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.

Hypoxia signaling and cholesterol/steroidogenic acute regulatory protein 1 axis: interplay and role in alcohol and non-alcohol-related liver diseases

Metabolic zonation in the liver carries out the maintenance of organ and body homeostasis. Hypoxia is an inherent physiological feature of the liver and contributes to the zonal properties of the hepatic parenchyma. As a master regulator of hypoxia, the transcription factor hypoxia-inducing factor (HIF) is stabilized primarily by oxygen availability, and it is thought to contribute to steatohepatitis due to alcohol-related (ASH) and non-alcohol-related liver disease (NASH). Cholesterol has emerged as an important player in both diseases, and hypoxia increases hepatic cholesterol levels. Steroidogenic acute regulatory protein 1 (STARD1) is a mitochondrial outer membrane protein that transfers cholesterol to mitochondrial inner membrane for metabolic processing and acts as the rate-limiting step in the alternative pathway of bile acid synthesis in hepatocytes. STARD1 expression increases in ASH and NASH and determines the accumulation of cholesterol in mitochondria, which impacts the physico-chemical mitochondrial membranes properties and as a consequence impairs the activity of specific mitochondrial solute carriers, such as the 2-oxoglutarate carrier (2-OGC), limiting the exchange between cytosolic glutathione and mitochondrial 2-oxoglutarate (2-OG). Although HIF-1 is stabilized in hypoxia largely due to the requirement of prolylhydroxylases (PHDs) for oxygen to signal HIF degradation, PHDs are also dependent on 2-OG, and therefore it is conceivable that impairment of 2-OGC by STARD1-mediated cholesterol accumulation may contribute to HIF-1 stabilization due in part to decreased availability of cytosolic 2-OG. In this perspective, this review explores the interplay between HIF-1 stabilization and STARD1 induction and the potential contribution of this functional relationship to ASH and NASH.

Selective PPARδ agonist seladelpar suppresses bile acid synthesis by reducing hepatocyte CYP7A1 via the fibroblast growth factor 21 signaling pathway

Peroxisome proliferator–activated receptor delta (PPARδ) agonists have been shown to exert beneficial effects in liver disease and reduce total bile acid levels. The mechanism(s) whereby PPARδ agonism reduces bile acid levels are, however, unknown, and therefore the aim of the present study was to investigate the molecular pathways responsible for reducing bile acid synthesis in hepatocytes, following treatment with the selective PPARδ agonist, seladelpar. We show that administration of seladelpar to WT mice repressed the liver expression of cholesterol 7 alpha-hydroxylase (Cyp7a1), the rate-limiting enzyme for bile acid synthesis, and decreased plasma 7α-hydroxy-4-cholesten-3-one (C4), a freely diffusible metabolite downstream of Cyp7a1. In primary mouse hepatocytes, seladelpar significantly reduced the expression of Cyp7a1 independent of the nuclear bile acid receptor, Farnesoid X receptor. In addition, seladelpar upregulated fibroblast growth factor 21 (Fgf21) in mouse liver, serum, and in cultured hepatocytes. We demonstrate that recombinant Fgf21 protein activated the c-Jun N-terminal kinase (JNK) signaling pathway and repressed Cyp7a1 gene expression in primary hepatocytes. The suppressive effect of seladelpar on Cyp7a1 expression was blocked by a JNK inhibitor as well as in the absence of Fgf21, indicating that Fgf21 plays an indispensable role in PPARδ-mediated downregulation of Cyp7a1. Finally, reduction of CYP7A1 expression by seladelpar was confirmed in primary human hepatocytes. In conclusion, we show that seladelpar reduces bile acid synthesis via an FGF21-dependent mechanism that signals at least partially through JNK to repress CYP7A1.

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.

Glucose stimulates cholesterol 7α-hydroxylase gene transcription in human hepatocytes

Bile acids play important roles in the regulation of lipid, glucose, and energy homeostasis. Recent studies suggest that glucose regulates gene transcription in the liver. The aim of this study was to investigate the potential role of glucose in regulation of bile acid synthesis in human hepatocytes. High glucose stimulated bile acid synthesis and induced mRNA expression of cholesterol 7alpha-hydroxylase (CYP7A1), the key regulatory gene in bile acid synthesis. Activation of an AMP-activated protein kinase (AMPK) decreased CYP7A1 mRNA, hepatocyte nuclear factor 4alpha (HNF4alpha) protein, and binding to CYP7A1 chromatin. Glucose increased ATP levels to inhibit AMPK and induce HNF4alpha to stimulate CYP7A1 gene transcription. Furthermore, glucose increased histone acetylation and decreased H3K9 di- and tri-methylation in the CYP7A1 chromatin. Knockdown of ATP-citrate lyase, which converts citrate to acetyl-CoA, decreased histone acetylation and attenuated glucose induction of CYP7A1 mRNA expression. These results suggest that glucose signaling also induces CYP7A1 gene transcription by epigenetic regulation of the histone acetylation status. This study uncovers a novel link between hepatic glucose metabolism and bile acid synthesis. Glucose induction of bile acid synthesis may have an important implication in metabolic control of glucose, lipid, and energy homeostasis under normal and diabetic conditions.

