Lipid Metabolism In Hepatocytes Research Articles

Endoplasmic reticulum stress and autophagy dysregulation in alcoholic and non-alcoholic liver diseases.

Alcoholic and non-alcoholic liver diseases begin from an imbalance in lipid metabolism in hepatocytes as the earliest response. Both liver diseases share common disease features and stages (i.e., steatosis, hepatitis, cirrhosis, and hepatocellular carcinoma). However, the two diseases have differential pathogenesis and clinical symptoms. Studies have elucidated the molecular basis underlying similarities and differences in the pathogenesis of the diseases; the factors contributing to the progression of liver diseases include depletion of sulfhydryl pools, enhanced levels of reactive oxygen and nitrogen intermediates, increased sensitivity of hepatocytes to toxic cytokines, mitochondrial dysfunction, and insulin resistance. Endoplasmic reticulum (ER) stress, which is caused by the accumulation of misfolded proteins and calcium depletion, contributes to the pathogenesis, often causing catastrophic cell death. Several studies have demonstrated a mechanism by which ER stress triggers liver disease progression. Autophagy is an evolutionarily conserved process that regulates organelle turnover and cellular energy balance through decomposing damaged organelles including mitochondria, misfolded proteins, and lipid droplets. Autophagy dysregulation also exacerbates liver diseases. Thus, autophagy-related molecules can be potential therapeutic targets for liver diseases. Since ER stress and autophagy are closely linked to each other, an understanding of the molecules, gene clusters, and networks engaged in these processes would be of help to find new remedies for alcoholic and non-alcoholic liver diseases. In this review, we summarize the recent findings and perspectives in the context of the molecular pathogenesis of the liver diseases.

Protective effects of Lactobacillus acidophilus NX2-6 against oleic acid-induced steatosis, mitochondrial dysfunction, endoplasmic reticulum stress and inflammatory responses

Lactobacillus acidophilus NX2-6 protected against oleic acid-induced steatosis through improving lipid metabolism, mitochondrial dysfunction, endoplasmic reticulum stress and inflammatory responses. • The cell-free extract of Lactobacillus acidophilus NX2-6 improved lipid profile. • The cell-free extract regulated lipid metabolism at the genetic and protein level. • The cell-free extract alleviated oxidative stress and endoplasmic reticulum stress. • The cell-free extract attenuated mitochondrial dysfunction. The cell-free extract (CFE) of Lactobacillus acidophilus NX2-6 was used to treat oleic acid (OA)-exposed HepG2 cells. Biochemical analysis showed that CFE could reduce high levels of triglyceride, LDL-c, LDH, and MDA caused by OA treatment. RT-PCR and western blot analysis revealed that CFE distinctly decreased levels of Srebp-1c and FAS but elevated expression of CPT1. Fluorimetric analysis showed that CFE augmented mitochondrial membrane potential and mitochondrial content, and reduced ROS production induced by OA. Meanwhile, RT-PCR analysis also exhibited that CFE contributed to mitochondrial respiration by increasing expressions of respiratory complex subunits MTND1 , MTCO1 , MTATP6 and MTCYB . CFE also downregulated GRP58 , ATF6 , IREα , XBP1 and CHOP to alleviate ERS. Results also showed that CFE inhibited inflammatory responses through decreasing the levels of IL-1β and IL-6 and downregulating expression of NF-κB . Therefore, L. acidophilus possessed potentials to improve lipid metabolism, oxidative stress, mitochondrial dysfunction, ERS and inflammation in hepatocytes.

Arachidyl amido cholanoic acid improves liver glucose and lipid homeostasis in nonalcoholic steatohepatitis via AMPK and mTOR regulation.

