Phosphorylation Status Of AMPK Research Articles

Chronic cortisol stimulation enhances hypothalamus-specific enrichment of metabolites in the rainbow trout brain.

The hypothalamus is a key integrating center that is involved in the initiation of the corticosteroid stress response, and in regulating nutrient homeostasis. Although cortisol, the principal glucocorticoid in humans and teleosts, plays a central role in feeding regulation, the mechanisms are far from clear. We tested the hypothesis that the metabolic changes to cortisol exposure signal an energy excess in the hypothalamus, leading to feeding suppression during stress in fish. Rainbow trout (Oncorhynchus mykiss) were administered a slow-release cortisol implant for 3 days, and the metabolite profiles in the plasma, hypothalamus, and the rest of the brain were assessed. Also, U-13C-glucose was injected into the hypothalamus by intracerebroventricular (ICV) route, and the metabolic fate of this energy substrate was followed in the brain regions by metabolomics. Chronic cortisol treatment reduced feed intake, and this corresponded with a downregulation of the orexigenic gene agrp, and an upregulation of the anorexigenic gene cart in the hypothalamus. The U-13C-glucose-mediated metabolite profiling indicated an enhancement of glycolytic flux and tricarboxylic acid intermediates in the rest of the brain compared with the hypothalamus. There was no effect of cortisol treatment on the phosphorylation status of AMPK or mechanistic target of rapamycin in the brain, whereas several endogenous metabolites, including leucine, citrate, and lactate were enriched in the hypothalamus, suggesting a tissue-specific metabolic shift in response to cortisol stimulation. Altogether, our results suggest that the hypothalamus-specific enrichment of leucine and the metabolic fate of this amino acid, including the generation of lipid intermediates, contribute to cortisol-mediated feeding suppression in fish.NEW & NOTEWORTHY Elevated cortisol levels during stress suppress feed intake in animals. We tested whether the feed suppression is associated with cortisol-mediated alteration in hypothalamus metabolism. The brain metabolome revealed a hypothalamus-specific metabolite profile suggesting nutrient excess. Specifically, we noted the enrichment of leucine and citrate in the hypothalamus, and the upregulation of pathways involved in leucine metabolism and fatty acid synthesis. This cortisol-mediated energy substrate repartitioning may modulate the feeding/satiety centers leading to the feeding suppression.

Exogenous ketosis increases circulating dopamine concentration and maintains mental alertness in ultra-endurance exercise.

Exogenous ketosis can improve psychocognitive functioning during exercise as well as stimulate postexercise muscular recovery. Therefore, we hypothesized that ketone ester (KE) supplementation can counteract the decline in psychocognitive functioning during ultra-endurance exercise and stimulate muscular recovery. Eighteen recreational runners participated in a full 100 km trail run (RUN, n = 8), or ran to premature exhaustion (80 km: n = 6; 60 km: n = 4). Before (25 g), during (25 g·h-1), and after (5 × 25 g in 24 h) RUN they received ketone ester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KE, n = 9) supplements or a noncaloric placebo (CON, n = 9). Blood samples and muscle biopsies were taken, and mental alertness was assessed by a psychocognitive test battery at different times before, during, and up to 36 h after RUN. Compared with CON (<0.3 mM), in KE blood d-β-hydroxybutyrate concentration was consistently elevated to ∼2-3 mM during RUN. In CON, RUN increased visual reaction times from 353 ± 53 to 419 ± 54 ms, and movement execution times from 174 ± 47 to 245 ± 64 ms. But this effect was fully negated by KE (P < 0.05). Plasma dopamine concentrations doubled in KE during RUN but remained stable in CON, resulting in higher concentrations after RUN in KE (4.1 ± 1.7 nM) than in CON (2.4 ± 0.8 nM, P = 0.048). KE also inhibited muscular infiltration of macrophages and suppressed AMPK phosphorylation status until 36-h postexercise (P < 0.05 KE vs. CON). In conclusion, KE increases circulating dopamine concentration and improves mental alertness, as well as improves postexercise muscular inflammation in ultra-endurance exercise.NEW & NOTEWORTHY Oral ketone ester ingestion elevates circulating dopamine concentration during ultra-endurance exercise. This is associated with improved mental alertness. Furthermore, ketone ester intake inhibits postexercise skeletal muscle macrophage infiltration, and counteracts the increase in AMPK phosphorylation after exercise, which indicates improved muscular energy status.

