Acetyl-coenzyme A Carboxylase Phosphorylation Research Articles

Medium- and long-chain triglyceride propofol reduces the activity of acetyl-coenzyme A carboxylase in hepatic lipid metabolism in HepG2 and Huh7 cells.

Medium- and long-chain triglyceride (MCT/LCT) propofol is widely used as an intravenous anesthetic, especially in the intensive care unit. The present study aimed to assess whether MCT/LCT propofol is safe in the hyperlipidemic population for long-term use. Free fatty acids (FFAs) were used to establish high-fat stimulation of HepG2 and Huh7 cells. Subsequently, these cells were treated with propofol at the concentration of 0, 4, or 8 µg/ml for 24 and 48 h. The results indicated that the cell viability was notably decreased when the cells were stimulated with 2 mmol/L FFAs and treated with 12 µg/ml MCT/LCT propofol. Accordingly, we chose 2 mmol/L FFAs along with 4 and 8 µg/ml MCT/LCT propofol for the subsequent experiments. Four and 8 µg/ml MCT/LCT propofol inhibited FFA-induced lipid accumulation in the cells and significantly reversed acetyl coenzyme A carboxylase (ACC) activity. In addition, MCT/LCT propofol not only significantly promoted the phosphorylation of AMPK and ACC, but also reversed the FFA-induced decreased phosphorylation of AMPK and ACC. In conclusion, MCT/LCT propofol reverses the negative effects caused by FFAs in HepG2 and Huh7 cells, indicating that MCT/LCT propofol might positively regulate lipid metabolism.

A novel gravity-induced blood flow restriction model augments ACC phosphorylation and PGC-1α mRNA in human skeletal muscle following aerobic exercise: a randomized crossover study.

This study tested the hypothesis that a novel, gravity-induced blood flow restricted (BFR) aerobic exercise (AE) model will result in greater activation of the AMPK-PGC-1α pathway compared with work rate-matched non-BFR. Thirteen healthy males (age: 22.4 ± 3.0 years; peak oxygen uptake: 42.4 ± 7.3 mL/(kg·min)) completed two 30-min work rate-matched bouts of cycling performed with their legs below (CTL) and above their heart (BFR) at ∼2 weeks apart. Muscle biopsies were taken before, immediately, and 3 h after exercise. Blood was drawn before and immediately after exercise. Our novel gravity-induced BFR model led to less muscle oxygenation during BFR compared with CTL (O2Hb: p = 0.01; HHb: p < 0.01) and no difference in muscle activation (p = 0.53). Plasma epinephrine increased following both BFR and CTL (p < 0.01); however, only norepinephrine increased more following BFR (p < 0.01). PGC-1α messenger RNA (mRNA) increased more following BFR (∼6-fold) compared with CTL (∼4-fold; p = 0.036). VEGFA mRNA increased (p < 0.01) similarly following BFR and CTL (p = 0.21), and HIF-1α mRNA did not increase following either condition (p = 0.21). Phosphorylated acetyl-coenzyme A carboxylase (ACC) increased more following BFR (p < 0.035) whereas p-PKA substrates, p-p38 MAPK, and acetyl-p53 increased (p < 0.05) similarly following both conditions (p > 0.05). In conclusion, gravity-induced BFR is a viable BFR model that demonstrated an important role of AMPK signalling on augmenting PGC-1α mRNA. Novelty Gravity-induced BFR AE reduced muscle oxygenation without impacting muscle activation, advancing gravity-induced BFR as a simple, inexpensive BFR model. Gravity-induced BFR increased PGC-1α mRNA and ACC phosphorylation more than work rate-matched non-BFR AE. This is the first BFR AE study to concurrently measure blood catecholamines, muscle activation, and muscle oxygenation.

