Fatty Acid Synthesis In Hepatocytes Research Articles

Dimethyloxalylglycine Suppresses SREBP1c and Lipogenic Gene Expressions in Hepatocytes Independently of HIF1A.

Dimethyloxalylglycine (DMOG) is a representative inhibitor of the prolyl hydroxylase domain (PHD), which mediates the degradation of hypoxia-inducible factor-1-alpha (HIF1A). DMOG exerts its pharmacological effects via the canonical pathway that involves PHD inhibition; however, it remains unclear whether DMOG affects lipogenic gene expression in hepatocytes. We aimed to elucidate the effects of DMOG on sterol regulatory element-binding protein-1c (SREBP1c), a master regulator of fatty acid synthesis in hepatocytes. DMOG treatment inhibited SREBP1c mRNA and protein expression in HepG2 and AML12 hepatocytes and reduced the transcript levels of SREBP1c-regulated lipogenic genes. A luciferase reporter assay revealed that DMOG inhibited the transcriptional activity of SREBP1c. Moreover, DMOG suppressed SREBP1c expression in mice liver. Mechanistically, treatment with DMOG enhanced the expression of HIF1A and insulin-induced gene 2 (INSIG2), which inhibits the activation of SREBP1c. However, HIF1A or INSIG2 knockdown failed to reverse the inhibitory effect of DMOG on SREBP1c expression, suggesting a redundant role of HIF1A and INSIG2 in terms of repressing SREBP1c. DMOG did not function through the canonical pathway involving inhibition of SREBP1c by PHD, highlighting the presence of non-canonical pathways that mediate its anti-lipogenic effect.

Phloretin ameliorates hepatic steatosis through regulation of lipogenesis and Sirt1/AMPK signaling in obese mice

BackgroundPhloretin is isolated from apple trees and could increase lipolysis in 3T3-L1 adipocytes. Previous studies have found that phloretin could prevent obesity in mice. In this study, we investigated whether phloretin ameliorates non-alcoholic fatty liver disease (NAFLD) in high-fat diet (HFD)-induced obese mice, and evaluated the regulation of lipid metabolism in hepatocytes.MethodsHepG2 cells were treated with 0.5 mM oleic acid to induce lipid accumulation, and then treated with phloretin to evaluate the molecular mechanism of lipogenesis. In another experiment, male C57BL/6 mice were fed normal diet or HFD (60% fat, w/w) for 16 weeks. After the fourth week, mice were treated with or without phloretin by intraperitoneal injection for 12 weeks.ResultsPhloretin significantly reduced excessive lipid accumulation and decreased sterol regulatory element-binding protein 1c, blocking the expression of fatty acid synthase in oleic acid-induced HepG2 cells. Phloretin increased Sirt1, and phosphorylation of AMP activated protein kinase to suppress acetyl-CoA carboxylase expression, reducing fatty acid synthesis in hepatocytes. Phloretin also reduced body weight and fat weight compared to untreated HFD-fed mice. Phloretin also reduced liver weight and liver lipid accumulation and improved hepatocyte steatosis in obese mice. In liver tissue from obese mice, phloretin suppressed transcription factors of lipogenesis and fatty acid synthase, and increased lipolysis and fatty acid β-oxidation. Furthermore, phloretin regulated serum leptin, adiponectin, triglyceride, low-density lipoprotein, and free fatty acid levels in obese mice.ConclusionsThese findings suggest that phloretin improves hepatic steatosis by regulating lipogenesis and the Sirt-1/AMPK pathway in the liver.

Mono-2-ethylhexyl phthalate induces the expression of genes involved in fatty acid synthesis in HepG2 cells

Mono-2-ethylhexyl phthalate (MEHP) is a major bioactive metabolite in the widely used industrial plasticizer diethylhexyl phthalate (DEHP) that has been found to be toxic to the liver. The aim of this study is to determine whether MEHP exposure can change the expression of fatty acid metabolism-related genes in HepG2 cells, which might be related to non-alcoholic fatty liver disease (NAFLD). The results revealed that exposure to MEHP promoted lipid accumulation in HepG2 cells. The levels of intracellular triglycerides in the hepatocytes increased after exposure to 0.8–100 μM MEHP for 24 h and 48 h. The genetic expressions of SREBP-1c, ChREBP, ACC1, FASN, and SCD significantly increased at 6 h after exposure to MEHP. At 24 h, the expression of the SREBP-1c and ChREBP genes remained increased, while the expression of the FASN and SCD genes decreased. At 48 h, the expression of SREBP-1c, ChREBP, ACC1, FASN, and SCD decreased. Furthermore, the levels of proteins including ACC1, FASN, SCD, and ChREBP (except SREBP-1c) increased at 24 h. These findings suggest that MEHP exposure can promote fatty acid synthesis in hepatocytes by regulating the expression of relevant genes and proteins, contributing to NAFLD.

