructose diets for 20weeks. In two models of HCC, either neonatal mice injected with streptozotocin (NASH/STAM) and high-fat fed or with diethylnitrosamine injection plus fibrogenic diet feeding, Tm6 LKO mice exhibited increased steatosis, greater tumor burden, and increased tumor area versus Tm6 flox controls. Additionally, diethylnitrosamine-injected and fibrogenic diet-fed Tm6 LKO mice administered wild-type Tm6 or E167K-mutant Tm6 AAV8 revealed significant tumor attenuation, with tumor burden inversely correlated with Tm6 protein levels. Liver-specific Tm6sf2 deletion impairs VLDL secretion, promoting hepatic steatosis, fibrosis, and accelerated development of HCC, which was mitigated with AAV8- mediated rescue.

The influence of RS738409 I148M polymorphism of patatin-like phospholipase domain containing 3 gene on the susceptibility of non-alcoholic fatty liver disease

We aimed to elucidate the frequency of polymorphic genotypes and alleles of patatin-like phospholipase domain containing 3 rs738409 polymorphism and its possible associations with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis in a cohort from Turkey.We enrolled 200 patients diagnosed with NAFLD and genotyped for rs738409 I148M polymorphism by real-time polymerase chain reaction, particularly by melting curve analysis. SPSS analysis software was used for statistical significance. Continuous variable values were expressed as mean ± standard deviation. Significant statistical level was chosen as p = 0.05.Our results demonstrate in a cohort from Turkey that rs738409 C > G polymorphism (I148M) of patatin-like phospholipase domain containing 3 gene is significantly able to affect individuals to have NAFLD in unadjusted regression model.Consistent with the previous studies in other populations, our study group showed a significantly higher risk of having NAFLD in unadjusted regression model but not in the adjusted model indicating that non-genetic factors such as age and sex may be responsible for the association. However, independent studies need to validate our findings with a larger group of NAFLD patients, as well as in different ethnic cohorts.