Melinjo (Gnetum gnemon) extract intake during lactation stimulates hepatic AMP-activated protein kinase in offspring of excessive fructose-fed pregnant rats

Excessive maternal fructose intake during pregnancy and in early postnatal life has metabolic consequences for the offspring. We investigated the effects of melinjo (Gnetum gnemon) extract (MeE) intake during lactation on the expression and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) in the liver of offspring from excessive fructose-fed pregnant dams. Pregnant Wistar rats received a normal diet and 100g/L fructose solution during gestation ad libitum. At delivery, dams were divided into two groups: a control diet (FC) or a 0.1% MeE-containing diet (FM) fed during lactation. The dams that were not treated with fructose were fed a control diet (CC). At postnatal week 3, some pups were sacrificed, while the remaining continued to receive a normal diet and were sacrificed at week 17. Blood chemistry and phosphorylation levels of AMPK and acetyl-coenzyme A carboxylase (ACC) were evaluated. Plasma glucose levels in FC female offspring increased compared to that receiving CC at weeks 3 and 17; however, the levels in FM female offspring decreased at week 17. The insulin levels in FM female offspring decreased significantly compared to that in FC female offspring at week 3. Hepatic AMPK phosphorylation was upregulated in FM offspring at week 3 and in female, but not male, offspring at week 17. ACC phosphorylation in FM female offspring was upregulated at week 17. Our results suggest that maternal MeE intake during lactation may modulate the hepatic AMPK pathways in female offspring.

Adipokines in Healthy Skeletal Muscle and Metabolic Disease.

Adipose tissue not only functions as a reserve to store energy but has become of major interest as an endocrine organ, releasing signalling molecules termed adipokines which impact on other tissues, such as skeletal muscle. Adipocytes, within skeletal muscle and adipose tissue, secrete adipokines to finely maintain the balance between feed intake and energy expenditure. This book chapter focuses on the three adipokines, adiponectin, leptin and IL-6, which have potent effects on skeletal muscle during rest and exercise. Similarly, adiponectin, leptin and IL-6 enhance glucose uptake and increase fatty acid oxidation in skeletal muscle. Fatty acid oxidation is increased through activation of AMPK (adenosine monophosphate-activated protein kinase signalling) causing phosphorylation and inhibition of ACC (acetyl-coenzyme A carboxylase), decreasing availability of malonyl CoA. Leptin and adiponectin also control feed intake via AMPK signalling in the hypothalamus. Adipokines function to maintain energy homeostasis, however, when feed intake exceeds energy expenditure adipokines can become dysregulated causing lipotoxicity in skeletal muscle and metabolic disease can prevail. Cross-talk between adipocytes and skeletal muscle via correct control by adipokines is important in controlling energy homeostasis during rest and exercise and can help prevent metabolic disease.

녹차씨 압착박 열수 추출물의 지방세포 분화 억제 효과

The effect of the hot water extract of defatted green tea seed cake (GTSE) on lipid metabolism and the underlying mechanisms of lipolysis in mature 3T3-L1 adipocytes were investigated. In this study, we found that the naringenin content of GTSE was 5.5 ㎎/g; however, catechins were not detected. The intracellular lipid droplets were stained with Oil Red O dye and quantified. Compared to the control, lipid accumulation was significantly decreased by 52%, and intracellular triglyceride (TG) level was reduced by 33% after treatment with GTSE at a concentration of 40 ㎍/mL. To determine the mechanism of reduction in TG content, we determined the level of fatty acid synthase (FAS), phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), and acetyl-coenzyme A carboxylase (ACC) in the cell model. Incubation of the 3T3-L1 adipocytes with GTSE stimulated AMPK and ACC phosphorylation in a dosedependent manner, and decreased the expression of FAS.

Estrogen Activates AMP-Activated Protein Kinase in Human Endothelial Cells via ERβ/Ca(2+)/Calmodulin-Dependent Protein Kinase Kinase β Pathway.

