Loss of Ncb5or results in impaired fatty acid desaturation, lipoatrophy, and diabetes

Targeted ablation of the novel flavoheme reductase Ncb5or knock-out (KO) results in progressive loss of pancreatic beta-cells and white adipose tissue over time. Lipoatrophy persisted in KO animals in which the confounding metabolic effects of diabetes were eliminated by islet transplantation (transplanted knockout (TKO)). Lipid profiles in livers prepared from TKO animals were markedly deficient in triglycerides and diacylglycerides. Despite enhanced expression of stearoyl-Co-A desaturase-1, levels of palmitoleic and oleic acids (Delta9 fatty acid desaturation) were decreased in TKO relative to wild type controls. Treatment of KO hepatocytes with palmitic acid reduced cell viability and increased apoptosis, a response blunted by co-incubation with oleic acid. The results presented here support the hypothesis that Ncb5or supplies electrons for fatty acid desaturation, offer new insight into the regulation of a crucial step in fatty acid biosynthesis, and provide a plausible explanation for both the diabetic and the lipoatrophic phenotype in Ncb5or(-/-) mice.     

The reductase NCB5OR is responsive to the redox status in beta-cells and is not involved in the ER stress response

The novel reductase NCB5OR (NADPH cytochrome b5 oxidoreductase) resides in the ER (endoplasmic reticulum) and may protect cells against ER stress. Levels of BiP (immunoglobulin heavy-chain-binding protein), CHOP (CCAAT/enhancer-binding protein homologous protein) and XBP-1 (X-box-binding protein-1) did not differ in WT (wild-type) and KO (Ncb5or-null) tissues or MEFs (mouse embryonic fibroblasts), and XBP-1 remained unspliced. MEFs treated with inducers of ER stress demonstrated no change in Ncb5or expression and expression of ER-stress-induced genes was not enhanced. Induction of ER stress in beta-cell lines did not change Ncb5or expression or promoter activity. Transfection with Ncb5or-specific siRNA (small interfering RNA) yielded similar results. Microarray analysis of mRNA from islets and liver of WT and KO animals revealed no significant changes in ER-stress-response genes. Induction of oxidative stress in betaTC3 cells did not alter Ncb5or mRNA levels or promoter activity. However, KO islets were more sensitive to streptozotocin when compared with WT islets. MEFs incubated with nitric oxide donors showed no difference in cell viability or levels of nitrite produced. No significant differences in mRNA expression of antioxidant enzymes were observed when comparing WT and KO tissues; however, microarray analysis of islets indicated slightly enhanced expression of some antioxidant enzymes in the KO islets. Short-term tBHQ (t-butylhydroquinone) treatment increased Ncb5or promoter activity, although longer incubation times yielded a dose-dependent decrease in activity. This response appears to be due to a consensus ARE (antioxidant-response element) present in the Ncb5or promoter. In summary, NCB5OR does not appear to be involved in ER stress, although it may be involved in maintaining or regulating the redox status in beta-cells.     

Comparison of metabolism of free fatty acid by isolated perfused livers from male and female rats.