Bile acids activate fibroblast growth factor 19 signaling in human hepatocytes to inhibit cholesterol 7α-hydroxylase gene expression

Mouse fibroblast growth factor 15 (FGF15) and human ortholog FGF19 have been identified as the bile acid-induced intestinal factors that mediate bile acid feedback inhibition of cholesterol 7alpha-hydroxylase gene (C YP7A1) transcription in mouse liver. The mechanism underlying FGF15/FGF19 inhibition of bile acid synthesis in hepatocytes remains unclear. Chenodeoxycholic acid (CDCA) and the farnesoid X receptor (FXR)-specific agonist GW4064 strongly induced FGF19 but inhibited CYP7A1 messenger RNA (mRNA) levels in primary human hepatocytes. FGF19 strongly and rapidly repressed CYP7A1 but not small heterodimer partner (SHP) mRNA levels. Kinase inhibition and phosphorylation assays revealed that the mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK/Erk1/2) pathway played a major role in mediating FGF19 inhibition of CYP7A1. However, small interfering RNA (siRNA) knockdown of SHP did not affect FGF19 inhibition of CYP7A1. Interestingly, CDCA stimulated tyrosine phosphorylation of the FGF receptor 4 (FGFR4) in hepatocytes. FGF19 antibody and siRNA specific to FGFR4 abrogated GW4064 inhibition of CYP7A1. These results suggest that bile acid-activated FXR is able to induce FGF19 in hepatocytes to inhibit CYP7A1 by an autocrine/paracrine mechanism. The hepatic FGF19/FGFR4/Erk1/2 pathway may inhibit CYP7A1 independent of SHP. In addition to inducing FGF19 in the intestine, bile acids in hepatocytes may activate the liver FGF19/FGFR4 signaling pathway to inhibit bile acid synthesis and prevent accumulation of toxic bile acid in human livers.

Vitamin D receptor is expressed in human livers and inhibits CYP7A1 gene transcription

Lithocholic acid (LCA) is a potent endogenous ligand of Vitamin D receptor (VDR). It is thought that VDR may induce intestinal CYP3A4 to detoxify bile acids and protective against colon cancer. It has been reported that VDR is expressed in the rat but not mouse liver. The objective of this study is to identify VDR in human hepatocytes and to test our hypothesis that VDR may inhibit transcription of the gene encoding CYP7A1, the rate‐limiting enzyme in bile acid synthesis in hepatocytes. Immunoblot and real‐time PCR analyses have detected VDR protein and mRNA expression in HepG2 and human primary hepatocytes. Confocal immunofluorescence has detected VDR in the cytoplasm of hepatocytes. Upon treatment with 1α, 25(OH)2‐VD3 or LCA, VDR is redistributed to the nucleus and cell membrane. Reporter assays showed that 1α, 25(OH)2VD3 and LCA‐acetate decreased human CYP7A1 reporter activities. The region conferring VDR inhibition was localized between −80 and −150 of the human CYP7A1 promoter. VDR decreased hepatocyte nuclear factor 4α (HNF4α) interaction with peroxisome proliferators‐activated receptor γ coactivator 1α (PGC‐1α)/glucocorticoid receptor interacting protein‐1 (GRIP‐1) and trans‐activation of CYP7A1. Co‐immunoprecipitation and GST pull down assays showed that VDR interacted with HNFα. Chromatin immunoprecipitation (CHIP) assay revealed that 1, 25(OH)2‐VD3‐activated VDR interacted with HNF4α and blocked its recruitment of PGC‐1α and GRIP‐1 to the human CYP7A1 chromatin, and resulted in inhibition of CYP7A1 gene transcription. This study suggests that VDR may play a role in protecting liver cells against bile acid toxicity and cholestatic liver injury.

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.

Cholesterol 7α-hydroxylase activity and bile acid synthesis in hepatocytes of unwearied and weaned pigs in monolayer culture

Activity of cholesterol 7α-hydroxylase (EC 1.14.13.17) in freshly isolated hepatocytes from unweaned piglets (2 to 3 weeks old) was 16-times lower as compared to hepatocytes from weaned piglets (7 to 8 weeks old). The monolayer culture activity of the enzyme remained low in unweaned piglet hepatocytes. In contrast, in cultured hepatocytes from weaned piglets, cholesterol 7α-hydroxylase activity declined during the first day of culture, but was restored during the next 2 culture days, provided that fetal bovine serum (10%) was added to the culture medium. Addition of dexamethasone (50 nM) and insulin (135 nM) to the medium, further enhanced cholesterol 7α-hydroxyease activity to values similar to those in freshly isolated hepatocytes and retarded the decline of enzyme activity after the 3rd culture day. Cultured hepatocytes from weaned and unweaned piglets synthesized similar types of bile acids from [ 14C]cholesterol. among which hyocholic acid (the most prominent), hyodeoxycholic acid, chenodeoxycholic acid, murocholic acid and lithocholic acid could be identified. 95% of radiolabelled bile acids synthesized was conjugated, mainly with glycine, but also with taurine, sulfate and glucuronic acid. The rate of mass production of bile acids by cultured hepatocytes of weaned piglets (as measured by gas-chromatography) parallelled cholesterol 7α-hydroxylase activity, and was low in the absence of serum, but increased in medium containing fetal bovine serum, dexamethasone and insulin to a rate lying in the range of 75% of the in vivo bile acid production during the 3rd culture day. Bile acid production by unweaned piglet hepatocytes was 3-times lower under these conditions. It is concluded that hepatocytes from young weaned pigs cultured in medium containing 10% fetal bovine serum, offer a suitable in vitro model for the study of bile acid synthesis, in view of the high cholesterol 7α-hydroxylase activities and bile acid production rates.

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.