BACKGROUNDArachidyl amido cholanoic acid (Aramchol) is a potent downregulator of hepatic stearoyl-CoA desaturase 1 (SCD1) protein expression that reduces liver triglycerides and fibrosis in animal models of steatohepatitis. In a phase IIb clinical trial in patients with nonalcoholic steatohepatitis (NASH), 52 wk of treatment with Aramchol reduced blood levels of glycated hemoglobin A1c, an indicator of glycemic control.AIMTo assess lipid and glucose metabolism in mouse hepatocytes and in a NASH mouse model [induced with a 0.1% methionine and choline deficient diet (0.1MCD)] after treatment with Aramchol.METHODSIsolated primary mouse hepatocytes were incubated with 20 μmol/L Aramchol or vehicle for 48 h. Subsequently, analyses were performed including Western blot, proteomics by mass spectrometry, and fluxomic analysis with 13C-uniformly labeled glucose. For the in vivo part of the study, male C57BL/6J mice were randomly fed a control or 0.1MCD for 4 wk and received 1 or 5 mg/kg/d Aramchol or vehicle by intragastric gavage for the last 2 wk. Liver metabolomics were assessed using ultra-high-performance liquid chromatography-time of flight-MS for the determination of glucose metabolism-related metabolites.RESULTSCombination of proteomics and Western blot analyses showed increased AMPK activity while the activity of nutrient sensor mTORC1 was decreased by Aramchol in hepatocytes. This translated into changes in the content of their downstream targets including proteins involved in fatty acid (FA) synthesis and oxidation [P-ACCα/β(S79), SCD1, CPT1A/B, HADHA, and HADHB], oxidative phosphorylation (NDUFA9, NDUFB11, NDUFS1, NDUFV1, ETFDH, and UQCRC2), tricarboxylic acid (TCA) cycle (MDH2, SUCLA2, and SUCLG2), and ribosome (P-p70S6K[T389] and P-S6[S235/S236]). Flux experiments with 13C-uniformely labeled glucose showed that TCA cycle cataplerosis was reduced by Aramchol in hepatocytes, as indicated by the increase in the number of rounds that malate remained in the TCA cycle. Finally, liver metabolomic analysis showed that glucose homeostasis was improved by Aramchol in 0.1MCD fed mice in a dose-dependent manner, showing normalization of glucose, G6P, F6P, UDP-glucose, and Rbl5P/Xyl5P.CONCLUSIONAramchol exerts its effect on glucose and lipid metabolism in NASH through activation of AMPK and inhibition of mTORC1, which in turn activate FA β-oxidation and oxidative phosphorylation.

CTRP12 inhibits triglyceride synthesis and export in hepatocytes by suppressing HNF-4α and DGAT2 expression.

C1q/TNF-related protein 12 (CTRP12) is an antidiabetic adipokine whose circulating levels are reduced in obesity and diabetes. Although partial and complete loss-of-function mouse models suggest a role for CTRP12 in modulating lipid metabolism and adiposity, its effect on cellular lipid metabolism remains poorly defined. Here, we demonstrate a direct action of CTRP12 in regulating lipid synthesis and secretion. In hepatoma cells and primary mouse hepatocytes, CTRP12 treatment inhibits triglyceride synthesis by suppressing glycerophosphate acyltransferase (GPAT) and diacylglycerol acyltransferase (DGAT) expression. CTRP12 treatment also downregulates the expression of hepatocyte nuclear factor-4α (HNF-4α) and its target gene microsomal triglyceride transfer protein (MTTP), leading to reduced very-low-density lipoprotein (VLDL)-triglyceride export from hepatocytes. Consistent with the invitro findings, overexpressing CTRP12 lowers fasting and postprandial serum triglyceride levels in mice. These results underscore the important function of CTRP12 in lipid metabolism in hepatocytes.

GS09 - Monoacylglycerol lipase reprograms lipid metabolism in macrophages and hepatocytes to promote liver regeneration

GS09 - Monoacylglycerol lipase reprograms lipid metabolism in macrophages and hepatocytes to promote liver regeneration

MiR-101b Regulates Lipid Deposition and Metabolism of Primary Hepatocytes in Teleost Yellow Catfish Pelteobagrus fulvidraco.