Livers from normal, fed male and female rats were perfused with different amounts of [1-14C]oleate under steady state conditions, and the rates of uptake and utilization of free fatty acid (FFA) were measured. The uptake of FFA by livers from either male or female rats was proportional to the concentration of FFA in the medium. The rate of uptake of FFA, per g of liver, by livers from female rats exceeded that of the males for the same amount of FFA infused. The incorporation by the liver of exogenous oleic acid into triglyceride, phospholipid, and oxidation products was proportional to the uptake of FFA. Livers from female rats incorporated more oleate into triglyceride (TG) and less into phospholipid (PL) and oxidation products than did livers from male animals. Livers from female rats secreted more TG than did livers from male animals when infused with equal quantities of oleate. The incorporation of endogenous fatty acid into TG of the perfusate was inhibite) by exogenous oleate. At low concentrations of perfusate FFA, however, endogenous fatty acids contributed substantially to the increased output of TG by livers from female animals. Production of 14CO2 and radioactive ketone bodies increased with increasing uptake of FFA. The partition of oleate between oxidative pathways (CO2 production and ketogenesis) was modified by the availability of the fatty acid substrate with livers from either sex. The percent incorporation of radioactivity into CO2 reached a maximum, whereas incorporation into ketone bodies continued to increase. The output of ketone bodies was dependent on the uptake of FFA, and output by livers from female animals was less than by livers from male rats. The increase in rate of ketogenesis was dependent on the influx of exogenous FFA, while ketogenesis from endogenous sources remained relatively stable. The output of glucose by the liver increased with the uptake of FFA, but no difference due to sex was observed. The output of urea by livers from male rats was unaffected by oleate, while the output of urea by livers from females decreased as the uptake of FFA increased. A major conclusion to be derived from this work is that oleate is not metabolized identically by livers from the two sexes, but rather, per gram of liver, livers from female rats take up and esterify more fatty acid to TG and oxidize less than do livers from male animals; livers from female animals synthesize and secrete more triglyceride than do livers from male animals when provided with equal quantities of free fatty acid.     

Palmitate protects hepatocytes from oxidative stress and triacylglyceride accumulation by stimulation of nitric oxide synthesis in the presence of high glucose and insulin concentration

Abstract

Excessive flux of free fatty acids (FFA) into the liver contributes to liver impairment in non-alcoholic fatty liver disease (NAFLD). It remains unclear how FFA contribute to impairment of hepatocytes. This study treated hepatocytes with linoleic acid and palmitate to investigate the early event triggering FFA-mediated impairment. It determined cell viability, content of nitrite/nitrate and triacylglycerides (TG), inducible nitric oxide synthase (iNOS) protein, oxidation of cardiolipin (CL) as well as formation of F₂-isoprostanes in the presence of insulin and glucose. Linoleic acid caused significant decrease in cell viability. It is shown that palmitate caused induction of iNOS resulting in increased nitrite/nitrate concentration and slight increase in TG content. Linoleic acid led to a decrease in nitrite/nitrate concentration parallelled by massive TG accumulation in combination with increased oxidation of CL and increased F₂-isoprostane levels. It is concluded that nitric oxide (NO) concentration regulates FFA-dependent TG accumulation and oxidative stress in rat hepatocytes.     

Insulin and insulin signaling play a critical role in fat induction of insulin resistance in mouse

The primary player that induces insulin resistance has not been established. Here, we studied whether or not fat can cause insulin resistance in the presence of insulin deficiency. Our results showed that high-fat diet (HFD) induced insulin resistance in C57BL/6 (B6) mice. The HFD-induced insulin resistance was prevented largely by the streptozotocin (STZ)-induced moderate insulin deficiency. The STZ-induced insulin deficiency prevented the HFD-induced ectopic fat accumulation and oxidative stress in liver and gastrocnemius. The STZ-induced insulin deficiency prevented the HFD- or insulin-induced increase in hepatic expression of long-chain acyl-CoA synthetases (ACSL), which are necessary for fatty acid activation. HFD increased mitochondrial contents of long-chain acyl-CoAs, whereas it decreased mitochondrial ADP/ATP ratio, and these HFD-induced changes were prevented by the STZ-induced insulin deficiency. In cultured hepatocytes, we observed that expressions of ACSL1 and -5 were stimulated by insulin signaling. Results in cultured cells also showed that blunting insulin signaling by the PI3K inhibitor LY-294002 prevented fat accumulation, oxidative stress, and insulin resistance induced by the prolonged exposure to either insulin or oleate plus sera that normally contain insulin. Finally, knockdown of the insulin receptor prevented the oxidative stress and insulin resistance induced by the prolonged exposure to insulin or oleate plus sera. Together, our results show that insulin and insulin signaling are required for fat induction of insulin resistance in mice and cultured mouse hepatocytes.     