Our previous studies suggested that Estrogen inhibits cytokine-induced expression of VCAM-1 and ICAM-1 in cultured human endothelial cells via AMP-activated protein kinase (AMPK) activation. Here, we sought to delineate the mechanisms underlying estrogen activation of AMPK. AMPK can be considered a 'fuel gauge' of cellular energy status in response to metabolic stress. It is controlled by upstream kinases such as Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) or LKB1. The present study of human endothelial cells demonstrates that AMPK is activated by estradiol (E2) through a Ca(2+)-dependent mechanism involving the estrogen receptor-β (ERβ) activation. Inhibition of CaMKK with STO-609, a specific inhibitor of CaMKKα and CaMKKβ, attenuated E2-induced AMPK activation, suggesting that CaMKKβ was the responsible AMPK kinase. Conversely, down-regulation of LKB1 did not affect E2-induced AMPK activation. E2 stimulation caused phosphorylation of acetyl coenzyme A carboxylase (ACC) and endothelial nitric oxide synthase (eNOS), two main targets of AMPK. Inhibition or down-regulation of CaMKKβ eliminated phosphorylation of ACC and eNOS in response to E2. Together, our data highlight the role of Ca(2+) as a regulator of AMPK activation in response to E2 stimulation. We demonstrate that E2 activates AMPK via an ERβ/Ca(2+)/CaMKKβ-dependent pathway in endothelial cells.

Aspirin regulates hepatocellular lipid metabolism by activating AMPK signaling pathway.

Aspirin has been reported to regulate lipid metabolism. However, the mechanism underlying the regulation is not clear. We presently investigated aspirin's promotion of AMP-activated protein kinase (AMPK) pathway activation in human hepatoma HepG2 cells by examining AMPK expression, the promotion of AMPK activation. Then we investigated the influence of aspirin-promoted AMPK signaling on fatty acid oxidation in HepG2 cells. The results demonstrated that aspirin treatment did not regulate the expression of AMPK and its downstream target, Acetyl-Coenzyme A Carboxylase (ACC), but activated the AMPK signaling pathway by promoting the phosphorylation of AMPK and ACC. And, interestingly, the promotion by aspirin is dependent of cellular esterase, which catalyzes aspirin to salicylate. Moreover, the activated AMPK signaling promoted the fatty acid oxidation, by promoting expression of Carnitine palmitoyltransferase I (CPT1) and Medium-Chain Acyl-CoA Dehydrogenase (MCAD) in both mRNA and protein levels. Thus, we confirmed in this study that aspirin promoted lipid oxidation by upregulating the AMPK signaling pathway.

AMPK-Dependent Inhibitory Phosphorylation of ACC Is Not Essential for Maintaining Myocardial Fatty Acid Oxidation

The energy sensor AMP-activated protein kinases (AMPK) is thought to play an important role in regulating myocardial fatty acid oxidation (FAO) via its phosphorylation and inactivation of acetyl coenzyme A carboxylase (ACC). However, studies supporting this have not directly assessed whether the maintenance of FAO rates and subsequent cardiac function requires AMPK-dependent inhibitory phosphorylation of ACC. To determine whether preventing AMPK-mediated inactivation of ACC influences myocardial FAO or function. A double knock-in (DKI) mouse (ACC-DKI) model was generated in which the AMPK phosphorylation sites Ser79 on ACC1 and Ser221 (Ser212 mouse) on ACC2 were mutated to prevent AMPK-dependent inhibitory phosphorylation of ACC. Hearts from ACC-DKI mice displayed a complete loss of ACC phosphorylation at the AMPK phosphorylation sites. Despite the inability of AMPK to regulate ACC activity, hearts from ACC-DKI mice displayed normal basal AMPK activation and cardiac function at both standard and elevated workloads. In agreement with the inability of AMPK in hearts from ACC-DKI mice to phosphorylate and inhibit ACC, there was a significant increase in cardiac malonyl-CoA content compared with wild-type mice. However, cardiac FAO rates were comparable between wild-type and ACC-DKI mice at baseline, during elevated workloads, and after a more stressful condition of myocardial ischemia that is known to robustly activate AMPK. Our findings show AMPK-dependent inactivation of ACC is not essential for the control of myocardial FAO and subsequent cardiac function during a variety of conditions involving AMPK-independent and AMPK-dependent metabolic adaptations.

