Phosphorylation Of AMP-activated Kinase Research Articles

Cooling of male rat skeletal muscle during endurance‐like contraction attenuates contraction‐induced PGC‐1α mRNA expression

AbstractThis study aimed to determine effects of cooling on contraction‐induced peroxisome proliferator‐activated receptor γ coactivator‐1α (PGC‐1α) and vascular endothelial growth factor (VEGF) gene expression, phosphorylations of its related protein kinases, and metabolic responses. Male rats were separated into two groups; room temperature (RT) or ice‐treated (COLD) on the right tibialis anterior (TA). The TA was contracted isometrically using nerve electrical stimulation (1‐s stimulation × 30 contractions, with 1‐s intervals, for 10 sets with 1‐min intervals). The TA was treated before the contraction and during 1‐min intervals with an ice pack for the COLD group and a water pack at RT for the RT group. The muscle temperature of the COLD group decreased to 19.42 ± 0.44°C (p < 0.0001, −36.4%) compared with the RT group after the experimental protocol. An increase in mRNA expression level of PGC‐1α, not VEGF, after muscle contractions was significantly lower in the COLD group than in the RT group (p < 0.0001, −63.0%). An increase in phosphorylated AMP‐activated kinase (AMPK) (p = 0.0037, −28.8%) and a decrease in glycogen concentration (p = 0.0231, +106.3%) after muscle contraction were also significantly inhibited by cooling. Collectively, muscle cooling attenuated the post‐contraction increases in PGC‐1α mRNA expression coinciding with decreases in AMPK phosphorylation and glycogen degradation.

Leflunomide alleviates obesity via activation of the TAK1-AMPK pathway and induction of lipophagy.

Lipophagy is a subset of selective autophagy that specifically degrades lipid droplets and plays an important role in obesity. Leflunomide treatment in rheumatoid arthritis (RA) patients has been associated with weight loss and decreased blood glucose levels, which cannot be attributed to its known side effects. Our prior studies showed that A77 1726, the active metabolite of leflunomide, acts as an inhibitor of S6K1 to sensitize the insulin receptor and control hyperglycemia. Whether the anti-obesity effect of leflunomide is mediated by targeting S6K1 and its underlying mechanisms remain unclear. Here, we report that A77 1726 induced LC3 lipidation and increased the formation of autophagosomes and lipoautolysosomes in 3T3-L1 adipocytes by activating TGF-β-activated kinase 1 (TAK1), AMP-activated kinase (AMPK), and Unc-51 like autophagy-activated kinase 1 (ULK1). A77 1726 reduced the content of lipid droplets in 3T3-L1 adipocytes, which was blocked by bafilomycin or by beclin-1 knockdown. Similar observations were made in murine adipocytes differentiated from S6K1-/- embryonic fibroblasts (MEFs). Leflunomide treatment restricted bodyweight gains in ob/ob mice and reduced the visceral fat deposit and the size of adipocytes. Leflunomide treatment induced autophagy in adipose and liver tissues and reduced hepatic lipid contents. Consistently, S6K1 knockout increased the levels of LC3 lipidation in the liver, muscle, and fat of S6K-/- mice. Leflunomide treatment and S6K1 deficiency both induced TAK1, AMPK, and ULK1 phosphorylation in these tissues. These observations collectively suggest that leflunomide controls obesity in part by activating AMPK and inducing lipophagy. Our study provides insights into the mechanisms of leflunomide-mediated anti-obesity activity.

Inhibition of Transforming Growth Factor-β Improves Primary Renal Tubule Cell Differentiation in Long-Term Culture.

Patient-oriented applications of cell culture include cell therapy of organ failure like chronic renal failure. Clinical deployment of a cell-based device for artificial renal replacement requires qualitative and quantitative fidelity of a cultured cell to its in vivo counterpart. Active specific apicobasal ion transport reabsorbs 90-99% of the filtered load of salt and water in the kidney. In a bioengineered kidney, tubular transport concentrates wastes and eliminates the need for hemodialysis, but renal tubule cells in culture transport little or no salt and water due to dedifferentiation that mammalian cells undergo in vitro thereby losing important cell-type specific functions. We previously identified transforming growth factor-β (TGF-β) as a signaling pathway necessary for in vitro differentiation of renal tubule cells. Inhibition of TGF-β receptor-1 led to active and inhibitable electrolyte and water transport by primary human renal tubule epithelial cells in vitro. Addition of metformin increased transport, in the context of a transient effect on 5'-AMP-activated kinase phosphorylation. These data motivated us to examine whether increased transport was an idiosyncratic effect of SB431542, probe pathways downstream of TGF-β receptors possibly responsible for the improved differentiation, evaluate whether TGF-β inhibition induced a range of differentiated tubule functions, and to explore crosstalk between the effects of SB431542 and metformin. In this study, we use multiple small-molecule inhibitors of canonical and noncanonical pathways to confirm that inhibition of canonical TGF-β signaling caused the increased apicobasal transport. Hallmarks of proximal tubule cell function, including sodium reabsorption, para-amino hippurate excretion, and glucose uptake increased with TGF-β inhibition, and the specificity of the response was shown using inhibitors of each transport protein. We did not find any evidence of crosstalk between metformin and SB431542. These data suggest that the TGF-β signaling pathway governs multiple features of differentiation in renal proximal tubule cells in vitro. Inhibition of TGF-β by pharmacologic or genome engineering approaches may be a viable approach to enhancing differentiated function of tubule cells in vitro. Impact statement Cell therapy of renal failure requires qualitative and quantitative fidelity between in vitro and in vivo phenotypes, which has been elusive. We show that control of transforming growth factor-β signaling can promote differentiation of renal tubule cells grown in artificial environments. This is a key enabling step for cell therapy of renal failure.

