A novel role for fatty acid transport protein 1 in the regulation of tricarboxylic acid cycle and mitochondrial function in 3T3-L1 adipocytes

Fatty acid transport proteins (FATPs) are integral membrane acyl-CoA synthetases implicated in adipocyte fatty acid influx and esterification. Whereas some FATP1 translocates to the plasma membrane in response to insulin, the majority of FATP1 remains within intracellular structures and bioinformatic and immunofluorescence analysis of FATP1 suggests the protein primarily resides in the mitochondrion. To evaluate potential roles for FATP1 in mitochondrial metabolism, we used a proteomic approach following immunoprecipitation of endogenous FATP1 from 3T3-L1 adipocytes and identified mitochondrial 2-oxoglutarate dehydrogenase. To assess the functional consequence of the interaction, purified FATP1 was reconstituted into phospholipid-containing vesicles and its effect on 2-oxoglutarate dehydrogenase activity evaluated. FATP1 enhanced the activity of 2-oxoglutarate dehydrogenase independently of its acyl-CoA synthetase activity whereas silencing of FATP1 in 3T3-L1 adipocytes resulted in decreased activity of 2-oxoglutarate dehydrogenase. FATP1 silenced 3T3-L1 adipocytes exhibited decreased tricarboxylic acid cycle activity, increased cellular NAD⁺/NADH, increased fatty acid oxidation, and increased lactate production indicative of altered mitochondrial energy metabolism. These results reveal a novel role for FATP1 as a regulator of tricarboxylic acid cycle activity and mitochondrial function.     

Abnormal metabolism flexibility in response to high palmitate concentrations in myotubes derived from obese type 2 diabetic patients

Insulin resistance in type 2 diabetes (T2D) is associated with intramuscular lipid (IMCL) accumulation. To determine whether impaired lipid oxidation is involved in IMCL accumulation, we measured expression of genes involved in mitochondrial oxidative metabolism or biogenesis, mitochondrial content and palmitate beta-oxidation before and after palmitate overload (600 μM for 16 h), in myotubes derived from healthy subjects and obese T2D patients. Mitochondrial gene expression, content and network were not different between groups. Basal palmitate beta-oxidation was not affected in T2D myotubes, whereas after 16 h of palmitate pre-treatment, T2D myotubes in contrast to control myotubes, showed an inability to increase palmitate beta-oxidation (p < 0.05). Interestingly, acetyl-CoA carboxylase (ACC) phosphorylation was increased with a tendency for statistical significance after palmitate pre-treatment in control myotubes (p = 0.06) but not in T2D myotubes which can explain their inability to increase palmitate beta-oxidation after palmitate overload. To determine whether the activation of the AMP activated protein kinase (AMPK)-ACC pathway was able to decrease lipid content in T2D myotubes, cells were treated with AICAR and metformin. These AMPK activators had no effect on ACC and AMPK phosphorylation in T2D myotubes as well as on lipid content, whereas AICAR, but not metformin, increased AMPK phosphorylation in control myotubes. Interestingly, metformin treatment and mitochondrial inhibition by antimycin induced increased lipid content in control myotubes. We conclude that T2D myotubes display an impaired capacity to respond to metabolic stimuli.     

TR4 activates FATP1 gene expression to promote lipid accumulation in 3T3-L1 adipocytes

We show that TR4 facilitates lipid accumulation in 3T3-L1 adipocytes via induction of the FATP1 gene. Further study showed that TR4 transactivated FATP1 5' promoter activity via direct binding to the TR4 responsive element located at the FATP1 5' promoter region. Constitutive overexpression of TR4 in 3T3-L1 adipocytes resulted in increased lipid accumulation, accompanied by an increase in fatty acid uptake. However, small interfering RNA knockdown of FATP1 abolished TR4-enhanced fatty acid uptake. Moreover, microRNA-mediated silencing of TR4 in 3T3-L1 adipocytes drastically reduced basal FATP1 5' promoter activity and FATP1 expression with reduced lipid accumulation.     