Obesity-induced oxidative stress and mitochondrial dysfunction negatively affect sperm quality.

Obesity is a systemic metabolic disease that can induce male infertility or subfertility through oxidative stress. The aim of this study was to determine how obesity impairs sperm mitochondrial structural integrity and function, and reduces sperm quality in both overweight/obese men and mice on a high-fat diet (HFD). Mice fed the HFD demonstrated higher body weight and increased abdominal fat content than those fed the control diet. Such effects accompanied the decline in antioxidant enzymes, such as glutathione peroxidase (GPX) and catalase and superoxide dismutase (SOD) in testicular and epidydimal tissues. Moreover, malondialdehyde (MDA) content significantly increased in sera. Mature sperm in HFD mice demonstrated higher oxidative stress, including increased mitochondrial reactive oxygen species (ROS) levels and decreased protein expression of GPX1, which may impair mitochondrial structural integrity and reduce mitochondrial membrane potential (MMP) and ATP production. Moreover, cyclic AMPK phosphorylation status increased, whereas sperm motility declined in the HFD mice. Clinical studies demonstrated that being overweight/obese reduced SOD enzyme activity in the seminal plasma and increased ROS in sperm, accompanied by lower MMP and low-quality sperm. Furthermore, ATP content in the sperm was negatively correlated with increases in the BMI of all clinical subjects. In conclusion, our results suggest that excessive fat intake had similar disruptive effects on sperm mitochondrial structure and function, as well as oxidative stress levels in humans and mice, which in turn induced lower sperm motility. This agreement strengthens the notion that fat-induced increases in ROS and impaired mitochondrial function contribute to male subfertility.

Ginsenoside F2 induces cellular toxicity to glioblastoma through the impairment of mitochondrial function

BackgroundGlioblastoma (GBM) is the most aggressive tumor residing within the central nervous system, with extremely poor prognosis. Although the cytotoxic effects of ginsenoside F2 (GF2) on GBM were previously suggested, the precise anti-GBM mechanism of GF2 remains unclear. The aim of this study was to explore the anti-cancer molecular mechanism of GF2 toward human GBM. MethodsGF2-driven cellular toxicity was confirmed in two different GBM cells, U373 and Hs683. To test mitochondrial impairment driven by GF2, we examined the mitochondrial membrane potential, OCR, and ATP production. An intracellular redox imbalance was identified by measuring the relative ratio of reduced glutathione to oxidized glutathione (GSH/GSSG), glutaredoxin (GLRX) mRNA expression, intracellular NAD+ level, and AMPK phosphorylation status. ResultsGF2 increased the percentage of cleaved caspase 3-positive cells and γH2AX signal intensities, confirming that GF2 shows the cytotoxicity against GBM. GO enrichment analysis suggested that the mitochondrial function could be negatively influenced by GF2. GF2 reduced the mitochondrial membrane potential, basal mitochondrial respiratory rate, and ATP production capacity. Our results showed that GF2 downregulated the relative GSH/GSSG, intracellular NAD+ level, and GLRX expression, suggesting that GF2 may alter the intracellular redox balance that led to mitochondrial impairment. ConclusionGF2 reduces mitochondrial membrane potential, inhibits cellular oxygen consumption, activates AMPK signaling, and induces cell death. Our study examined the potential vulnerability of mitochondrial activity in GBM, and this may hold therapeutic promise.

MicroRNA-141 is associated with hepatic steatosis by downregulating the sirtuin1/AMP-activated protein kinase pathway in hepatocytes.