Excessive fat deposition in the hepatocytes, associated with excess dietary fat intake, was related to the occurrence of fatty livers in fish. miR-101b plays the important roles in controlling lipid metabolism, but the underlying mechanism at the post-transcriptional level remains unclear. The purpose of this study is to explore the roles and mechanism of miR-101b-mediating lipid deposition and metabolism in yellow catfish Pelteobagrus fulvidraco. We found that miR-101b directly targeted fatty acid translocase (cd36), caspase9 (casp9) and autophagy-related gene 4A (atg4a). Furthermore, using palmitic acid (PA) or oleic acid (OA) to incubate the primary hepatocytes of yellow catfish, we demonstrated that miR-101b inversely regulated cd36, casp9, and atg4a expression at the transcriptional level; the inhibition of miR-101b aggravated fatty acids (FAs, PA or OA)-induced lipid accumulation, indicating that miR-101b mediated FAs-induced variations of lipid metabolism in yellow catfish. Taken together, our study gave novel insight into the regulatory mechanism of lipid deposition and metabolism and might provide potential targets for the prevention and treatment of fatty livers in fish.

Epigallocatechin-3-gallate activates the AMP-activated protein kinase signaling pathway to reduce lipid accumulation in canine hepatocytes.

Epigallocatechin-3-gallate (EGCG) plays a crucial role in hepatic lipid metabolism. However, the underlying regulatory mechanism of hepatic lipid metabolism by EGCG in canine is unclear. Primary canine hepatocytes were treated with EGCG (0.01, 0.1, or 1 μM) and BML-275 (an AMP-activated protein kinase [AMPK] inhibitor) to study the effects of EGCG on the gene and protein expressions associated with AMPK signaling pathway. Data showed that treatment with EGCG had greater activation of AMPK, as well as greater expression levels and transcriptional activity of peroxisome proliferator activated receptor-α (PPARα) along with upregulated messenger RNA (mRNA) abundance and protein abundance of PPARα-target genes. EGCG decreased the expression levels and transcriptional activity of sterol regulatory element-binding protein 1c (SREBP-1c) along with downregulated mRNA abundance and protein abundance of SREBP-1c target genes. Of particular interest, exogenous BML-275 could reduce or eliminate the effects of EGCG on lipid metabolism in canine hepatocytes. Furthermore, the content of triglyceride was significantly decreased in the EGCG-treated groups. These results suggest that EGCG might be a potential agent in preventing high-fat diet-induced lipid accumulation in small animals.

(−)-Hydroxycitric acid regulates energy metabolism by activation of AMPK - PGC1α - NRF1 signal pathway in primary chicken hepatocytes

As the most important bioactive substance in Garcinia cambogia, (−)-hydroxycitric acid (HCA) is widely used in food additives to regulate obesity and diabetes in animals or humans, while the mechanism is poorly understood. The purpose of this study was to elucidate the regulatory effect and mechanism of (−)-HCA in regulating glucose and lipid metabolism in chicken primary hepatocytes. The results showed that (−)-HCA obviously decreased triglyceride content through inhibiting the fatty acid synthase protein level, and enhancing the protein level of phosphorylated acetyl CoA carboxylase, enoyl coenzyme A hydratase short chain 1 and carnitine palmitoyltransferase 1A in hepatocytes. Moreover, (−)-HCA markedly enhanced the protein level of phosphofructokinase-1, pyruvate dehydrogenase, succinate dehydrogenase A and complex IV, and which led to the enhancing of glucose uptake and catabolism in hepatocytes. Importantly, the regulation of (−)-HCA on these key factors associated with lipid and glucose metabolism in hepatocytes was mainly achieved through activation of AMP-activated protein kinase/peroxisome proliferator-activated receptor gamma coactivator 1α-nuclear respiratory factor 1 signaling pathway. These results convincingly demonstrated the mechanism of (−)-HCA's regulating on glucose and lipid metabolism, and provided a strategy in prevention of diseases associated with glycolipid metabolic abnormalities in animals, even in humans.

High Glucose Induces Lipid Accumulation via 25-Hydroxycholesterol DNA-CpG Methylation.