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside-induced AMP-activated protein kinase phosphorylation inhibits basal and insulin-stimulated glucose uptake, lipid synthesis, and fatty acid oxidation in isolated rat adipocytes

The objective of this study was to investigate the effects of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR)-induced AMP-activated protein kinase (AMPK) activation on basal and insulin-stimulated glucose and fatty acid metabolism in isolated rat adipocytes. AICAR-induced AMPK activation profoundly inhibited basal and insulin-stimulated glucose uptake, lipogenesis, glucose oxidation, and lactate production in fat cells. We also describe the novel findings that AICAR-induced AMPK phosphorylation significantly reduced palmitate (32%) and oleate uptake (41%), which was followed by a 50% reduction in palmitate oxidation despite a marked increase in AMPK and acetyl-CoA carboxylase phosphorylation. Compound C, a selective inhibitor of AMPK, not only completely prevented the inhibitory effect of AICAR on palmitate oxidation but actually caused a 2.2-fold increase in this variable. Compound C also significantly increased palmitate oxidation in the presence of inhibitory concentrations of malonyl-CoA and etomoxir indicating an increase in CPT1 activity. In contrast to skeletal muscle in which AMPK stimulates fatty acid oxidation to provide ATP as a fuel, we propose that AMPK activation inhibits lipogenesis and fatty acid oxidation in adipocytes. Inhibition of lipogenesis would conserve ATP under conditions of cellular stress, although suppression of intra-adipocyte oxidation would spare fatty acids for exportation to other tissues where their utilization is crucial for energy production. Additionally, the stimulatory effect of compound C on long chain fatty acid oxidation provides a novel pharmacological approach to promote energy dissipation in adipocytes, which may be of therapeutic importance for obesity and type II diabetes.     

Fatty acid metabolism in adipocytes: functional analysis of fatty acid transport proteins 1 and 4

The role of fatty acid transport protein 1 (FATP1) and FATP4 in facilitating adipocyte fatty acid metabolism was investigated using stable FATP1 or FATP4 knockdown (kd) 3T3-L1 cell lines derived from retrovirus-delivered short hairpin RNA (shRNA). Decreased expression of FATP1 or FATP4 did not affect preadipocyte differentiation or the expression of FATP1 (in FATP4 kd), FATP4 (in FATP1 kd), fatty acid translocase, acyl-coenzyme A synthetase 1, and adipocyte fatty acid binding protein but did lead to increased levels of peroxisome proliferator-activated receptor gamma and CCAAT/enhancer binding protein alpha. Both FATP1 and FATP4 kd adipocytes exhibited reduced triacylglycerol deposition and corresponding reductions in diacylglycerol and monoacylglycerol levels compared with control cells. FATP1 kd adipocytes displayed an approximately 25% reduction in basal (3)H-labeled fatty acid uptake and a complete loss of insulin-stimulated (3)H-labeled fatty acid uptake compared with control adipocytes. In contrast, FATP4 kd adipocytes as well as HEK-293 cells overexpressing FATP4 did not display any changes in fatty acid influx. FATP4 kd cells exhibited increased basal lipolysis, whereas FATP1 kd cells exhibited no change in lipolytic capacity. Consistent with reduced triacylglycerol accumulation, FATP1 and FATP4 kd adipocytes exhibited enhanced 2-deoxyglucose uptake compared with control adipocytes. These findings define unique and distinct roles for FATP1 and FATP4 in adipose fatty acid metabolism.     