Sirtuin1 (SIRT1) is a crucial regulator of metabolism and it is implicated in the metabolic pathophysiology of several disorders inclusive of Type 2 diabetes and fatty liver disease (NAFLD). The aim of this study was to investigate the role of miR-141 in hepatic steatosis via regulation of SIRT1/AMP-activated protein kinase (AMPK) pathway in hepatocytes. Liver hepatocellular cells (HepG2) were treated with high concentration of glucose to be subsequently used for the assessment of miR-141 and SIRT1 levels in a model of hepatic steatosis. On the other hand, cells were transfected with miR-141 to investigate its effect on hepatocyte steatosis and viability as well as SIRT1 expression and activity along with AMPK phosphorylation. Targeting of SIRT1 by miR-141 was evaluated by bioinformatics tools and confirmed by luciferase reporter assay. Following the intracellular accumulation of lipids in HepG2 cells, the level of miR-141 was increasedwhile SIRT1 mRNA and protein levels, as well as AMPK phosphorylation, was decreased. Transfection with miR-141 mimic significantly downregulated SIRT1 expression and activity while miR-141 inhibitor had the opposite effects. Additionally, modulation of miR-141 levels significantly influenced AMPK phosphorylation status. The results of luciferase reporter assay verified SIRT1 to be directly targeted by miR-141. miR-141 could effectively suppress SIRT1 and lead to decreased AMPK phosphorylation in HepG2 cells. Thus, miR-141/SIRT1/AMPK signaling pathway may be considered a potential target for the therapeutic management of NAFLD.

Predictive and preventive significance of AMPK activation on hepatocarcinogenesis in patients with liver cirrhosis

Metformin has been demonstrated to prevent hepatocellular carcinoma (HCC). Metformin acts mainly by phosphorylation of AMPK. However, the phosphorylation status of AMPK and its role in the prediction and prevention of HCC in cirrhotic patients remains unclear. The phosphorylation status of AMPK (Thr172) was determined by immunostaining in tissue microarrays of 426 cirrhotic liver tissues. Low expression of p-AMPK was observed in 94 (22.1%) cases. The median follow-up time was 87 months. HCC occurrence probability at 1/3/5/10 years after Hassab procedure was 3.1/9.6/13.8/30.6% in patients with p-AMPK low expression and 0/0.3/0.3/8% in patients with p-AMPK high expression, respectively. HCC occurrence risk was significantly higher in patients with p-AMPK low expression in univariable analysis (HR, 6.25; 95% CI: 3.36–11.60; P < 0.001) and multivariable analysis (HR, 6.0; 95% CI: 3.24–11.10; P < 0.001). An independent external cohort validated the significance of p-AMPK low expression. In addition, in vivo experiments demonstrated that AMPK activation status was negatively related to HCC occurrence and blocking autophagy by chloroquine counteracted the protective effect of AMPK phosphorylation. These results present novel insight into a critical predictive role of AMPK activation in hepatocarcinogenesis and AMPK activation seems to be a potential target for the prevention of hepatocellular carcinoma in patients with liver cirrhosis.

Ceramides are involved in the regulation of food intake in rainbow trout (Oncorhynchus mykiss).

We hypothesize that ceramides are involved in the regulation of food intake in fish. Therefore, we assessed in rainbow trout (Oncorhynchus mykiss) the effects of intracerebroventricular treatment with C6:0 ceramide on food intake. In a second experiment, we assessed the effects in brain areas of ceramide treatment on neuropeptide expression, fatty acid-sensing systems, and cellular signaling pathways. Ceramide treatment induced a decrease in food intake, a response opposed to the orexigenic effect described in mammals, which can be related to enhanced mRNA abundance of cocaine and amphetamine-related transcript and proopiomelanocortin and decreased mRNA abundance of Agouti-related protein and neuropeptide Y. Fatty acid-sensing systems appear to be inactivated by ceramide treatment. The mRNA abundance of integrative sensors AMPK and sirtuin 1, and the phosphorylation status of cellular signaling pathways dependent on protein kinase B, AMPK, mammalian target of rapamycin (mTOR), and forkhead box protein O1 (FoxO1) are generally activated by ceramide treatment. However, there are differences between hypothalamus and hindbrain in the phosphorylation status of AMPK (decreased in hypothalamus and increased in hindbrain), mTOR (decreased in hypothalamus and increased in hindbrain), and FoxO1 (increased in hypothalamus and decreased in hindbrain) to ceramide treatment. The results suggest that ceramides are involved in the regulation of food intake in rainbow trout through mechanisms comparable to those characterized previously in mammals in some cases.

AKT and AMPK activation after high-fat and high-glucose in vitro treatment of prostate epithelial cells.