Differential effects of estrogen/androgen on the prevention of nonalcoholic fatty liver disease in the male rat

It is important to clarify the distinct contributions of estrogen/estrogen receptor (ER) and androgen/androgen receptor (AR) signaling and their reciprocal effects on the regulation of hepatic lipid homeostasis. We studied the molecular mechanisms underlying the preventive effects of estradiol (E2), dihydrotestosterone (DHT), or E2+DHT on high-fat diet-induced nonalcoholic fatty liver disease (NAFLD) in an orchidectomized Sprague-Dawley (SD) rat model. E2 is shown to be associated with decreased fatty acid synthesis in hepatic zone 3-specific manner by increasing the phosphorylation of acetyl coenzyme-A carboxylase via an ERα-mediated pathway. DHT is shown to be associated with decreased lipid accumulation and cholesterol synthesis in a hepatic zone 1-specific manner by increasing expression of carnitine palmitotyltransferase1 and phosphorylation of 3-hydroxy-3-methyl-glutaryl-CoA reductase via an AR-mediated pathway. E2+DHT showed an additive positive effect and normalized all three impaired zones of the liver. Gene expression changes in human severe liver steatosis were similar to those of experimental rat NAFLD. Steroids reversed the histopathological NAFLD changes, likely by decreasing fatty acid and cholesterol synthesis and increasing β-oxidation. The diverse steroid effects (ER/AR) on NAFLD prevention in male rats indicate the potential applicability of ER/AR modulators for NAFLD treatment.

Azuki bean polyphenols intake during lactation upregulate AMPK in male rat offspring exposed to fetal malnutrition

ObjectiveFetal malnutrition is an early-life inducer of dyslipidemia and glucose intolerance. The aim of this study was to examine whether maternal azuki bean (Vigna angularis) polyphenol (AP) intake during lactation affects the adenosine monophosphate–activated protein kinase (AMPK) pathway and lipid metabolism in offspring exposed to fetal malnutrition. MethodsPregnant Wistar rats were divided into three groups: a control diet offered during gestation and lactation (CC), a low-protein diet during gestation and a control diet during lactation (LPC); and a low-protein diet during gestation and a 1.0% AP-containing control diet during lactation (LPAP). Male pups were randomly selected for the study; half the pups were sacrificed at 3 wk of age and the other half were fed a standard diet and sacrificed at 23 wk. Hepatic triacylglycerol levels, phosphorylation levels of AMPK and acetyl-coenzyme A carboxylase (ACC), and mRNA levels of sterol regulatory element-binding protein-1c (SREBP-1c) were evaluated. ResultsSignificant decreases in body weights and hepatic triacylglycerol levels were found in the LPAP compared with the LPC group. Plasma adiponectin levels in the LPAP group were higher than those in the LPC group. AMPK phosphorylation was upregulated in the livers and skeletal muscles in young and adult LPAP compared with LPC rats. ACC phosphorylation was upregulated in skeletal muscles of LPAP rats. SREBP-1c mRNA expression was decreased in the livers of LPAP rats. ConclusionOur results suggest that maternal AP intake during lactation upregulates AMPK phosphorylation not only in young but also in adult offspring exposed to fetal malnutrition and may lead to decreased hepatic lipid accumulation by ACC phosphorylation and downregulation of SREBP-1c expression.