Acyl-CoAs are functionally channeled in liver: potential role of acyl-CoA synthetase

Acyl-CoA synthetase (ACS) catalyzes the activation of long-chain fatty acids to acyl-CoAs, which can be metabolized to form CO(2), triacylglycerol (TAG), phospholipids (PL), and cholesteryl esters (CE). To determine whether inhibiting ACS affects these pathways differently, we incubated rat hepatocytes with [(14)C]oleate and the ACS inhibitor triacsin C. Triacsin inhibited TAG synthesis 70% in hepatocytes from fed rats and 40% in starved rats, but it had little effect on oleate incorporation into CE, PL, or beta-oxidation end products. Triacsin blocked [(3)H]glycerol incorporation into TAG and PL 33 and 25% more than it blocked [(14)C]oleate incorporation, suggesting greater inhibition of de novo TAG synthesis than reacylation. Triacsin did not affect oxidation of prelabeled intracellular lipid. ACS1 protein was abundant in liver microsomes but virtually undetectable in mitochondria. Refeeding increased microsomal ACS1 protein 89% but did not affect specific activity. Triacsin inhibited ACS specific activity in microsomes more from fed than from starved rats. These data suggest that ACS isozymes may be functionally linked to specific metabolic pathways and that ACS1 is not associated with beta-oxidation in liver.     

NAD(P)H Cytochrome b5 Oxidoreductase Deficiency in Leishmania major Results in Impaired Linoleate Synthesis Followed by Increased Oxidative Stress and Cell Death

NAD(P)H cytochrome b₅ oxidoreductase (Ncb5or), comprising cytochrome b₅ and cytochrome b₅ reductase domains, is widely distributed in eukaryotic organisms. Although Ncb5or plays a crucial role in lipid metabolism of mice, so far no Ncb5or gene has been reported in the unicellular parasitic protozoa Leishmania species. We have cloned, expressed, and characterized Ncb5or gene from Leishmania major. Steady state catalysis and spectral studies show that NADH can quickly reduce the ferric state of the enzyme to the ferrous state and is able to donate an electron(s) to external acceptors. To elucidate its exact physiological role in Leishmania, we attempted to create NAD(P)H cytochrome b₅ oxidoreductase from L. major (LmNcb5or) knock-out mutants by targeted gene replacement technique. A free fatty acid profile in knock-out (KO) cells reveals marked deficiency in linoleate and linolenate when compared with wild type (WT) or overexpressing cells. KO culture has a higher percentage of dead cells compared with both WT and overexpressing cells. Increased O₂ uptake, uncoupling and ATP synthesis, and loss of mitochondrial membrane potential are evident in KO cells. Flow cytometric analysis reveals the presence of a higher concentration of intracellular H₂O₂, indicative of increased oxidative stress in parasites lacking LmNcb5or. Cell death is significantly reduced when the KO cells are pretreated with BSA bound linoleate. Real time PCR studies demonstrate a higher Δ12 desaturase, superoxide dismutase, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA with a concomitant fall in Δ9 desaturase mRNA expression in LmNcb5or null cell line. Together these findings suggest that decreased linoleate synthesis, and increased oxidative stress and apoptosis are the major consequences of LmNcb5or deficiency in Leishmania.     