Considering the increasing consumption of saturated fat and glucose in diets worldwide and its possible association to carcinogenesis, this investigation analysed the proliferation profile of nonmalignant human prostate epithelial cells after exposure to elevated levels of fat and glucose. PNT1A cells were cultured with palmitate (100 or 200 μM) and/or glucose (450 mg/dl) for 24 or 48 h. Treated cells were evaluated for viability test and cell proliferation (MTS assay). AKT and AMPK phosphorylation status were analysed by Western blotting. After 24 h of high-fat alone or associated with high-glucose treatment, there was an increase in AMPK and AKT activation associated to unchanged MTS-cell proliferation. Following 48 h of high-fat but not high-glucose alone, cells decreased AMPK activation and maintained elevated AKT levels. These data were associated to increased cell proliferation after further high-fat treatment. After longer high-fat exposure, MTS revealed that cells remained proliferating. High-glucose alone or associated to high-fat treatment was not able to increase cell proliferation and AKT activation. A high-fat medium containing 100 μM of palmitate stimulates proliferation in PNT1A cells by decreasing the activation of AMPK and increasing activation of AKT after longer exposure time. These findings improve the knowledge about the negative effect of high levels of this saturated fatty acid on proliferative disorders of prostate.

Abstract OT1-4-03: Phase I study of erlotinib and metformin in triple negative breast cancer

Abstract Background: Triple negative breast cancers (TNBC) are enriched for increased phosphorylated EGFR expression and decreased expression of PTEN. We found that combined treatment of metformin and erlotinib induces cell death in a subset of TNBC cell lines with evidence of combined enhanced reduction of EGFR, AKT, S6 and 4EBP1 phosphorylation. Analysis of TNBC cell line alterations led to the hypothesis that loss of PTEN sensitized cells to the drug combination, which was confirmed using isogenic cell line models with and without PTEN expression. The inhibitory effects of combined erlotinib and metformin were confirmed in mouse xenografts of PTEN null TNBC breast cancer cell lines. These data provided a rationale for clinical translation and testing in advanced TNBC patients. Trial Design: Single institution expanded Phase I design in patients with advanced TNBC. Erlotinib dosing will start and remain at 150 mg. Due to frequent GI upset in patients starting metformin, the dose of metformin will be titrated up to the assigned dose level over 2 weeks. The first metformin dose level will be 850 mg twice daily and will be escalated to its maximum FDA approved dose of 850 mg three times daily. Dose escalation will follow the standard 3 + 3 design. Dose limiting toxicities will be determined during the first 5 weeks of therapy. One metformin dose de-escalation level to 500 mg twice daily is allowed. Cycles will be defined as three weeks of treatment, and response assessments will be performed after every three cycles. Main Eligibility Criteria: 1. Pathologic diagnosis of TNBC. 2. Measurable or non-measurable disease. 3. At least one prior treatment for metastatic disease. 4. ECOG PS = 0-2. 5. Prior metformin or EGFR targeted therapy. 6. Fasting blood glucose level &amp;lt; 126 mg/dl. 7. Normal renal function. 8. Available archived tumor tissue. Specific Aims: The primary endpoints are defining the maximum tolerated dose (MTD) of metformin in combination with 150 mg erlotinib, and for the extended phase, determining the potential for clinical benefit by treating 14 patients at the MTD with the goal of rejecting the null hypothesis that the regimen is futile. Clinical benefit is defined as either partial response or lack of progression after 18 weeks of therapy (stable disease). Secondary endpoints include: 1) progression free survival, and 2) pharmacodynamic assessment of the phosphorylation status of AMPK and EGFR from pre- and on-treatment skin biopsies. The exploratory endpoint will be assessment of the status of EGFR, PTEN, LKB1, and 4EBP1 from available patient tumor samples to correlate against treatment response. Statistical Methods: If one patient meets the primary endpoint, among the 14 patients treated at the MTD, then the regimen will be considered worthy of further investigation. Target Accrual: 17-20 patients. Four patients accrued as of June 2013. Accrual completion expected June 2015. Contact: Matthew Maurer, Columbia University Medical Center, mm2058@columbia.edu. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr OT1-4-03.