Men Have Greater Basal Skeletal Muscle Ampk Phosphorylation Than Women

AMP-activated protein kinase (AMPK) plays a central role in the regulation of cellular metabolism. It is activated when the energy charge of the cell is low. Acetyl-coenzyme A carboxylase (ACC) is a downstream target for AMPK wich produces its phosphorylation and inhibition, leading to reduced malonyl-coenzyme A and increased fatty acid transport into the mitochondria. Since women have greater leptin levels and higher capacity to oxide fat during exercise we hypothesized that women will have greater basal skeletal muscle AMPK and ACC phosphorylation compared to men. PURPOSE. To determine if there is an influence of fiber type and gender in skeletal muscle basal phosphorylation of AMPK and ACC. METHODS. Muscle biopsies were obtained in twenty five healthy men and nine women (age: 24.1 ± 3.3, 25.3 ± 4 yrs, and weight: 78.6 ± 8.8, 56.9 ± 6.7 kg, respectively). Protein expression of AMPK and ACC was measured by Western Blot. MHC distribution was determined by SDS-PAGE electrophoresis. RESULTS. Thr172-AMPKα phosphorylation was 2.4-fold higher in men compared to women (2.33 ± 1.4 and 0.99 ± 0.5 a.u., respectively, P < 0.01). However, ACC phosphorylation was similar in both genders (P=0.8). There was a significant positive association between type I MHC and basal Thr172-AMPKα phosphorylation (r=0.54; P<0.01), but only in men. CONCLUSION. Skeletal muscle basal AMPKα phosphorylation is higher in men compared to women, but both sexes have similar basal ACC phosphorylation. This could be in part explained by the lower percentage of type II muscle fibres in women compared to men. Our results suggest that other mechanisms in addition to AMPK regulate the basal ACC phosphorylation state in human skeletal muscle. Granted by ULPGC2009-07, INNOVA2020, proyecto 2010/01RM Cátedra del Real Madrid de la UEM.

Abstract 3576: Lapatinib in combination with doxorubicin is highly damaging to cardiac myocytes

Abstract Lapatinib (Tykerb) is dual tyrosine kinase inhibitor targeting the HER2 (ERBB2) and EGFR (ERBB1, HER1) pathways. It is used to treat ER+/EGFR+/HER2+ breast cancer patients and patients who have HER2-positive advanced breast cancer that has progressed after previous treatment with other chemotherapeutic agents. Lapatinib, which was initially rated as having no cardiotoxicity, has recently been shown in a clinical trial to produce low levels of cardiotoxicity that is reversible. Using a neonatal rat cardiac myocyte model we have investigated the ability of lapatinib alone and in combination with doxorubicin to damage myocytes. Lapatinib alone at pharmacological concentrations did not significantly cause myocyte damage. However, doxorubicin-induced myocyte damage was greatly increased by the addition of low nanomolar lapatinib concentrations. Previous clinical studies have shown that the combination of trastuzumab (Herceptin) and doxorubicin was highly cardiotoxic. Because trastuzumab also targets HER2 receptors, the lapatinib/doxorubicin combination provides a good model to probe the mechanism of this cardiotoxicity using a rat myocyte model. Lapatinib treatment alone had little or no effect on myocyte mitochondrial membrane potential, on oxidation of intracellular DCF (dichlorofluorescein), on intracellular ATP levels or on caspase 3/7 induction. The doxorubicin cardioprotective agent dexrazoxane was able to greatly reduce doxorubicin-induced myocyte damage. However, dexrazoxane was not able to reduce the increase in myocyte damage produced by adding lapatinib to the doxorubicin treatment. The effect of lapatinib on the change in phosphorylation of ACC (acetyl-coenzyme A carboxylase), which is a measure of a change in cellular energy status, was also determined. Doxorubicin-induced growth inhibitory effects on K562 cells, which contain very low levels of ERBB1/2 protein, were not affected by the addition of lapatinib. This result suggests that inhibition of ERBB1/2 is required for the synergistic toxic effects seen with doxorubicin in myocytes. In conclusion these results suggest that combination therapy employing doxorubicin and lapatinib may produce increased cardiotoxicity. Support: CIHR and a Canada Research Chair in Drug Development to BBH. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3576. doi:10.1158/1538-7445.AM2011-3576

Activation of acetyl-coenzyme A carboxylase is involved in Taxol-induced ovarian cancer cell death

Acetyl-coenzyme A carboxylase (ACC) is an attractive target for research into the treatment of a variety of human diseases, including diabetes, obesity and cancer. Mounting evidence suggests that the inhibition of ACC induced of cancer cell apoptosis. However, whether the inhibition of ACC regulates apoptosis in CaOV3 cancer cells has yet to be addressed. This study investigated the cytotoxic mechanism of action of ACC inhibition. Results showed that 5-(tetradecyloxy)-2-furoic acid (TOFA), an ACC inhibitor, enhanced Taxol-induced CaOV3 human ovarian cancer cell apoptosis. Notably, when TOFA was administered as a monotherapy, it induced CaOV3 cell apoptosis. Pre-treatment with the EGFR inhibitor PD153035 was found to markedly enhance ACC phosphorylation, whereas AMP-activated protein kinase (AMPK) activator AICAR was found to marginally enhance ACC phosphorylation. Taken together, the data showed ACC is a potential novel molecular target of Taxol. Additionally, ACC inhibition partially contributed to the cytotoxic effect of Taxol in ovarian cancer cells.