Functional Analysis of Long-chain Acyl-CoA Synthetase 1 in 3T3-L1 Adipocytes

ACSL1 (acyl-CoA synthetase 1), the major acyl-CoA synthetase of adipocytes, has been proposed to function in adipocytes as mediating free fatty acid influx, esterification, and storage as triglyceride. To test this hypothesis, ACSL1 was stably silenced (knockdown (kd)) in 3T3-L1 cells, differentiated into adipocytes, and evaluated for changes in lipid metabolism. Surprisingly, ACSL1-silenced adipocytes exhibited no significant changes in basal or insulin-stimulated long-chain fatty acid uptake, lipid droplet size, or tri-, di-, or monoacylglycerol levels when compared with a control adipocyte line. However, ACSL1 kd adipocytes displayed a 7-fold increase in basal and a ∼15% increase in forskolin-stimulated fatty acid efflux without any change in glycerol release, indicating a role for the protein in fatty acid reesterification following lipolysis. Consistent with this proposition, ACSL1 kd cells exhibited a decrease in activation and phosphorylation of AMP-activated protein kinase and its primary substrate acetyl-CoA carboxylase. Moreover, ACSL1 kd adipocytes displayed an increase in phosphorylated protein kinase Cθ and phosphorylated JNK, attenuated insulin signaling, and a decrease in insulin-stimulated glucose uptake. These findings identify a primary role of ACSL1 in adipocytes not in control of lipid influx, as previously considered, but in lipid efflux and fatty acid-induced insulin resistance.Fatty acid influx and efflux mechanisms and their regulation affect lipid storage and metabolism in adipocytes. Imbalances in adipose lipid metabolism have been shown to significantly contribute to the development of obesity and associated metabolic diseases, such as type 2 diabetes, hypertension, and cardiovascular disease (1–3). Although the molecular mechanisms involved in fatty acid efflux are still undefined, several proteins implicated in fatty acid influx have been proposed: CD36 (fatty acid translocase), acyl-CoA synthetases (fatty acid transport protein (FATP)² and acyl-CoA synthetase (ACSL) family members), plasma membrane fatty acid-binding protein, and caveolin-1 (4–9).FATPs and long-chain ACSLs are membrane-bound enzymes that catalyze the ATP-dependent esterification of long chain (ACSL) and very long-chain (FATP) fatty acids to their acyl-CoA derivatives (10, 11). Both types of CoA synthetases have common ATP/AMP binding and fatty acid binding signature motifs. In mammals, six different isoforms of FATP (FATP1–FATP6) and five different isoforms of ACSL (ACSL1, -3, -4, -5, and -6) have been identified with tissue-specific expression patterns (12). White adipose tissue predominantly express FATP1, FATP4, and ACSL1, whereas brown adipose tissue in addition expresses ACSL5. Our recent results have confirmed a major role of FATP1 and CD36, but not FATP4, in insulin-stimulated LCFA uptake in 3T3-L1 adipocytes (6).ACSL1 is a ∼78-kDa intrinsic membrane protein localized to multiple sites in a variety of different cells. In liver, ACSL1 has been shown to be localized to the endoplasmic reticulum and mitochondria-associated membranes, whereas in adipocytes, ACSL1 was also found associated with the plasma membrane, the lipid droplet surface (13), and glucose transporter 4-containing vesicles (14, 15). Recent studies have postulated a cooperative role of FATP1 and ACSL1 in the movement of LCFAs across the plasma membrane via a process termed vectoral acylation (16), in which the CoA- and ATP-dependent esterification of internalized fatty acid provides the thermodynamic force necessary for net lipid influx. Evidence supporting this hypothesis came from a functional cloning strategy that identified mouse ACSL1 along with FATP1 as proteins involved in LCFA transport (17). In contrast to the role of ACSL1 in LCFA uptake and triglyceride synthesis in adipocytes, overexpression of ACSL1 in rat primary hepatocytes channeled fatty acids toward diacylglycerol and phospholipids synthesis and increased reacylation of hydrolyzed fatty acids into triglyceride (18).Since lipid flux is defined by the location and activity of its regulatory enzymes and proteins, overexpression strategies can result in changes in metabolism potentially distinct from the endogenous function. To that end, our laboratory has recently undertaken a gene silencing approach to the evaluation of proteins implicated in adipocyte fatty acid influx and efflux, and prior studies have focused on FATP1, FATP4, and CD36 (6). In this report, we evaluated the adipose-specific role(s) of ACSL1 using stable gene-silencing strategies in 3T3-L1 adipocytes using lentiviral delivery of shRNA. We report herein that, contrary to previous reports, in 3T3-L1 adipocytes, ACSL1 does not facilitate the basal or insulin-stimulated component of LCFA uptake. ACSL1 is, however, involved in the reesterification of hydrolyzed fatty acids released during basal and forskolin-stimulated lipolysis, thereby regulating their availability and efflux from the cell. Additionally, fatty acid reesterification by ACSL1 during lipolysis plays a major role in regulating the AMP-activated protein kinase (AMPK) as well as the PKCθ and JNK pathways leading to insulin resistance. Such findings bring to light a new interpretation of the role of ACSL1 and other acyl-CoA synthetases in the control of intermediary metabolism and lipid-mediated signal transduction.     