SummaryThis work investigates the relationship between high-glucose (HG) culture, CpG methylation of genes involved in cell signaling pathways, and the regulation of carbohydrate and lipid metabolism in hepatocytes. The results indicate that HG leads to an increase in nuclear 25-hydroxycholesterol (25HC), which specifically activates DNA methyltransferase-1 (DNMT1), and regulates gene expression involved in intracellular lipid metabolism. The results show significant increases in 5mCpG levels in at least 2,225 genes involved in 57 signaling pathways. The hypermethylated genes directly involved in carbohydrate and lipid metabolism are of PI3K, cAMP, insulin, insulin secretion, diabetic, and NAFLD signaling pathways. The studies indicate a close relationship between the increase in nuclear 25HC levels and activation of DNMT1, which may regulate lipid metabolism via DNA CpG methylation. Our results indicate an epigenetic regulation of hepatic cell metabolism that has relevance to some common diseases such as non-alcoholic fatty liver disease and metabolic syndrome.

Inhibition of stearoyl-coenzyme A desaturase 1 ameliorates hepatic steatosis by inducing AMPK-mediated lipophagy.

SCD1 is a key enzyme controlling lipid metabolism and a link between its activity and NAFLD has been proposed. Lipophagy is a novel regulatory approach to lipid metabolism regulation, which is involved in the development of NAFLD. However, the possible functional connection between SCD1 and lipophagy in NAFLD remains unknown. To investigate the molecular mechanisms through which SCD1 regulates lipophagy in hepatic steatosis, the model of hepatic steatosis was established by inducing mouse primary hepatocytes with sodium palmitate and feeding C57BL/6 mice with HFD. Our results indicated that sodium palmitate-treated hepatocytes exhibited increased SCD1 expression, AMPK inactivation and defective lipophagy. Inhibition of SCD1 expression in hepatocytes resulted in enhanced AMPK activity and lipophagy, and reduced lipid deposition. Although SCD1 overexpression led to decreased AMPK activity and lipophagy, lipid deposition was increased in hepatocytes. SCD1 regulated lipophagy through AMPK to affect lipid metabolism in mouse primary hepatocytes. Additionally, compared to HFD-fed mice, CAY10566(an SCD1-specific inhibitor)-treated mice exhibited significantly decreased hepatic steatosis and hepatic lipid droplet accumulation, as well as enhanced AMPK activity and lipophagy. This study elucidated that SCD1 inhibition ameliorates hepatic steatosis by inducing AMPK-mediated lipophagy, suggesting that the SCD1-AMPK-lipophagy pathway is a potential therapeutic target for NAFLD.

Genetic variants in the FAM3C gene are associated with lipid traits in Chinese children.

Previous studies have related FAM3C gene with childhood bone health, and the regulation of lipid metabolism in hepatocytes. The present case-control study aimed to analyze the association of FAM3C genetic variants with overweight/obesity and lipid traits among Chinese children. Two genetic variants (rs7776725 and rs7793554) within the FAM3C gene were genotyped in 3305 Chinese children aged 6-18 years. In the whole study population, the T-allele of rs7776725 and A-allele of rs7793554 within the FAM3C gene were associated with 40.2% (95% CI: 11.6-76.1%; P = 0.004) and 29.1% (6.9-56.0%; P = 0.008) increased risk of dyslipidemia, higher triglyceride (P = 0.014 and P = 0.001) and lower HDL-C (P = 0.015 and P = 0.003). In addition, we found that rs7776725 interacted with sex on dyslipidemia (Pfor interaction = 0.004), and sex-stratified analyses showed that it was significantly associated with dyslipidemia only in girls (P = 8.78 × 10-5). The variant also showed nominally significant interactions with sex on total cholesterol and LDL-C (Pfor interaction = 0.012 and 0.008). We found that FAM3C genetic variants were associated with dyslipidemia and lipid traits among Chinese children. In addition, we found significant gene-by-sex interactions. Our findings provided evidence supporting the role of FAM3C gene in regulating lipid metabolism in humans. FAM3C genetic variants were associated with dyslipidemia and lipid traits among Chinese children. In addition, we found significant gene-by-sex interactions. FAM3C/rs7776725 was associated with dyslipidemia and lipid traits only in girls. Our findings provided evidence supporting the role of FAM3C gene in regulating lipid metabolism in humans.