SIRT3 reduces lipid accumulation via AMPK activation in human hepatic cells

Sirtuin 3 (SIRT3) is a nicotinamide adenine dinucleotide (NAD)(+)-dependent protein deacetylase localized on mitochondria and regulates the adaptive thermogenesis in brown adipocytes. This study aims to investigate the role of SIRT3 in hepatic lipid accumulation, and whether the activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) is required. A retroviral system was used for overexpressing of SIRT3 in HepG2 cells, whereas a lentivirus-mediated vector encoding SIRT3 small interfering RNA (siRNA) was used to infect these cells for knocking down endogenous SIRT3 expression. The cells were treated with oleate to induce lipid accumulation and Nile red staining was used to assess the number of lipid droplets in HepG2 cells. The AMPK signaling pathway was facilitated with the administrating of isoproterenol and an immunoblot analysis was performed to assess the phosphorylation of AMPK and acetyl coenzyme A carboxylase (ACC). Compound C was adopted to inhibit AMPK activity. The number of lipid droplets in HepG2 cells overexpressing SIRT3 was significantly lower than that in the control cells (P < 0.05). SIRT3-infected cells exhibited significantly more phosphorylation of AMPK and ACC (P < 0.05), which was reversed by the treatment of compound C, an inhibitor of AMPK. Knocking down SIRT3 downregulated phosphorylation of AMPK and ACC by 60-80% (P < 0.05) and promoted lipid accumulation. The activation of AMPK by SIRT3 was dependent on SIRT3 deacetylase activity. SIRT3 reduces lipid accumulation via AMPK activation in human hepatic cells.

Cessation of daily exercise dramatically alters precursors of hepatic steatosis in Otsuka Long‐Evans Tokushima Fatty (OLETF) rats

The purpose of this study was to delineate potential mechanisms initiating the onset of hepatic steatosis following the cessation of daily physical activity. Four-week-old, hyperphagic/obese Otsuka Long-Evans Tokushima Fatty rats were given access to voluntary running wheels for 16 weeks to prevent the development of hepatic steatosis. The animals were then suddenly transitioned to a sedentary condition as wheels were locked (wheel lock; WL) for 5 h (WL5), 53 h (WL53) or 173 h (WL173). Importantly after the cessation of daily exercise (5-173 h), no changes occurred in body weight, fat pad mass (omental and retroperitoneal), food intake, serum insulin, hepatic triglycerides or in the exercise-suppressed hepatic stearoyl-CoA desaturase-1 and peroxisome proliferator-activated receptor-gamma protein content. However, complete hepatic fatty acid oxidation and mitochondrial enzyme activities were highest at WL5 and WL53 and dropped significantly to SED levels by WL173. In addition, cessation of daily exercise quickly increased the hepatic protein contents of fatty acid synthase and acetyl-coenzyme A carboxylase (ACC), reduced ACC phosphorylation status, and dramatically increased hepatic malonyl-CoA concentration. This study is the first to show that the sudden cessation of daily exercise in a hyperphagic/obese model activates a subgroup of precursors and processes known to initiate hepatic steatosis, including decreased hepatic mitochondrial oxidative capacity, increased hepatic expression of de novo lipogenesis proteins, and increased hepatic malonyl CoA levels; each probably increasing the susceptibility to non-alcoholic fatty liver disease.