Oxidative stress in newborn infants with and without asphyxia as measured by plasma antioxidants and free fatty acids

A rapid perfusion of oxygen in infants at birth may cause an increase of oxidative stress. To assess this possibility, we measured levels of blood plasma antioxidants and free fatty acids in 20 normal infants at 0, 1, 3, and 5 days after birth. Plasma levels of the most reactive antioxidant, ascorbic acid, decreased daily to equilibrium values at days 3 and 5. Percentages of oxidized form of coenzyme Q-10 (%CoQ-10) in total coenzyme Q, another good marker of oxidative stress, in infants (25-31%) were significantly higher than those in healthy young adults (4.5%). Plasma levels of total free fatty acids (FFA) in normal infants were highest at day 1 and decreased rapidly thereafter. The content of polyunsaturated fatty acids (PUFA) in total FFA was lowest at day 1 and then increased. Since PUFA are susceptible to oxidation, these changes in FFA composition suggest that oxidative stress is most evident at the initial day of neonatal life. Furthermore, it appears that mono-unsaturated fatty acids such as oleic and palmitoleic acids increase in response to the oxidative loss of PUFA. Similar changes in plasma antioxidants, FFA levels, and FFA compositions were observed in 9 infants with asphyxia. Values of %CoQ-10 in infants with asphyxia were significantly greater than those in normal infants, suggesting that infants with asphyxia have elevated oxidative stress.     

不同来源肝细胞在体外降脂药物评价中药效反应的对比

目的:通过对比不同来源的人肝癌细胞系HepG2和原代大鼠肝细胞在体外降脂药物评价中药效反应,指导两种肝细胞在体外降脂药物评价中的实际应用。方法:用游离脂肪酸(油酸/棕榈酸,2:1)诱导HepG2细胞、原代大鼠肝细胞脂肪变性,并用100μmol·L-1苯扎贝特干预,检测细胞内甘油三酯(TG)、总胆固醇(TC)、活性氧(ROS)含量,细胞内脂滴数目、并检测细胞上清液中丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性。结果:FFA刺激使HepG2细胞和原代大鼠肝细胞脂质沉积(TG、脂滴)和氧化应激(ROS、MDA、SOD)水平上升。苯扎贝特对HepG2细胞1 mmol·L-1FFA造模组和原代大鼠肝细胞0.5 mmol·L-1FFA造模组脂质沉积和氧化应激水平改善显著;而HepG2细胞0.5 mmol·L-1FFA造模组和原代大鼠肝细胞1 mmol·L-1FFA造模组脂质沉积和氧化应激水平在苯扎贝特干预后变化不明显。结论:在相同FFA造模浓度,原代大鼠肝细胞病理特征变化更为明显;苯扎贝特对两种肝细胞在脂质沉积和氧化应激水平的作用也不完全相同。因而HepG2细胞和原代大鼠肝细胞在体外降脂药物评价中药效反应是不完全相同的。     

Selective inhibition of long-chain fatty acid uptake in short-term cultured rat hepatocytes by an antibody to the rat liver plasma membrane fatty acid-binding protein

Uptake of long-chain fatty acids by short-term cultured hepatocytes was studied. Rat hepatocytes, which were cultured for 16 h on plastic dishes (3.6 X 10(6) cells/dish), were incubated with [3H]oleate in the presence of various concentrations of bovine serum albumin as a function of the concentration of unbound [3H]oleate in the medium. At 37 degrees C initial uptake velocity (V0) was saturable (Km = 9 X 10(-8) M; Vmax = 835 pmol/min per mg protein). V0 was temperature dependent with an optimum at 37 degrees C and markedly reduced at 4 degrees C and 70 degrees C. To evaluate the biologic significance of a previously isolated rat liver plasma membrane fatty acid-binding protein as putative carrier protein in the hepatocellular uptake of fatty acids, cultured hepatocytes were treated with a monospecific rabbit antibody (IgG-fraction) to this membrane protein or the IgG-fraction of the pre-immune serum as controls. Uptake kinetics of [3H]oleate in antibody pretreated short-term cultured hepatocytes revealed a depression of Vmax by 70%, while Km was only reduced by 16% compared to controls, indicating a predominant non-competitive type of inhibition. V0 of a variety of long-chain fatty acids (oleic acid, arachidonic acid, palmitic acid, stearic acid) was reduced by 56-69%, while V0 of [35S]sulfobromophthalein, [3H]cholic acid and [14C]taurocholic acid remained unaltered. These data support the concept that in the system of cultured hepatocytes, uptake of long-chain fatty acids is mediated by the rat liver plasma membrane fatty acid-binding protein.     