Inhibition of Folate Metabolism Drives Autophagy-Dependent Differentiation and Reduces Survival of Therapy-Resistant Leukaemic Stem Cells

Metabolic rewiring is an important hallmark of cancer. The folate metabolism pathway, also known as one-carbon (1C) metabolism, allows for transfer of 1C units through folate intermediates for biosynthetic processes, including precursors for DNA synthesis. Recent studies have shown that enzymes involved in the mitochondrial arm of 1C metabolism are overexpressed in a subset of aggressive cancers and that their expression affects responses to anti-metabolite drug treatments. However, the role of 1C metabolism in therapy resistant leukemic stem cells (LSCs) is currently unknown. Therefore, we aimed to investigate the activity and the impact of genetic and pharmacological inhibition of folate enzymes in primitive chronic myeloid leukaemia (CML) cells.We initially performed transcriptomic analysis of CD34+38- cells, from individuals with chronic phase CML (E-MTAB-2581). This revealed a significant upregulation of folate metabolism genes in CML LSCs, including serine hydroxymethyltransferase (SHMT2; p≤0.05), a key mitochondrial enzyme. To assess the activity of 1C metabolism in primitive cells we performed gas chromatography-mass spectrometry-mediated secretomic analysis using patient-derived, c-KIT enriched CML cells, which revealed a significant increase in the exchange rate of formate (folate intermediate necessary for purine synthesis) in CML cells, when compared to the secretome of normal counterparts (p<0.05). This reinforced the idea that 1C metabolism may be a metabolic dependency in CML.Following CRISPR-Cas9-mediated SHMT2 knockout (KO) in CML cell line, we observed a significant decrease in growth rate, together with a decrease in glycolytic capacity and oxygen consumption rate (p<0.01), suggesting impairment in proliferation and central carbon metabolism. Further metabolic characterisation of CML SHMT2 KO cells using liquid chromatography-mass spectrometry demonstrated a significant increase in AICAR, a purine biosynthesis intermediate and an AMP activated kinase (AMPK) activator. This prompted us to investigate the effect of 1C metabolism inhibition on AMPK. We found that AMPK phosphorylation on the conserved Thr 172 (a site that is phosphorylated under energy stress) was increased in SHMT2 KO cells, with similar effect seen following pharmacological inhibition of both SHMT2 and its cytosolic counterpart SHMT1 using SHIN1,which also promoted AMPK-dependent phosphorylation of the autophagy-inducing kinase ULK1 and downstream ULK1 target ATG13. Moreover, analysis of mitochondrial fraction revealed accumulation of mitochondrial fission related protein DRP1 and the mitophagy receptor NIX on mitochondria, hinting towards cellular interplay between 1C metabolism and mitochondrial homeostasis.Phenotypically, both pharmacological and genetic inhibition of SHMT1/2 induced the expression of erythropoiesis markers CD71 and Glycophorin A, which was reversed following formate supplementation. CRISPR-Cas9-mediated double AMPKα1/α2 KO revealed that the increased expression of these erythropoiesis markers following SHMT1/2 inhibition was independent of AMPK activity. Conversely, while NIX KO had no effect, pharmacological inhibition of ULK1 kinase activity, or genetic inhibition of ULK1 and ATG7 (protein important for autophagosome formation), prevented increased expression of CD71/Glycophorin A following SHMT1/2 inhibition.We next investigated the effect of 1C metabolism inhibition on differentiation and survival of primary CML cells. Of clinical relevance, pharmacological inhibition of SHMT1/2 promoted erythroid maturation of CD34+ CML cells (measured by expression of CD71, CD44, CD36 and Glycophorin A) when challenged with erythropoietin, which sensitises primitive cells to erythroid lineage commitment. Lastly, pharmacological inhibition of 1C metabolism decreased the colony formation capacity of CD34+ CML by 50%, with minimum effect on normal CD34+ cells. Moreover, combination treatment of SHIN1 with imatinib, a frontline treatment for CML patients, further increased the sensitivity of primary CML cells to imatinib by 40%.Overall, our novel findings indicate that disruption of the folate metabolism pathway inhibits central carbon metabolism in CML cells, promotes autophagy dependent, but AMPK independent maturation phenotype and has detrimental effect on the survival of primitive CML cells. DisclosuresNo relevant conflicts of interest to declare.