Deficient Chaperone-Mediated Autophagy Promotes Lipid Accumulation in Macrophage

Chaperone-mediated autophagy (CMA) serves as a critical upstream regulator of lipophagy and lipid metabolism in hepatocyte. However, the role of CMA in lipid metabolism of macrophage, the typical component of atherosclerotic plaque, remains unclear. In our study, LAMP-2A (L2A, a CMA marker) was reduced in macrophages exposed to high dose of oleate, and lipophagy was impaired in advanced atherosclerosis in ApoE (−/−) mice. Primary peritoneal macrophages isolated from macrophage-specific L2A-deficient mice exhibited pronounced intracellular lipid accumulation. Lipid regulatory enzymes, including long-chain-fatty-acid-CoA ligase 1 (ACSL1) and lysosomal acid lipase (LAL), were increased and reduced in L2A-KO macrophage, respectively. Other lipid-related proteins, such as SR-A, SR-B (CD36), ABCA1, or PLIN2, were not associated with increased lipid content in L2A-KO macrophage. In conclusion, deficient CMA promotes lipid accumulation in macrophage probably by regulating enzymes involved in lipid metabolism. CMA may represent a novel therapeutic target to alleviate atherosclerosis by promoting lipid metabolism.Graphical abstract

The Vitamin D Receptor Regulates Glycerolipid and Phospholipid Metabolism in Human Hepatocytes

The vitamin D receptor (VDR) must be relevant to liver lipid metabolism because VDR deficient mice are protected from hepatosteatosis. Therefore, our objective was to define the role of VDR on the overall lipid metabolism in human hepatocytes. We developed an adenoviral vector for human VDR and performed transcriptomic and metabolomic analyses of cultured human hepatocytes upon VDR activation by vitamin D (VitD). Twenty percent of the VDR responsive genes were related to lipid metabolism, including MOGAT1, LPGAT1, AGPAT2, and DGAT1 (glycerolipid metabolism); CDS1, PCTP, and MAT1A (phospholipid metabolism); and FATP2, SLC6A12, and AQP3 (uptake of fatty acids, betaine, and glycerol, respectively). They were rapidly induced (4–6 h) upon VDR activation by 10 nM VitD or 100 µM lithocholic acid (LCA). Most of these genes were also upregulated by VDR/VitD in mouse livers in vivo. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) metabolomics demonstrated intracellular accumulation of triglycerides, with concomitant decreases in diglycerides and phosphatidates, at 8 and 24 h upon VDR activation. Significant alterations in phosphatidylcholines, increases in lyso-phosphatidylcholines and decreases in phosphatidylethanolamines and phosphatidylethanolamine plasmalogens were also observed. In conclusion, active VitD/VDR signaling in hepatocytes triggers an unanticipated coordinated gene response leading to triglyceride synthesis and to important perturbations in glycerolipids and phospholipids.

Regulation of lipid-induced macrophage polarization through modulating peroxisome proliferator-activated receptor-gamma activity affects hepatic lipid metabolism via a Toll-like receptor 4/NF-κB signaling pathway.

Chronic inflammation links closely to insulin resistance and lipid metabolism in nonalcoholic fatty liver disease (NAFLD). Macrophage M1 activation plays an important role in the initiation and continuing of pro-inflammatory response of NAFLD. Our study was to investigate whether macrophage M1/M2 polarization switching would affect hepatic inflammation and lipid metabolism through modulation of peroxisome proliferator-activated receptor-gamma (PPAR-γ) activity in vivo and in vitro. RAW264.7 macrophages were treated with different fatty acids, and cell culture supernatants were collected to prepare conditioned media (CM). Different co-culture systems between primary hepatocytes and CM from macrophages were established. A PPAR-γ agonist or antagonist was administered to regulate PPAR-γ activity and macrophage polarization. M1/M2 phenotype markers, inflammatory signaling pathway, and lipid-related genes expression were determined. Wild-type C57BL/6 mice were fed a high-fat diet to induce NAFLD and given rosiglitazone to regulate PPAR-γ activity in vivo. Saturated fatty acids induced M1-polarized macrophages while polyunsaturated fatty acids induced M2-polarized macrophages. M1-polarized macrophages significantly promoted lipid synthesis and accumulation in primary hepatocytes through upregulation of a toll-like receptor 4 (TLR4)/NF-κB signaling pathway. The PPAR-γ agonist made lipid-induced M1-polarized macrophages switch to an M2-predominant phenotype, while PPAR-γ antagonist had the opposite effect. Macrophage polarization shifting subsequently affected lipid metabolism in primary hepatocytes. Administration of rosiglitazone improved high-fat diet induced hepatic steatosis and lipid metabolism through reducing hepatic TLR4/NF-κB expression and M1-polarized Kupffer cells. Lipid-induced macrophage M1 polarization promoted hepatic lipid metabolism. Modulation of PPAR-γ activity could shift macrophage polarization and subsequently affect lipid metabolism. Upregulation of the TLR4/NF-κB signaling pathway is closely linked to dysregulated lipid metabolism in NAFLD.