Bile acids override steatosis in farnesoid X receptor deficient mice in a model of non-alcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases, and the pathogenesis is still not well known. The farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily and plays an essential role in maintaining bile acid and lipid homeostasis. In this study, we study the role of FXR in the pathogenesis of NFALD. We found that FXR deficient (FXR^−/−) mice fed methionine- and choline-deficient (MCD) diet had higher serum ALT and AST activities and lower hepatic triglyceride levels than wild-type (WT) mice fed MCD diet. Expression of genes involved in inflammation (VCAM-1) and fibrosis (α-SMA) was increased in FXR^−/− mice fed MCD diet (FXR^−/−/MCD) compared to WT mice fed MCD diet (WT/MCD). Although MCD diet significantly induced hepatic fibrosis in terms of liver histology, FXR^−/−/MCD mice showed less degree of hepatic steatosis than WT/MCD mice. Moreover, FXR deficiency synergistically potentiated the elevation effects of MCD diet on serum and hepatic bile acids levels. The super-physiological concentrations of hepatic bile acids in FXR^−/−/MCD mice inhibited the expression of genes involved in fatty acid uptake and triglyceride accumulation, which may be an explanation for less steatosis in FXR^−/−/MCD mice in contrast to WT/MCD mice. These results suggest that hepatic bile acids accumulation could override simple steatosis in hepatic injury during the progression of NAFLD and further emphasize the role of FXR in maintaining hepatic bile acid homeostasis in liver disorders and in hepatic protection.     

Diets enriched in menhaden fish oil, seal oil, or shark liver oil have distinct effects on the lipid and fatty-acid composition of guinea pig heart

The purpose of this investigation was to determine whether diets supplemented with oils from three different marine sources, all of which contain high proportions of long-chain n-3 polyunsaturated fatty acids (PUFA), result in qualitatively distinct lipid and fatty acid profiles in guinea pig heart. Albino guinea pigs (14 days old) were fed standard, nonpurified guinea pig diets (NP) or NP supplemented with menhaden fish oil (MO), harp seal oil (SLO) or porbeagle shark liver oil (PLO) (10%, w/w) for 4-5 weeks. An n-6 PUFA control group was fed NP supplemented with corn oil (CO). All animals appeared healthy, with weight gains marginally lower in animals fed the marine oils. Comparison of relative organ weights indicated that only the livers responded to the diets, and that they were heavier only in the marine-oil fed guinea pigs. Heart total cholesterol levels were unaffected by supplementing NP with any of the oils, whereas all increased the triacylglycerol (TAG) content. The fatty-acid profiles of totalphospholipid (TPL), TAG and free fatty acid (FFA) fractions of heart lipids showed that feeding n-3 PUFA significantly altered the proportions of specific fatty-acid classes. For example, all marine-oil-rich diets were associated with increases in total monounsaturated fatty acids in TPL (p < 0.05), and with decreases in total saturates in TAG (p < 0.05). Predictably, the n-3 PUFA enriched regimens significantly increased the cardiac content of n-3 PUFA and decreased that of n-6 PUFA, although the extent varied among the diets. As a result, n-6/n-3 ratios were significantly lower in all myocardial lipid classes of marine-oil-fed guinea pigs. Analyses of the profiles of individual PUFA indicated that quantitatively, the fatty acids of the three marine oils were metabolized and/or incorporated into TPL, TAG and FFA in a diet-specific manner. In animals fed MO-enriched diets in which eicosapentaenoic acid (EPA) > docosahexacnoic acid (DHA), ratios of DHA /EPA in the hearts were 1.2, 2.2 and 1.5 in TPL, TAG and FFA, respectively. In SLO-fed guinea pigs in which dietary EPA DHA, ratios of DHA/EPA were 0.9, 3.4 and 2.1 in TPL, TAG and FFA, respectively. Feeding NP + PLO (DHA/EPA = 4.8), resulted in values for DHA/EPA in cardiac tissue of 2.1, 10.6 and 2.9 in TPL, TAG and FFA, respectively. In the TAG and FFA, proportions of n-3 docosapentaenoic acid (n-3 DPA) were equal to or higher than EPA in the SLO- and PLO-fed animals. The latter group exhibited the greatest difference between the DHA/n-3 DPA ratio in the diet and in cardiac TAG and FFA fractions (7, 3.4 and 3.1, respectively). Quantitative analysis indicated that 85% of the n-3 PUFA were in TPL, 7-11% were in TAG, and 2-6% were FFA. Specific patterns of distribution of EPA, DPA and DHA depended on the dietary oil. Both the qualitative and quantitative results of this study demonstrated that in guinea pigs, n-3 PUFA in different marine oils are metabolized and/or incorporated into cardiac lipids in distinct manners. In support of the concept that the diet-induced alterations reflect changes specifically in cardiomyocytes, we observed that direct supplementation of cultured guinea pig myocytes for 2-3 weeks with EPA or DHA produced changes in the PUFA profiles of their TPL that were qualitatively similar to those observed in tissue from the dietary study. The factors that regulate specific deposition of n-3 PUFA from either dietary oils or individual PUFA are not yet known, however the differences that we observed could in some manner be related to cardiac function and thus their relative potentials as health-promoting dietary fats.     