Mechanism of thrombin mediated eNOS phosphorylation in endothelial cells is dependent on ATP levels after stimulation

Conflicting results have been reported concerning the role of AMP-activated protein kinase (AMPK) in mediating thrombin stimulation of endothelial NO-synthase (eNOS). We examined the involvement of two upstream kinases in AMPK activation in cultured human umbilical endothelial cells, LKB1 stimulated by a rise in intracellular AMP/ATP ratio, and Ca+2/CaM kinase kinase (CaMKK) responding to elevation of intracellular Ca+2. We also studied the effects of AMPK activation on the downstream target eNOS. In culture medium 1640 the level of intracellular ATP was unchanged after thrombin stimulation and the CaMKK inhibitor STO-609 totally inhibited phosphorylation of AMPK and acetyl coenzyme A carboxylase (ACC) but not eNOS. In Morgan's medium 199 thrombin caused a significant lowering of intracellular ATP and STO-609 only partially inhibited the phosphorylation of AMPK, ACC and eNOS. Inhibition of AMPK by Compound C or AMPK downregulation using siRNA partially inhibited the phosphorylation of eNOS in medium 199 but not in 1640, underscoring a clear difference in the pathways mediating thrombin-stimulated eNOS phosphorylation in different culture media. Thus, conditions subjecting endothelial cells to a fall in ATP after thrombin stimulation facilitate activation of pathways partly dependent on AMPK causing downstream phosphorylation of eNOS. In contrast, under culture conditions that do not facilitate a fall in ATP after stimulation, AMPK activation is exclusively mediated by CaMKK and does not contribute to the phosphorylation of eNOS.

Daily exercise increases hepatic fatty acid oxidation and prevents steatosis in Otsuka Long-Evans Tokushima Fatty rats

Exercise training is commonly prescribed for treatment of nonalcoholic fatty liver disease (NAFLD). We sought to determine whether exercise training prevents the development of NAFLD in Otsuka Long-Evans Tokushima Fatty (OLETF) rats and to elucidate the molecular mechanisms underlying the effects of exercise on hepatic steatosis. Four-week-old OLETF rats were randomly assigned to either a sedentary control group (Sed) or a group given access to voluntary running wheels for 16 wk (Ex). Wheels were locked 2 days before euthanasia in the Ex animals, and both groups were euthanized at 20 wk old. Voluntary wheel running attenuated weight gain and reduced serum glucose, insulin, free fatty acids, and triglycerides in Ex animals compared with Sed (P < 0.001). Ex animals exhibited significantly reduced hepatic triglyceride levels and displayed fewer lipid droplets (Oil Red O staining) and reduced lipid droplet size compared with Sed. Wheel running increased by threefold the percent of palmitate oxidized completely to CO(2) in the Ex animals but did not alter AMP-activated protein kinase-alpha (AMPKalpha) or AMPK phosphorylation status. However, fatty acid synthase and acetyl-coenzyme A carboxylase (ACC) content were significantly reduced (approximately 70 and approximately 35%, respectively), and ACC phosphorylation and cytochrome c content were significantly elevated (approximately 35 and approximately 30%, respectively) in the Ex animals. These results unequivocally demonstrate that daily physical activity attenuates hepatic steatosis and NAFLD in an obese rodent model and suggest that this effect is likely mediated, in part, through enhancement of hepatic fatty acid oxidation and reductions in key protein intermediates of fatty acid synthesis.

Methionine restriction effects on 11β-HSD1 activity and lipogenic/lipolytic balance in F344 rat adipose tissue

Methionine restriction (MR) limits age-related adiposity in Fischer 344 (F344) rats. To assess the mechanism of adiposity resistance, the effect of MR on adipose tissue (AT) 11beta-hydroxysteroid dehydrogenase-1 (11beta-HSD1) was examined. MR induced 11beta-HSD1 activity in all ATs, correlating with increased tissue corticosterone. However, an inverse relationship between 11beta-HSD1 activity and adipocyte size was observed. Because dietary restriction controls lipogenic and lipolytic rates, MR's effects on lipogenic and lipolytic enzymes were evaluated. MR increased adipose triglyceride lipase and acetyl-coenzyme A carboxylase (ACC) protein levels but induced ACC phosphorylation at serine residues that render the enzyme inactive, suggesting alterations of basal lipolysis and lipogenesis. In contrast, no changes in basal or phosphorylated hormone-sensitive lipase levels were observed. ACC-phosphorylated sites were specific for AMP-activated protein kinase (AMPK); therefore, AMPK activation was evaluated. Significant differences in AMPKalpha protein, phosphorylation, and activity levels were observed only in retroperitoneal fat from MR rats. No differences in protein kinase A phosphorylation and intracellular cAMP levels were detected. In vitro studies revealed increased lipid degradation and a trend toward increased lipid synthesis, suggesting the presence of a futile cycle. In conclusion, MR disrupts the lipogenic/lipolytic balance, contributing importantly to adiposity resistance in F344 rats.