Fatty acid transport protein 1 and long-chain acyl coenzyme A synthetase 1 interact in adipocytes

The fatty acid transport proteins (FATP) and long-chain acyl coenzyme A synthetase (ACSL) proteins have been shown to play a role in facilitating long-chain fatty acid (LCFA) transport in mammalian cells under physiologic conditions. The involvement of both FATP and ACSL proteins is consistent with the model of vectorial acylation, in which fatty acid transport is coupled to esterification. This study was undertaken to determine whether the functions of these proteins are coordinated through a protein-protein interaction that might serve as a point of regulation for cellular fatty acid transport. We demonstrate for the first time that FATP1 and ACSL1 coimmunoprecipitate in 3T3-L1 adipocytes, indicating that these proteins form an oligomeric complex. The efficiency of FATP1 and ACSL1 coimmunoprecipitation is unaltered by acute insulin treatment, which stimulates fatty acid uptake, or by treatment with isoproterenol, which decreases fatty acid uptake and stimulates lipolysis. Moreover, inhibition of ACSL1 activity in adipocytes impairs fatty acid uptake, suggesting that esterification is essential for fatty acid transport. Together, our findings suggest that a constitutive interaction between FATP1 and ACSL1 contributes to the efficient cellular uptake of LCFAs in adipocytes through vectorial acylation.     

Overexpressed FATP1, ACSVL4/FATP4 and ACSL1 Increase the Cellular Fatty Acid Uptake of 3T3-L1 Adipocytes but Are Localized on Intracellular Membranes

Long chain acyl-CoA synthetases are essential enzymes of lipid metabolism, and have also been implicated in the cellular uptake of fatty acids. It is controversial if some or all of these enzymes have an additional function as fatty acid transporters at the plasma membrane. The most abundant acyl-CoA synthetases in adipocytes are FATP1, ACSVL4/FATP4 and ACSL1. Previous studies have suggested that they increase fatty acid uptake by direct transport across the plasma membrane. Here, we used a gain-of-function approach and established FATP1, ACSVL4/FATP4 and ACSL1 stably expressing 3T3-L1 adipocytes by retroviral transduction. All overexpressing cell lines showed increased acyl-CoA synthetase activity and fatty acid uptake. FATP1 and ACSVL4/FATP4 localized to the endoplasmic reticulum by confocal microscopy and subcellular fractionation whereas ACSL1 was found on mitochondria. Insulin increased fatty acid uptake but without changing the localization of FATP1 or ACSVL4/FATP4. We conclude that overexpressed acyl-CoA synthetases are able to facilitate fatty acid uptake in 3T3-L1 adipocytes. The intracellular localization of FATP1, ACSVL4/FATP4 and ACSL1 indicates that this is an indirect effect. We suggest that metabolic trapping is the mechanism behind the influence of acyl-CoA synthetases on cellular fatty acid uptake.     

Eicosapentaenoic acid increases lipolysis through up-regulation of the lipolytic gene expression and down-regulation of the adipogenic gene expression in 3T3-L1 adipocytes

In this study, we investigated the lipolytic effects of eicosapentaenoic acid (EPA) in 3T3-L1 adipocytes. The differentiated 3T3-L1 adipocytes were treated in a serum-free medium with 300 muM of EPA for 3, 6, 12, and 24 h. In comparison with the control, intracellular lipid accumulation was significantly decreased by 24% at 24 h following the addition of EPA (P < 0.05). Under the same experimental conditions, there was an increase of glycerol and free fatty acids (FFAs). The mRNA level of carnitine palmitoyltransferase I-a, a component of the fatty-acid shuttle system involved in the mitochondrial oxidation of long-chain fatty acids, was also significantly elevated by EPA (P < 0.05). However, the expression of peroxisome proliferator-activated receptor-gamma and acetyl-CoA carboxylase (ACC), which are involved in adipogenesis, was significantly down-regulated by EPA (P < 0.05). These results suggest that EPA may modulate lipid metabolism by stimulation of lipolysis, which was associated with induction of lipolytic gene expression and suppression of adipogenic gene expression in 3T3-L1 adipocytes.     

Covalent activation of heart AMP-activated protein kinase in response to physiological concentrations of long-chain fatty acids.