Ginkgolide C reduced oleic acid-induced lipid accumulation in HepG2 cells

Ginkgolide C, isolated from Ginkgo biloba, is a diterpene lactone that has multiple biological functions and can improve Alzheimer disease and platelet aggregation. Ginkgolide C also inhibits adipogenesis in 3T3-L1 adipocytes. The present study evaluated whether ginkgolide C reduced lipid accumulation and regulated the molecular mechanism of lipogenesis in oleic acid-induced HepG2 hepatocytes. HepG2 cells were treated with 0.5 mM oleic acid for 48 h to induce a fatty liver cell model. Then, the cells were exposed to various concentrations of ginkgolide C for 24 h. Staining with Oil Red O and the fluorescent dye BODIPY 493/503 revealed that ginkgolide C significantly reduced excessive lipid accumulation in HepG2 cells. Ginkgolide C decreased peroxisome proliferator-activated receptor γ and sterol regulatory element-binding protein 1c to block the expression of fatty acid synthase. Ginkgolide C treatment also promoted the expression of adipose triglyceride lipase and the phosphorylation level of hormone-sensitive lipase to enhance the decomposition of triglycerides. In addition, ginkgolide C stimulated CPT-1 to activate fatty acid β-oxidation, significantly increased sirt1 and phosphorylation of AMP-activated protein kinase (AMPK), and decreased expression of acetyl-CoA carboxylase for suppressed fatty acid synthesis in hepatocytes. Taken together, our results suggest that ginkgolide C reduced lipid accumulation and increased lipolysis through the sirt1/AMPK pathway in oleic acid-induced fatty liver cells.

Characterization of novel mechanisms for steatosis from global protein hyperacetylation in ethanol-induced mouse hepatocytes

Steatosis is the earliest and most common disease of the liver due to chronic ethanol consumption, and stems from alterations in the function of transcription factors related to lipid metabolism. Protein acetylation at the lysine residue (Kac) is known to have diverse functions in cell metabolism. Recent studies showed that ethanol exposure induces global protein hyperacetylation by reducing the deacetylase activities of SIRT1 and SIRT3. Although global acetylome analyses have revealed the involvement of a variety of lysine acetylation sites, the exact sites directly regulated by ethanol exposure are unknown. In this study, to elucidate the exact hyperacetylation sites that contribute to SIRT1 and SIRT3 downregulation, we identified and quantified a total of 1285 Kac sites and 686 Kac proteins in AML-12 cells after ethanol treatment (100 mM) for 3 days. All quantified Kac sites were divided into four quantiles: Q1 (0–15%), Q2 (15–50%), Q3 (50–85%), and Q4 (85–100%). Q4 had 192 Kac sites indicating ethanol-induced hyperacetylation. Using the Motif-x program, the [LXKL], [KH], and [KW] motifs were included in the Q4 category, where [KW] was a specific residue for SIRT3. We also performed gene ontology term and KEGG pathway enrichment analyses. Hyperacetylation sites were significantly enriched in biosynthetic processes and ATPase activities within the biological process and molecular function categories, respectively. In conclusion, ethanol regulates the acetylation of proteins in a variety of metabolic pathways mediated by SIRT1 and SIRT3. As a result, ethanol stimulates increased de novo fatty acid synthesis in hepatocytes.

Yamogenin in fenugreek inhibits lipid accumulation through the suppression of gene expression in fatty acid synthesis in hepatocytes.