Orai calcium release-activated calcium modulator 1 (ORAI1) plays a role in endoplasmic reticulum stress in bovine mammary epithelial cells challenged with physiological levels of ketone bodies

The Orai calcium release-activated calcium modulator 1 (ORAI1) is a key component of the store-operated Ca2+ entry mechanism regulating cellular Ca2+ balance in nonruminants. Alterations in ORAI1 abundance have been associated with endoplasmic reticulum (ER) stress and changes in lipid metabolism in hepatocytes, an important lipogenic organ in nonruminants. Objectives were to (1) determine abundance of ORAI1 and components of the ER stress response in mammary tissue of ketotic cows, and (2) the potential role of ORAI1 on mammary cell responses to high levels of β-hydroxybutyrate (BHB). Healthy (n = 6, plasma BHB < 0.60 mmol/L) and clinically ketotic (n = 6, plasma BHB > 2.0 mmol/L) Holstein cows (days in milk = 10.13 ± 1.90) were used for mammary gland tissue and blood sample collection. Although milk production (22.5 ± 1.26, 33 ± 1.59, kg of milk/cow per day) and dry matter intake (19.5 ± 1.05, 21.9 ± 0.95, kg/d) were lower in ketotic cows, abundance of ORAI1 protein was greater and was associated with greater mRNA abundance of ER stress proteins (PERK, IRE1, ATF6, and GRP78) and lipogenic genes (FASN, SREBP1, and ACACA). Cellular mechanisms to establish links between BHB and mammary cell responses were evaluated using the immortalized cell line bovine mammary epithelial cells (MAC-T). First, a dose response study was performed with 0, 0.6, 1.2, 1.8, 2.4, or 4.8 mM BHB for 24 h. The mRNA abundance of FASN, SREBP1, and ACACA and lipid droplet formation peaked at 1.2 mM BHB. A subsequent study involved transfecting MAC-T with small interfering Orai 1 (siORAI1) or the ORAI1 inhibitor BTP2 for 24 h followed by a challenge with 1.2 mM BHB for 24 h. Transcription and protein abundance of FASN, SREBP1, ACACA, and ER stress proteins returned to basal levels when ORAI1 was silenced or inhibited. Furthermore, the Ca2+ ionophore ionomycin (raises the intracellular level of Ca2+) also increased abundance of ORAI1, FASN, SREBP1, ACACA, and ER stress proteins. Data suggest that the mammary gland experiences ER stress during ketosis, partly due to the greater supply of BHB originating from ketogenesis in the liver. Intracellular Ca2+ signaling and ORAI1 seem to mediate in part the BHB-induced ER stress in mammary cells.

Increased Expression of the Leptin Gene in Adipose Tissue of Patients with Chronic Kidney Disease-The Possible Role of an Abnormal Serum Fatty Acid Profile.