MCT1 and MCT4 Expression During Myocardial Ischemic-Reperfusion Injury in the Isolated Rat Heart

Background/Aims: Myocardium ischemia-reperfusion (I/R) injury can be caused by imbalances in cellular metabolism. Lactate, transported by monocarboxylate transporters (MCTs), has been implicated as a mechanism in this process. The present study was designed to investigate the expression and functional role of MCTs in rat hearts during ischemia and reperfusion. Methods: Langendorff-perfused rat hearts were subjected to 20 minutes stabilization, 30 minutes of global ischemia and 60 minutes reperfusion. Hearts were collected serially for detecting expression changes in MCT1, MCT4 during myocardial I/R injury and lactate concentration was measured. Post-ischemic left ventricular function and infract size were determined at end-point, followed by the pretreatment of D-lactate, a competitive inhibitor of MCTs. Results: MCT4 was significantly increased following global ischemia and MCT1 expression was increased during the early stages of reperfusion in isolated rat hearts, while the expression of the ancillary protein CD147 was increased during I/R injury. We determined increases in AMPK phosphorylation status, which was significantly elevated following ischemia and early reperfusion. Blocking monocarboxylate transport by competitive inhibition with D-lactate caused decreased left ventricular performance and increased infarct size. Conclusion: Increased MCT4 expression facilitates lactate extrusion during the ischemic period, while increased MCT1 may facilitate lactate transport into and out of cells simultaneously during early reperfusion, with increases in AMPK phosphorylation status during the myocardial I/R period. Lactate transport by MCTs has a profound protective effect during myocardial ischemia reperfusion injury.

The Early Mtor Signaling Responses To Resistance Exercise Stimuli In Skeletal Muscle In Well-trained Athletes

INTRODUCTION: Muscle contractile activity associated with physical exercise stimulates multiple signaling pathways and induces transient changes in gene transcription. The bulk of skeletal muscle is regulated by the control of hypertrophy (anabolic) and atrophy (catabolic) processes. Muscle protein synthesis (MPS) is stimulated, while a small suppression of muscle protein breakdown (MPB) occurs, such that protein balance becomes positive (MPS > MPB). Recently several line of studies has been focused on the role played by the mammalian target of rapamycin (mTOR) signal pathway, i.e., activation of mTOR and its downstream signaling proteins p70 ribosomal protein S6 kinase (p70S6k) and eukaryotic initiation factor (eIF) 4E-binding protein (4E-BP1), in regulating translation initiation to syntheses of muscle protein. Furthermore, more recent work has shown that artificial activation of AMPK suppresses muscle protein synthesis by inhibiting the mTOR signaling pathway. Therefore, we hypothesized that the cellular mechanism for the decrease in muscle protein synthesis during an acute bout of resistance exercise in humans would be associated with an activation of AMPK in addition to an inhibition of the downstream components of the mTOR signaling pathway (4E-BP1 Thr37/46 and S6K1 Thr421/Ser424). METHODS: Eight healthy elite male (n=4) and female (n=4) athletes (22.8±1yrs) performed 10 sets of 10 leg extensions at 60°deg/sec. Muscle biopsies were taken from the vastus lateralis at rest, and 30 s after stopping the exercise. Western blotting with phosphospecific and pan antibodies was used to determine the phosphorylation status of AMPK, 4E-BP1, mTOR, and p70S6k. Blood samples were analysed for serum estradiol(E2), testosterone, insulin, GH and IGF- 1. RESULTS: The phosphorylation states of p70S6k on Thr421/Ser424 and AMPK on Thr172 were increased by 137% and 27% (p<.05). However, high intensity of resistance exercise did not induce any significant changes in mTOR and 4E-BP1 phosphorylations after acute exercise. In addition, estradiol(E2) and growth hormone were increased by 21% (p<.05) and 1784% (p<.001), whereas insulin was decreased by 112% (p<.05) after exercise. CONCLUSION: The present study demonstrates that resistance exercise increases AMPK activity and inhibit mTOR, 4E-BP1 phosphorylation and protein synthesis in human skeletal muscle. Thus, we postulated that one session of resistance exercise activates rather p70S6k in human muscle via an Akt-independent pathway as reported in the previous studies. KEY WORDS: resistance exercise, mTOR, AMPK, 4E-BP1, p70S6K and Western blotting *[email protected]

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.