Yamogenin is a diastereomer of diosgenin, which we have identified as the compound responsible for the anti-hyperlipidemic effect of fenugreek. Here, we examined the effects of yamogenin on the accumulation of triacylglyceride (TG) in hepatocytes, because yamogenin is also contained in fenugreek. It was demonstrated that yamogenin also inhibited TG accumulation in HepG2 hepatocytes and suppressed the mRNA expression of fatty acid synthesis-related genes such as fatty acid synthase and sterol response element-binding protein-1c. Indeed, yamogenin also antagonized the activation of the liver X receptor (LXR) in luciferase ligand assay similar to diosgenin. However, yamogenin could not exert such effects in the presence of T0901713, a potent agonist of LXR. These findings indicate that the effects of yamogenin on TG accumulation would be weaker than those of diosgenin, suggesting that the structural difference between yamogenin and diosgenin would be important for the inhibition of LXR activation.

Triacylglycerol and Fatty Acid Synthesis in Hepatocytes in Suspension Isolated from Rats Fed Soybean Phospholipid

The rates of triacylglycerol and fatty acid synthesis in hepatocytes in suspension isolated from rats fed dietary soybean phospholipid were compared with those from the animals fed soybean oil. The rats were fed the experimental diets containing either soybean phospholipid or soybean oil (the dietary levels correspond to 3% fatty acids, 5.1 and 3.2% on weight bases for the phospholipid and the oil, respectively) for 10 days. Dietary soybean phospholipid compared to the oil decreased the rate of incorporation of [2-3H]glycerol into triacylglycerol in isolated hepatocytes. However, the difference disappeared when the incubation mixture was fortified with oleate substrate (1 mm). The dietary phospholipid compared to the oil greatly decreased the rate of incorporation of [1-14C]acetate into fatty acids while it increased that of the tracer into cholesterol. The rate of ketone body production from oleate substrate was decreased in rats fed soybean phospholipid compared to those fed the oil. Thus, dietary soybe...

Inhibition of fatty acid synthesis in rat hepatocytes by exogenous polyunsaturated fatty acids is caused by lipid peroxidation

Rat hepatocyte long-term cultures were utilized to investigate the impact of different polyunsaturated fatty acids (PUFA) on the insulin-induced de novo fatty acid synthesis in vitro. The addition of 0.5 mM albumin-complexed oleic, linoleic, columbinic, arachidonic, eicosapentaenoic or docosahexaenoic acid resulted in a marked suppression of fatty acid synthesis. By evaluation of cell viability (determined as the leakage of lactate dehydrogenase (LDH)) it turned out, that the antioxidant used (50 μM α-tocopherol phosphate) had a low antioxidant activity, resulting in cytotoxic effects by the peroxidized PUFA. Arachidonic acid and eicosapentaenoic acid showed a dose- and time-dependent cytotoxicity. Two other antioxidants: 50 μM α-tocopherol acid succinate and 1 μM, N, N'-diphenyl-1,4-phenylenediamine, both proved more efficient than α-tocopherol phosphate. There was a significant correlation between LDH-leakage and inhibition of fatty acid synthesis. Lipid peroxidation, measured as thiobarbituric acid-reactive substances, also showed a significant correlation with the degree of inhibition of fatty acid synthesis. Furthermore, PUFA had no inhibitory effect on fatty acid synthesis when peroxidation was minimized by the use of proper antioxidants. These data indicate that PUFA in vitro inhibit the insulin-induced de novo fatty acid synthesis in hepatocytes from starved rats, due to cytotoxic effects caused by lipid peroxidation.

Acute effects of interleukin 1α and 6 on intermediary metabolism in freshly isolated rat hepatocytes

Using hepatocytes in suspension, freshly isolated from adult male fed rats, we studied the acute influence of recombinant human interleukins 1α, 2 and 6 on glycogen and fatty acid metabolism. By far the largest effects were observed with interleukin-1α: short incubations (up to 60 min) sufficed to depress glycogen synthesis in a dose-dependent manner, while the rates of glycogenolysis and glycolysis were increased as indicated by the release of glucose and lactate. Interleukin-6 acted similarly, though being much less effective on a molar basis, whereas interleukin-2 only caused a small increase in lactate production. In hepatocytes from 24h-starved rats interleukin-1α caused a minor stimulation of gluconeogenesis. Although neither fatty acid synthesis nor oxidation of fatty acids in quiescent hepatocytes from fed rats was significantly affected by interleukins, interleukin-1α was able to cause appreciable inhibition of fatty acid synthesis in hepatocytes from regenerating liver (isolated 22h after partial hepatectomy). It is concluded (i) that interleukins, in particular interleukin-1α, acutely promote hepatic glucose release, and (ii) that transition of adult hepatocytes from a quiescent into a proliferatory state allows the occurrence of rapid effects of interleukin-1α on fatty acid metabolism.