Rat hearts were perfused for 1 h with 5 mm glucose with or without palmitate or oleate at concentrations characteristic of the fasting state. The inclusion of fatty acids resulted in increased activities of the alpha-1 or the alpha-2 isoforms of AMP-activated protein kinase (AMPK), increased phosphorylation of acetyl-CoA carboxylase and a decrease in the tissue content of malonyl-CoA. Activation of AMPK was not accompanied by any changes in the tissue contents of ATP, ADP, AMP, phosphocreatine or creatine. Palmitate increased phosphorylation of Thr172 within AMPK alpha-subunits and the activation by palmitate of both AMPK isoforms was abolished by protein phosphatase 2C leading to the conclusion that exposure to fatty acid caused activation of an AMPK kinase or inhibition of an AMPK phosphatase. In vivo, 24 h of starvation also increased heart AMPK activity and Thr172 phosphorylation of AMPK alpha-subunits. Perfusion with insulin decreased both alpha-1 and alpha-2 AMPK activities and increased malonyl-CoA content. Palmitate prevented both of these effects. Perfusion with epinephrine decreased malonyl-CoA content without an effect on AMPK activity but prevented the activation of AMPK by palmitate. The concept is discussed that activation of AMPK by an unknown fatty acid-driven signalling process provides a mechanism for a 'feed-forward' activation of fatty acid oxidation.     

Berberine attenuates cAMP-induced lipolysis via reducing the inhibition of phosphodiesterase in 3T3-L1 adipocytes

Berberine, a hypoglycemic agent, has been shown to decrease plasma free fatty acids (FFAs) level in insulin-resistant rats. In the present study, we explored the mechanism responsible for the antilipolytic effect of berberine in 3T3-L1 adipocytes. It was shown that berberine attenuated lipolysis induced by catecholamines, cAMP-raising agents, and a hydrolyzable cAMP analog, but not by tumor necrosis factor α and a nonhydrolyzable cAMP analog. Unlike insulin, the inhibitory effect of berberine on lipolysis in response to isoproterenol was not abrogated by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, but additive to that of PD98059, an extracellular signal-regulated kinase kinase inhibitor. Prior exposure of adipocytes to berberine decreased the intracellular cAMP production induced by isoproterenol, forskolin, and 3-isobutyl-1-methylxanthine (IBMX), along with hormone-sensitive lipase (HSL) Ser-563 and Ser-660 dephosphorylation, but had no effect on perilipin phosphorylation. Berberine stimulated HSL Ser-565 as well as adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. However, compound C, an AMPK inhibitor, did not reverse the regulatory effect of berberine on HSL Ser-563, Ser-660, and Ser-565 phosphorylation, nor the antilipolytic effect of berberine. Knockdown of AMPK using RNA interference also failed to restore berberine-suppressed lipolysis. cAMP-raising agents increased AMPK activity, which was not additive to that of berberine. Stimulation of adipocytes with berberine increased phosphodiesterase (PDE) 3B and PDE4 activity measured by hydrolysis of ³[H]cAMP. These results suggest that berberine exerts an antilipolytic effect mainly by reducing the inhibition of PDE, leading to a decrease in cAMP and HSL phosphorylation independent of AMPK pathway.     

Fatty acids revert the inhibition of respiration caused by the antidiabetic drug metformin to facilitate their mitochondrial β-oxidation

While metformin has been widely used to treat type 2 diabetes for the last fifty years, its mode of action remains unclear. Hence, we investigated the short-term alterations in energy metabolism caused by metformin administration in 3T3-L1 adipocytes. We found that metformin inhibited mitochondrial respiration, although ATP levels remained constant as the decrease in mitochondrial production was compensated by an increase in glycolysis. While AMP/ATP ratios were unaffected by metformin, phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase augmented. The inhibition of respiration provoked a rapid and sustained increase in superoxide levels, despite the increase in UCP2 and superoxide dismutase activity. The inhibition of respiration was rapidly reversed by fatty acids and thus respiration was lower in treated cells in the presence of pyruvate and glucose while rates were identical to control cells when palmitate was the substrate. We conclude that metformin reversibly inhibits mitochondrial respiration, it rapidly activates AMPK without altering the energy charge, and it inhibits fatty acid synthesis. Mitochondrial β-oxidation is facilitated by reversing the inhibition of complex I and, presumably, by releasing the inhibition of carnitine palmitoyltransferase. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Fargesin improves lipid and glucose metabolism in 3T3-L1 adipocytes and high-fat diet-induced obese mice