Cardiac expression of adiponectin and its receptors in streptozotocin-induced diabetic rats

Adiponectin can improve both glucose metabolism and insulin resistance via the adenosine monophosphate–activated protein kinase (AMPK) signaling pathway. Activated AMPK phosphorylates a variety of intracellular proteins, including acetyl coenzyme A carboxylase (ACC) that is involved in fatty acid oxidation. Adenosine monophosphate–activated protein kinase increases glucose transport by stimulating the translocation of glucose transporter 4 (GLUT4) to the sarcolemma in the heart. Adiponectin exerts its effect through adiponectin receptors, which are predominantly expressed in the liver and skeletal muscle. It is unknown whether the cardiac expression of adiponectin and its receptors is changed in diabetic rats. In the present study, we investigated the protein expression of adiponectin and its receptors in streptozotocin (STZ)–induced diabetic rat hearts. We also explored whether the levels of AMPK, ACC, and GLUT4 will be altered with the changed adiponectin and its receptors in STZ diabetic rat hearts. Plasma and cardiac adiponectin levels were measured by radioimmunoassay. Plasma and cardiac interleukin 6 and plasma tumor necrosis factor α (TNF- α) were assayed by enzyme-linked immunosorbent assay. Cardiac adiponectin receptors, AMPK- α, ACC, GLUT4, and TNF- α were analyzed by Western blot in control and STZ diabetic rats. The plasma adiponectin level was decreased, but the cardiac protein expression of adiponectin receptor 1 was increased in diabetic rats. There was no difference in the cardiac adiponectin level and the cardiac adiponectin receptor 2 protein expression between control and diabetic rats. The phosphorylation of AMPK- α and protein expression of GLUT4 were decreased, but the phosphorylation of ACC was unchanged in diabetic rat hearts. Plasma and cardiac levels of interleukin 6 and TNF- α were increased in diabetic rats. In conclusion, STZ-induced diabetes up-regulates adiponectin receptors in the heart. Despite an increase in cardiac adiponectin receptor 1 expression, there is an increased cardiac inflammatory response and a decreased GLUT4 protein expression associated with a reduction in circulating adiponectin.

Apolipoprotein A-I stimulates AMP-activated protein kinase and improves glucose metabolism

In humans, one of the hallmarks of type 2 diabetes is a reduced plasma concentration of HDL and its major protein component, apolipoprotein A-I (APOA-I). However, it is unknown whether APOA-I directly protects against diabetes. The aim of this study was to characterise the functional role of APOA-I in glucose homeostasis. The effects of APOA-I on phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC), glucose uptake and endocytosis were analysed in C2C12 myocytes. Glucose metabolism was investigated in Apoa-I knockout (Apoa-I (-/-)) mice. APOA-I was able to stimulate the phosphorylation of AMPK and ACC, and elevated glucose uptake in C2C12 myocytes. APOA-I could be endocytosed into C2C12 myotubes through a clathrin-dependent endocytotic process. Inhibition of endocytosis abrogated APOA-I-stimulated AMPK phosphorylation. In Apoa-I (-/-) mice, AMPK phosphorylation was reduced in skeletal muscle and liver, and expression of gluconeogenic enzymes was increased in liver. In addition, the Apoa-I (-/-) mice had increased fat content and compromised glucose tolerance. Our data indicate that APOA-I has a protective effect against diabetes via activation of AMPK. ApoA-I deletion in the mouse led to increased fat mass and impaired glucose tolerance.