Chronic kidney disease (CKD) is associated with an increased level of leptin and an abnormal fatty acid (FA) profile in the serum. However, there are no data on the associations between them, and the reason for increased serum levels in patients with CKD is not well elucidated. Recently, we found that a CKD-related abnormal FA profile caused significant changes in the expression of genes involved in lipid metabolism in hepatocytes. The aim of this study was to examine whether leptin gene expression in subcutaneous adipose tissue (SAT) of patients with CKD may contribute to increased serum levels of this adipokine and whether the abnormal serum FA profile observed in CKD patients has an impact on leptin gene expression in adipocytes. The FA profile was measured in serum samples from patients with CKD and controls by GC–MS. The relative mRNA levels of leptin were measured in SAT by Real-Time PCR. Moreover, the effect of the CKD-related abnormal FA profile on leptin gene expression was studied in in vitro cultured 3T3-L1 adipocytes. Patients with CKD had higher concentrations of serum leptin than controls and higher expression level of the leptin gene in SAT. They also had increased serum monounsaturated FAs and decreased polyunsaturated FAs. The incubation of adipocytes with FAs isolated from CKD patients resulted in an increase of the levels of leptin mRNA. Increased leptin gene expression in SAT may contribute to elevated concentrations of these adipokine in patients with CKD. CKD-related alterations of the FA profile may contribute to elevated serum leptin concentrations in patients with CKD by increasing the gene expression of this adipokine in SAT.

Scutellarin, a modulator of mTOR, attenuates hepatic insulin resistance by regulating hepatocyte lipid metabolism via SREBP-1c suppression.

High levels of consumption of saturated lipids have been largely associated with the increasing prevalence of metabolic diseases. In particular, saturated fatty acids such as palmitic acid (PA) have been implicated in the development of insulin resistance (IR). Scutellarin (Scu) is one of the effective traditional Chinese medicines considered beneficial for liver diseases and diabetes. In this study, we investigated the effect of Scu on IR and lipid metabolism disorders in vitro and in high fat diet (HFD)-fed mice. In vitro, we found that Scu decreased insulin-dependent lipid accumulation and the mRNA expression of CD36, Fasn, and ACC in PA-treated HepG2 cells. Additionally, Scu upregulated Akt phosphorylation and improved the insulin signalling pathway. Moreover, Scu downregulated mammalian target of rapamycin (mTOR) phosphorylation and the n-SREBP-1c protein level and also reduced lipid accumulation via the mTOR-dependent pathway, as confirmed by the molecular docking of Scu to mTOR. In HFD-fed C57BL/6 mice, Scu improved oral glucose tolerance, pyruvate tolerance and the IR index and also increased the Akt phosphorylation level. Moreover, Scu reduced hepatocyte steatosis, decreased lipid accumulation and triglyceride levels, inhibited mTOR phosphorylation, and decreased the SREBP-1c level in the liver. Taken together, these findings suggest that Scu ameliorates hepatic IR by regulating hepatocyte lipid metabolism via the mTOR-dependent pathway through SREBP-1c suppression.

Acupuncture on ST36, CV4 and KI1 Suppresses the Progression of Methionine- and Choline-Deficient Diet-Induced Nonalcoholic Fatty Liver Disease in Mice.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide, and its treatment remain a constant challenge. A number of clinical trials have shown that acupuncture treatment has beneficial effects for patients with NAFLD, but the molecular mechanisms underlying its action are still largely unknown. In this study, we established a mouse model of NAFLD by administering a methionine- and choline-deficient (MCD) diet and selected three acupoints (ST36, CV4, and KI1) or nonacupoints (sham) for needling. We then investigated the effects of acupuncture treatment on the progression of NAFLD and the underlying mechanisms. After two weeks of acupuncture treatment, the liver in the needling-nonapcupoint group (NG) mice appeared pale and yellowish in color, while that in the needling-acupoint group (AG) showed a bright red color. Histologically, fewer lipid droplets and inflammatory foci were observed in the AG liver than in the NG liver. Furthermore, the expression of proinflammatory signaling factors was significantly downregulated in the AG liver. A lipid analysis showed that the levels of triglyceride (TG) and free fatty acid (FFA) were lower in the AG liver than in the NG liver, with an altered expression of lipid metabolism-related factors as well. Moreover, the numbers of 8-hydroxy-2′-deoxyguanosine (8-OHdG)-positive hepatocytes and levels of hepatic thiobarbituric acid reactive substances (TBARS) were significantly lower in AG mice than in NG mice. In line with these results, a higher expressions of antioxidant factors was found in the AG liver than in the NG liver. Our results indicate that acupuncture repressed the progression of NAFLD by inhibiting inflammatory reactions, reducing oxidative stress, and promoting lipid metabolism of hepatocytes, suggesting that this approach might be an important complementary treatment for NAFLD.