This study examined the effects of fargesin, a neolignan isolated from Magnolia plants, on obesity and insulin resistance and the possible mechanisms involved in these effects in 3T3-L1 adipocytes and high-fat diet (HFD)-induced obese mice. Fargesin promoted the glucose uptake in 3T3-L1 adipocytes. In HFD-induced obese mice, fargesin decreased the body weight gain, white adipose tissue (WAT), and plasma triglyceride, non-esterified fatty acid and glucose levels, and improved the glucose tolerance. Fargesin increased glucose transporter 4 (GLUT4) protein expression and phosphorylation of Akt, AMP-activated protein kinase (AMPK), and acetyl-CoA carboxylase (ACC) in both 3T3-L1 adipocytes and WAT of HFD-induced obese mice. Fargesin also decreased the mRNA expression levels of fatty acid oxidation-related genes, such as peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase-1 (CPT-1), uncoupling protein-2 (UCP-2) and leptin in WAT. Taken together, the present findings suggest that fargesin improves dyslipidemia and hyperglycemia by activating Akt and AMPK in WAT. ? 2012 International Union of Biochemistry and Molecular Biology, Inc.     

Intracellular fatty acid downregulates ob gene expression in 3T3-L1 adipocytes

The effect of intracellular free fatty acid (FFA) accumulation on ob gene expression in adipocytes was examined. In fully differentiated 3T3-L1 adipocytes, triacsin C, a specific acyl CoA synthetase inhibitor with a K(i) of 8.97 microM, inhibited ob gene expression by 20% at 5 x 10(-5)M. At this concentration, triacsin C induced accumulation of intracellular FFA. Treatment with both chylomicron and triacsin C reduced ob gene expression more than treatment with triacsin C alone. Treatment with 2-bromopalmitate, a poorly metabolizable palmitate analog, reduced ob gene expression by 50% at 10(-4)M, but palmitate at the same concentration had no effect. This is the first demonstration that the ob gene is downregulated by intracellular FFA accumulation, thereby raising the possibility that ob product is regulated in response to lipolysis.     

TR4 promotes fatty acid synthesis in 3T3-L1 adipocytes by activation of pyruvate carboxylase expression

We show that testicular orphan nuclear receptor 4 (TR4) increases the expression of pyruvate carboxylase (PC) gene in 3T3-L1 adipocytes by direct binding to a TR4 responsive element in the murine PC promoter. While TR4 overexpression increased PC activity, oxaloacetate (OAA) and glycerol levels with enhanced incorporation of ¹⁴C from ¹⁴C-pyruvate into fatty acids in 3T3-L1 adipocytes, PC knockdown by short interfering RNA (siRNA) or inhibition of PC activity by phenylacetic acid (PAA) abolished TR4-enhanced fatty acid synthesis. Moreover, TR4 microRNA reduced PC expression with decreased fatty acid synthesis in 3T3-L1 adipocytes, suggesting that TR4-mediated enhancement of fatty acid synthesis in adipocytes requires increased expression of PC gene.     

Differential effects of palmitate on glucose uptake in rat-1 fibroblasts and 3T3-L1 adipocytes.

Non-esterified fatty acids are thought to be one of the causes for insulin resistance. However, the molecular mechanism of fatty acid-induced insulin resistance is not clearly known. In this study, we first examined the effect of palmitate on insulin signaling in 3T3-L1 adipocytes. We found that 1h treatment with 1 mmol/l palmitate had no effect on insulin binding, tyrosine phosphorylation of insulin receptors, 185 kDa proteins and Shc, and PI3 kinase activity in 3T3-L1 adipocytes. Then, the effects of palmitate on MAP kinase activity and glucose uptake in fully differentiated 3T3-L1 adipocytes were compared with those in poorly differentiated 3T3-L1 cells and in HIRc-B cells. Palmitate treatment had no effect on MAP kinase activity in fully differentiated 3T3-L1 adipocytes, while it inhibited MAP kinase in poorly differentiated 3T3-L1 cells and HIRc-B cells. Glucose transport in 3T3-L1 adipocytes treated with palmitate for 1 h, 4 h and 16 h was higher than that in control cells, but palmitate treatment caused a rightward shift of the insulin-dose responsive curve for glucose uptake in HIRc-B cells. Palmitate treatment did not significantly affect basal and insulin-stimulated GLUT4 translocation. When the cells were treated with PD98059, a specific MEK inhibitor, insulin-stimulated glucose uptake was not affected in 3T3-L1 adipocytes, while it was almost completely inhibited in HIRc-B cells. These results suggest the primary effect of palmitate on adipocytes may not involve insulin resistance of adipocytes themselves.     