Measurement of acetyl-CoA carboxylase activity in isolated hepatocytes

An assay is described for acetyl-CoA carboxylase activity in isolated hepatocytes. The assay is based on two principles: (a) The hepatocytes are made permeable by digitonin. 64 μg of digitonin per mg of cellular protein were most effective in exposing enzyme activity without a significant effect on mitochondrial permeability. (b) Enzyme activity is measured by coupling the carboxylase reaction to the fatty acid synthase reaction. The advantages offered by this procedure over existing assays are: (i) rapidity, (ii) no need to prepare cell extracts, (iii) absence of product inhibition, (iv) no interference by mitochondrial enzymes, (v) useful in systems with bicarbonate buffers, and (vi) simple separation of radioactive substrate from labelled products. Using this coupled enzyme assay a good correlation was observed between changes in the activity of acetyl-CoA carboxylase and changes in the rate of fatty acid synthesis in hepatocytes as effected by short-term modulators.

Effect of insulin on ketogenesis and fatty acid synthesis in rat hepatocytes incubated with dichloroacetate

In parenchymal liver cells isolated from fed rats, insulin increased the formation of 14CO 2 from [1- 14C]pyruvate (and presumably the flux through pyruvate dehydrogenase) by 14%. Dichloroacetate, an activator of the pyruvate dehydrogenase complex, stimulated this process by 133%. As judged from the conversion of [2- 14C]pyruvate to 14CO 2, the tricarboxylic acid cycle activity was not affected by insulin, but it was depressed by dichloroacetate. In hepatocytes from fed rats, incubated with glucose as the only carbon source, dichloroacetate caused a stimulation (31%) of fatty acid synthesis, measured as 3H incorporation from 3H 2O into fatty acid, and an increased (134%) accumulation of ketone bodies ( acetoacetate + d -3- hydroxybutyrate ). Dichloroacetate did not affect ketone body formation from [ 14C]palmitate, suggesting that the increased accumulation of ketone bodies resulted from acetyl-CoA derived from pyruvate. Insulin stimulated fatty acid synthesis in hepatocytes from fed rats. In the combined presence of insulin plus dichloroacetate, fatty acid synthesis was more rapid than in the presence of either insulin or dichloroacetate, whereas the accumulation of ketone bodies was smaller than in the presence of dichloroacetate alone. Although pyruvate dehydrogenase activity, which is rate-limiting for fatty acid synthesis in hepatocytes from fed rats, is stimulated both by insulin and by dichloroacetate, the reciprocal changes in fatty acid synthesis and ketone body accumulation brought about by insulin in the presence of dichloroacetate suggest that insulin is also involved in the regulation of fatty acid synthesis at a mitochondrial site after pyruvate dehydrogenase, possibly at the partitioning of acetyl-CoA between citrate and ketone body formation.

Both insulin and epidermal growth factor stimulate fatty acid synthesis and increase phosphorylation of acetyl-CoA carboxylase and ATP-citrate lyase in isolated hepatocytes

Insulin and EGF cause identical stimulation(≅40%) of fatty acid synthesis in hepatocytes isolated from rats which have been starved and then refed a low-fat diet. In both cases this stimulation is associated with increased phosphorylation of ATP-citrate lyase and of a specific site on acetyl-CoA carboxylase. However, the altered phosphorylation of acetyl-CoA carboxylase is not associated with a change in kinetic parameters which is detectable in the purified enzyme. Whatever the mechanism involved, stimulation of fatty acid synthesis by growth factors may have a role in providing new phospholipid for growth of membranes.

Age-related decrease in sensitivity to glucagon and dibutyryl cyclic AMP inhibition of fatty acid synthesis in hepatocytes isolated from obese female Zucker rats.