Accumulation of arachidonic acid-rich triacylglycerols in the microalga Parietochloris incisa (Trebuxiophyceae,Chlorophyta)

The freshwater green microalga Parietochloris incisa is the richest known plant source of the polyunsaturated fatty acid (PUFA), arachidonic acid (20:4omega6, AA). While many microalgae accumulate triacylglycerols (TAG) in the stationary phase or under certain stress conditions, these TAG are generally made of saturated and monounsaturated fatty acids. In contrast, most cellular AA of P. incisa resides in TAG. Using various inhibitors, we have attempted to find out if the induction of the biosynthesis of AA and the accumulation of TAG are codependent. Salicylhydroxamic acid (SHAM) affected a growth reduction that was accompanied with an increase in the content of TAG from 3.0 to 6.2% of dry weight. The proportion of 18:1 increased sharply in all lipids while that of 18:2 and its down stream products, 18:3omega6, 20:3omega6 and AA, decreased, indicating an inhibition of the Delta12 desaturation of 18:1. Treatment with the herbicide SAN 9785 significantly reduced the proportion of TAG. However, the proportion of AA in TAG, as well as in the polar lipids, increased. These findings indicate that while there is a preference for AA as a building block of TAG, the latter can be produced using other fatty acids, when the production of AA is inhibited. On the other hand, inhibiting TAG construction did not affect the production of AA. In order to elucidate the possible role of AA in TAG we have labeled exponential cultures of P. incisa kept at 25 degrees C with [1-14C]arachidonic acid and cultivated the cultures for another 12 h at 25, 12 or 4 degrees C. At the lower temperatures, labeled AA was transferred from TAG to polar lipids, indicating that TAG of P. incisa may have a role as a depot of AA that can be incorporated into the membranes, enabling the organism to quickly respond to low temperature-induced stress.     