PSXIII-41 Fibroblast growth factor 21 promotes lipid oxidation and transportation while inhibits lipogenesis via AMP-activated protein kinase signaling pathway in bovine hepatocytes

Abstract Background: Fibroblast growth factor 21 (FGF21) plays essential roles in the regulation of whole body energy metabolism. However, it is not entirely clear for dairy cows whether FGF21 activates AMPK signaling pathway and what will be affected for lipid metabolism in bovine hepatocytes. Methods: Bovine hepatocytes were isolated from caudate lobes by using three-step of perfusion and collagenase digestion method. The accumulation of TG and the secretion of VLDL were examined in hepatocytes and supernatant, respectively. The expressions of the metabolic key factors were detected by Real-time PCR and Western Blot.Results: The 4th hour is the optimal time that FGF21 activates AMPK. FGF21 has significant dose-dependent inhibition of TG in bovine hepatocytes; and high concentration (1800 pg/mL) significantly promoted VLDL secretion at 4 h. The protein expression of APOB 100, APOE and MTTP, which are components of VLDL, were stimulated by FGF21, and all the mRNA expression reached the peak point (P &amp;lt; 0.01) at medium concentration (900 pg/mL). Interestingly, the proteins associated with lipid transportation were promoted too, such as LDLR, CD36, L-FABP. To some extent, meanwhile, it could be observed that some genes regulating lipid oxidation were strengthened following FGF21 treatment. In detail, ACOX1 and SIRT1 were very sensitive to the concentration of FGF21, showing remarkable difference at low concentration (P &amp;lt; 0.01); PGC-α, PPAR-α and CPT-1 showed significant changes at 900 pg/mL (P &amp;lt; 0.01); CPT-2 required a higher concentration to achieve significant enhancement. However, the results had a negative impact on lipogenesis. SREBP1c, ACC, FASN and ACLY were inhibited after treatment with low or medium doses of FGF21 (P &amp;lt; 0.01). Conclusion: FGF21 can promote lipid oxidation and transport, while inhibit lipid synthesis via activating AMPK signalling pathway in primary hepatocytes of dairy cows, thereby participation in the adaptive regulation of energy metabolism.

Choline and methionine regulate lipid metabolism via the AMPK signaling pathway in hepatocytes exposed to high concentrations of nonesterified fatty acids.

High concentrations of nonesterified fatty acids (NEFAs) and β-hydroxybutyric acid (BHBA) induce lipid peroxidation, resulting in liver damage. Choline and methionine (Met) can promote energy balance and benefit liver health in transition dairy cows; however, the regulating mechanism remains unclear. In the present study, we established the hepatocytedamage model by 1.5 mM NEFAs or BHBA treatment, and examined lipid metabolism in hepatocytes. The results showed that 1.5 mM NEFAs and 1.5 mM BHBA significantly decreased the messenger RNA (mRNA) expression of AMP-activated protein kinase (AMPK)-α as well as its target genes carnitine palmitoyltransferase-1α (CPT-1α), acetyl-CoA carboxylase, fatty acid synthetase, and Apolipoprotein B100 (ApoB100). Choline and Met upregulated the phosphorylation level of AMPK-α, which was blocked by BML (an AMPK-α inhibitor). The mRNA expression level of peroxisome proliferator-activated receptor-α (PPAR-α), CPT-1α, and ApoB100 showed a similar trend. The expressions of liver X recptoer α (LXR-α) and sterol regulatory element-binding protein 1c (SREBP-1c) were decreased by choline and Met, while only the decrease of LXR-α was blocked by BML. These findings indicate that the high-level NEFAs and BHBA weaken the lipid metabolism by impairing the fatty acid oxidation, synthesis, and transport proteins. Choline and Met regulate PPAR-α and LXR-α transcriptional activity through AMPK-α phosphorylation and regulate SREBP-1c independently of AMPK-α to promote lipid oxidation and transport in NEFAs-treated hepatocytes.