Combined metadoxine and garlic oil treatment efficaciously abrogates alcoholic steatosis and CYP2E1 induction in rat liver with restoration of AMPK activity

Alcoholic steatosis is the earliest and most common response to heavy alcohol intake, and may precede more severe forms of liver injury. Accumulation of fat, largely triglyceride, in hepatocytes results from the inhibition of fatty acid oxidation and excessive oxidative stress involving CYP2E1. This study evaluated the therapeutic effects of metadoxine, garlic oil or their combination on alcoholic steatosis. Feeding rats an alcohol-containing diet for 4 weeks elicited an increase in hepatic triglyceride content and induced CYP2E1. The concurrent administration of metadoxine and garlic oil (MG) to rats during the last week of the diet feeding efficaciously abrogated both fat accumulation and CYP2E1 induction as compared to the individual treatment at higher doses. Histopathology confirmed the ability of MG combination to inhibit lipid accumulation. Blood biochemistry verified improvement of liver function in rats treated with MG. Alcohol administration resulted in a decrease in AMP-activated protein kinase-alpha (AMPKalpha) phosphorylation, which was restored by MG treatments. Recovery of AMPK activity by MG was supported by an increase in acetyl-CoA carboxylase phosphorylation. Hepatic fatty acid synthase (FAS) expression was markedly decreased after alcohol consumption, which correlated with a decrease in AMPK activity and a commensurate increase in lipid content. Combined MG treatments caused restoration of the FAS level. These results demonstrate that the combination of MG effectively treats alcoholic steatosis with CYP2E1 inhibition, which may be associated with the recovery of AMPK activity, promising that the combination therapy may constitute an advance in the development of clinical candidates for alcoholic steatosis.     

Regulation of AMP-activated protein kinase and acetyl-CoA carboxylase phosphorylation by palmitate in skeletal muscle cells

The purpose of this study was to investigate the effects of long-chain fatty acids (LCFAs) on AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) phosphorylation and beta-oxidation in skeletal muscle. L6 rat skeletal muscle cells were exposed to various concentrations of palmitate (1-800 microM). Subsequently, ACC and AMPK phosphorylation and fatty acid oxidation were measured. A 2-fold increase in both AMPK and ACC phosphorylation was observed in the presence of palmitate concentrations as low as 10 microM, which was also accompanied by a significant increase in fatty acid oxidation. The effect of palmitate on AMPK and ACC phosphorylation was dose-dependent, reaching maximum increases of 3.5- and 4.5-fold, respectively. Interestingly, ACC phosphorylation was coupled with AMPK activation at palmitate concentrations ranging from 10 to 100 microM; however, at concentrations >200 microM, ACC phosphorylation and fatty acid oxidation remained high even after AMPK phosphorylation was completely prevented by the use of a selective AMPK inhibitor. This indicates that LCFAs regulate ACC activity by AMPK-dependent and -independent mechanisms, based on their abundance in skeletal muscle cells. Here, we provide novel evidence that the AMPK/ACC pathway may operate as a mechanism to sense and respond to the lipid energy charge of skeletal muscle cells.     

Gelatin enhances 2-keto-L-gulonic acid production based on Ketogulonigenium vulgare genome annotation

In the two-step fermentative production of vitamin C, its precursor 2-keto-l-gulonic acid (2-KLG) was synthesized by Ketogulonicigenium vulgare through co-culture with Bacillus megaterium. The reconstruction of the amino acid metabolic pathway through completed genome sequence annotation demonstrated that K. vulgare was deficient in one or more key enzymes in the de novo biosynthesis pathways of eight different amino acids (l-histidine, l-glycine, l-lysine, l-proline, l-threonine, l-methionine, l-leucine, and l-isoleucine). Among them, l-glycine, l-proline, l-threonine, and l-isoleucine play vital roles in K. vulgare growth and 2-KLG production. The addition of those amino acids increased the 2-KLG productivity by 20.4%, 17.2%, 17.2%, and 11.8%, respectively. Furthermore, food grade gelatin was developed as a substitute for the amino acids to increase the cell concentration, 2-KLG productivity, and l-sorbose consumption rate by 10.2%, 23.4%, and 20.9%, respectively. As a result, the fermentation period decreased to 43 h in a 7-L fermentor.