Hepatocytes were isolated from 3 and 5 month old female genetically obese Zucker rats and their lean littermate controls. An age-dependent loss in sensitivity of fatty acid synthesis to inhibition by both glucagon and dibutyryl cyclic AMP was observed with hepatocytes from the obese rats. Hepatocytes from lean animals were much more sensitive to these agents, regardless of age. Low concentrations of glucagon and dibutyryl cyclic AMP actually produced some stimulation of fatty acid synthesis with hepatocytes prepared from the older obese rats. 5-Tetradecyloxy-2-furoic acid, a compound which inhibits fatty acid synthesis, was a very effective inhibitor of fatty acid synthesis by hepatocytes isolated from all rats used in the study. An inhibition of lactate plus pyruvate accumulation and a strong stimulation of glycogenolysis occurred in response to both glucagon and dibutyryl cyclic AMP with hepatocytes from both age groups of lean and obese rats. The results suggest that with aging of the obese female Zucker rat some step of hepatic fatty acid synthesis becomes progressively less sensitive to inhibition by glucagon and dibutyryl cyclic AMP. This may play an important role in maintenance of obesity in these animals.

Regulation of fatty acid synthesis in isolated hepatocytes by intestinal chylomicrons and their remnants

Dietary fatty acids are incorporated into chylomicrons and the chylomicron triglycerides are degraded by the action of lipoprotein lipase. The resultant particles, remnants, are selectively metabolized by the liver [ 11. Sherrill and Dietschy demonstrated that unmetabolized chylomicrons were taken up at very low rates, whereas chylomicron remnants were taken up by the perfused liver at much higher velocities [2]. Recently, Carrella and Cooper demonstrated that there was a high affinity receptor for the chylomicron remnant on the surface of the hepatocytes [3]. This study was undertaken to examine the effects of chylomicrons and their remnants on the fatty acid synthesis in isolated rat hepatocytes. Chylomicrons in the medium containing postheparin rat plasma significantly inhibited fatty acid synthesis in hepatocytes. On the other hand, chylomicrons in the medium containing pre-heparin rat plasma caused inhibition to a much smaller extent. Remnants prepared from the chylomicrons injected into functionally hepatectomized rats inhibited fatty acid synthesis to a greater extent than unmetabolized chylomicrons. Thus the chylomicron remnants produced by the action of lipoprotein lipase may play an important role in the regulation of hepatic fatty acid synthesis.

Comparison of C 18 −, C 20 − and C 22 −unsaturated fatty acids in reducing fatty acid synthesis in isolated rat hepatocytes

C 18 −, C 20 − and C 22 −unsaturated acids were tested for inhibition of fatty acid synthesis in hepatocytes isolated from essential fatty acid-deficient rats. Fatty acid synthesis was measured by incorporation of radioactivity from [1- 14C]-acetate or 3H 2O into fatty acids. C 20 −polyunsaturated fatty acids included arachidonic acid (20 : 4 ( n−6)) and 4 other fatty acids formed from linoleic acid (18 : 2 ( n−6)) or linolenic acid (18 : 3 ( n−3)). These were (11,14)-icosadienoic acid (20 : 2 ( n−6)), (8,11,14)-icosatrienoic acid (20 : 3 ( n−6)), (11,14,17)-icosatrienoic acid (20 : 3 ( n−3)) and (5,8,11,14,17)-icosapentaenoic acid (20 : 5 ( n−3)). All of these have essential fatty acid activity. The fatty acid (5,8,11)-icosatrienoic acid (20 : 3 ( n−9)) was also tested. This fatty acid is formed from oleic acid (18 : 1 ( n−9)) and is not an essential fatty acid or a prostaglandin precursor. C 20 −unsaturated fatty acids and (22 : 6 ( n−3)) were as effective as stearic acid in inhibiting fatty acid synthesis and were more inhibitory than their precursor C 18-unsaturated fatty acids. These results are evidence that C 20 −unsaturated fatty acids of the linoleic and linolenic acid series can act in short-term inhibition as well as in adaptive inhibition of fatty acid synthesis (Bloch, K. and Vance, D. (1977) Annu. Rev. Biochem. 46, 263–298). The effectiveness of (5,8,11)-icosatrienoic acid indicates that short-term inhibition by C 20 −unsaturated fatty acids is not limited to those fatty acids which have essential fatty acid activity.