Inactivation of microsomal triglyceride transfer protein impairs the normal redistribution but not the turnover of newly synthesized glycerolipid in the cytosol,endoplasmic reticulum and Golgi of primary rat hepatocytes

The requirements for microsomal triglyceride transfer protein (MTP) during the turnover and transfer of glycerolipids from intracellular compartments into secretory very low-density lipoprotein (VLDL) were studied by pre-labelling lipids with [³H]glycerol and [¹⁴C]oleate in primary cultures of rat hepatocytes. The intracellular redistribution of pre-labelled glycerolipids was then compared at the end of subsequent chase periods during which the MTP inhibitor BMS-200150 was either present or absent in the medium. Inhibition of MTP resulted in a decreased output of VLDL triacylglycerol (TAG) and a delayed removal of labelled TAG from the cytosol and from the membranes of the smooth endoplasmic reticulum (SER), the cis- and the trans-Golgi. Inactivation of MTP did not decrease the bulk lipolytic turnover of cellular TAG as reflected by changes in its [³H]glycerol:[¹⁴C]oleate ratios. However, a larger proportion of the resultant TAG fatty acids was re-esterified and remained with the membranes of the various subcellular fractions rather than emerging as VLDL. The effects of BMS-200150 on the pattern of phospholipid (PL) mechanism and redistribution suggested that inhibition of MTP prevented the normal lipolytic transfer of PL-derived fatty acids out of the SER, cis- and trans-Golgi membrane pools. Finally, changes in the ¹⁴C specific radioactivities of the cytosolic and membrane pools of TAG suggested that inhibition of MTP prevented a normal influx of relatively unlabelled fatty acids into these pools during the chase period.     

A human hepatocellular in vitro model to investigate steatosis

The present study was designed to define an experimental model of hepatocellular steatosis with a fat overaccumulation profile in which the metabolic and cytotoxic/apoptotic effects could be separated. This was accomplished by defining the experimental conditions of lipid exposure that lead to significant intracellular fat accumulation in the absence of overt cytotoxicity, therefore allowing to differentiate between cytotoxic and apoptotic effects. Palmitic (C16:0) and oleic (C18:1) acids are the most abundant fatty acids (FFAs) in liver triglycerides in both normal subjects and patients with nonalcoholic fatty liver disease (NAFLD). Therefore, human hepatocytes and HepG2 cells were incubated with a mixture of different proportions of saturated (palmitate) and unsaturated (oleate) FFAs to induce fat-overloading. Similar intracellular levels of lipid accumulation as in the human steatotic liver were achieved. Individual FFAs have a distinct inherent toxic potential. Fat accumulation, cytotoxicity and apoptosis in cells exposed to the FFA mixtures were investigated. The FFA mixture containing a low proportion of palmitic acid (oleate/palmitate, 2:1 ratio) is associated with minor toxic and apoptotic effects, thus representing a cellular model of steatosis that mimics benign chronic steatosis. On the other hand, a high proportion of palmitic acid (oleate/palmitate, 0:3 ratio) might represent a cellular model of steatosis in which saturated FFAs promote an acute harmful effect of fat overaccumulation in the liver. These hepatic cellular models are apparently suitable to experimentally investigate the impact of fat overaccumulation in the liver excluding other factors that could influence hepatocyte behaviour.     

Rat long chain acyl-CoA synthetase 5 increases fatty acid uptake and partitioning to cellular triacylglycerol in McArdle-RH7777 cells

Long chain acyl-CoA synthetase (ACSL) catalyzes the initial step in long chain fatty acid metabolism. Of the five mammalian ACSL isoforms cloned and characterized, ACSL5 is the only isoform found to be located, in part, on mitochondria and thus was hypothesized to be involved in fatty acid oxidation. To elucidate the specific roles of ACSL5 in fatty acid metabolism, we used adenoviral-mediated overexpression of ACSL5 (Ad-ACSL5) in rat hepatoma McArdle-RH7777 cells. Confocal microscopy revealed that Ad-ACSL5 colocalized to both mitochondria and endoplasmic reticulum. When compared with cells infected with Ad-GFP, Ad-ACSL5-infected cells at 24 h after infection had 2-fold higher acyl-CoA synthetase activities and 30% higher rates of fatty acid uptake when incubated with 500 microM [1-(14)C]oleic acid. Metabolism of [1-(14)C]oleic acid to cellular triacylglycerol (TAG) increased 42% in Ad-ACSL5-infected cells, but when compared with control cells, metabolism to acid-soluble metabolites, phospholipids, and medium TAG did not differ substantially. The incorporation of [1-(14)C]oleate and [1,2,3-(3)H]glycerol into TAG was similar in Ad-ACSL5-infected cells, thus indicating that Ad-ACSL5 increased TAG synthesis through both de novo and reacylation pathways. However, [1-(14)C]acetic acid incorporation into cellular lipids showed that, when compared with control cells, Ad-ACSL5-infected cells did not increase the metabolism of fatty acids that were derived from de novo synthesis. These results suggest that uptake of fatty acids into cells is regulated by metabolism and that overexpressed ACSL5 partitions exogenously derived fatty acids toward TAG synthesis and storage.     

Fatty acids inhibit N-methylation of phosphatidylethanolamine in rat hepatocytes and liver microsomes

Supplementation of rat hepatocytes with various fatty acids in the culture medium reduced the conversion of [3H]phosphatidylethanolamine into phosphatidylcholine. Unsaturated fatty acids were the most effective inhibitors of phospholipid methylation. The inhibition of phosphatidylethanolamine methylation by oleate (2 mM) was reversed within 1 h after replacement with fatty acid-deficient medium. Fatty acids and their CoA derivatives (0.15-0.5 mM) produced 50% inhibition of phosphatidylethanolamine methyltransferase in rat liver microsomes. The first methylation reaction was the site of fatty acid inhibition, as methylation of phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine was not reduced in the presence of oleate. The inhibition by oleate was reversed by inclusion of bovine serum albumin or by addition of phospholipid liposomes. Thus, while fatty acids stimulate phosphatidylcholine biosynthesis in hepatocytes via the CDP-choline pathway, the methylation pathway is inhibited.     

GLP-1 analogs reduce hepatocyte steatosis and improve survival by enhancing the unfolded protein response and promoting macroautophagy

Background

Nonalcoholic fatty liver disease (NAFLD) is a known outcome of hepatosteatosis. Free fatty acids (FFA) induce the unfolded protein response (UPR) or endoplasmic reticulum (ER) stress that may induce apoptosis. Recent data indicate ER stress to be a major player in the progression of fatty liver to more aggressive lesions. Autophagy on the other hand has been demonstrated to be protective against ER stress- induced cell death. We hypothesized that exendin-4 (GLP-1 analog) treatment of fat loaded hepatocytes can reduce steatosis by autophagy which leads to reduced ER stress-related hepatocyte apoptosis.

Methodology/Principal Findings

Primary human hepatocytes were loaded with saturated, cis- and trans-unsaturated fatty acids (palmitic, oleic and elaidic acid respectively). Steatosis, induced with all three fatty acids, was significantly resolved after exendin-4 treatment. Exendin-4 sustained levels of GRP78 expression in fat-loaded cells when compared to untreated fat-loaded cells alone. In contrast, CHOP (C/EBP homologous protein); the penultimate protein that leads to ER stress-related cell death was significantly decreased by exendin-4 in hepatocytes loaded with fatty acids. Finally, exendin-4 in fat loaded hepatocytes clearly promoted gene products associated with macroautophagy as measured by enhanced production of both Beclin-1 and LC3B-II, markers for autophagy; and visualized by transmission electron microscopy (TEM). Similar observations were made in mouse liver lysates after mice were fed with high fat high fructose diet and treated with a long acting GLP-1 receptor agonist, liraglutide.

Conclusions/Significance

GLP-1 proteins appear to protect hepatocytes from fatty acid-related death by prohibition of a dysfunctional ER stress response; and reduce fatty acid accumulation, by activation of both macro-and chaperone-mediated autophagy. These findings provide a novel role for GLP-1 proteins in halting the progression of more aggressive lesions from underlying steatosis in humans afflicted with NAFLD.