The hepatocyte IKK:NF-κB axis promotes liver steatosis by stimulating de novo lipogenesis and cholesterol synthesis

ObjectiveObesity-related chronic inflammation plays an important role in the development of Metabolic Associated Fatty Liver Disease (MAFLD). Although the contribution of the pro-inflammatory NF-κB signaling pathway to the progression from simple steatosis to non-alcoholic steatohepatitis (NASH) is well-established, its role as an initiator of hepatic steatosis and the underlying mechanism remains unclear. Here, we investigated the hypothesis that the hepatocytic NF-κB signaling pathway acts as a metabolic regulator, thereby promoting hepatic steatosis development. MethodsA murine model expressing a constitutively active form of IKKβ in hepatocytes (Hep-IKKβca) was used to activate hepatocyte NF-κB. In addition, IKKβca was also expressed in hepatocyte A20-deficient mice (IKKβca;A20LKO). A20 is an NF-κB-target gene that inhibits the activation of the NF-κB signaling pathway upstream of IKKβ. These mouse models were fed a sucrose-rich diet for 8 weeks. Hepatic lipid levels were measured and using [1–13C]-acetate de novo lipogenesis and cholesterol synthesis rate were determined. Gene expression analyses and immunoblotting were used to study the lipogenesis and cholesterol synthesis pathways. ResultsHepatocytic NF-κB activation by expressing IKKβca in hepatocytes resulted in hepatic steatosis without inflammation. Ablation of hepatocyte A20 in Hep-IKKβca mice (IKKβca;A20LKO mice) exacerbated hepatic steatosis, characterized by macrovesicular accumulation of triglycerides and cholesterol, and increased plasma cholesterol levels. Both De novo lipogenesis (DNL) and cholesterol synthesis were found elevated in IKKβca;A20LKO mice. Phosphorylation of AMP-activated kinase (AMPK) - a suppressor in lipogenesis and cholesterol synthesis - was decreased in IKKβca;A20LKO mice. This was paralleled by elevated protein levels of hydroxymethylglutaryl-CoA synthase 1 (HMGCS1) and reduced phosphorylation of HMG-CoA reductase (HMGCR) both key enzymes in the cholesterol synthesis pathway. Whereas inflammation was not observed in young IKKβca;A20LKO mice sustained hepatic NF-κB activation resulted in liver inflammation, together with elevated hepatic and plasma cholesterol levels in middle-aged mice. ConclusionsThe hepatocytic IKK:NF-κB axis is a metabolic regulator by controlling DNL and cholesterol synthesis, independent of its central role in inflammation. The IKK:NF-κB axis controls the phosphorylation levels of AMPK and HMGCR and the protein levels of HMGCS1. Chronic IKK-mediated NF-κB activation may contribute to the initiation of hepatic steatosis and cardiovascular disease risk in MAFLD patients.

Thyroid hormone receptor phosphorylation regulates acute fasting-induced suppression of the hypothalamic–pituitary–thyroid axis

Fasting induces profound changes in the hypothalamic-pituitary-thyroid (HPT) axis. After binding thyroid hormone (TH), the TH receptor beta 2 isoform (THRB2) represses Trh and Tsh subunit genes and is the principle negative regulator of the HPT axis. Using mass spectrometry, we identified a major phosphorylation site in the AF-1 domain of THRB2 (serine 101, S101), which is conserved among many members of the nuclear hormone receptor superfamily. More than 50% of THRB2 is phosphorylated at S101 in cultured thyrotrophs (TαT1.1) and in the mouse pituitary. All other THR isoforms lack this site and exhibit limited overall levels of phosphorylation. To determine the importance of THRB2 S101 phosphorylation, we used the TαT1.1 cell line and S101A mutant knock-in mice (Thrb2S101A ). We found that TH promoted S101 THRB2 phosphorylation and was essential for repression of the axis at physiologic TH concentrations. In mice, THRB2 phosphorylation was also increased by fasting and mimicked Trh and Tshb repression by TH. In vitro studies demonstrated that a master metabolic sensor, AMP-activated kinase (AMPK) induced phosphorylation at the same site and caused Tshb repression independent of TH. Furthermore, we identified cyclin-dependent kinase 2 (CDK2) as a direct kinase phosphorylating THRB2 S101 and propose that AMPK or TH increase S101 phosphorylation through the activity of CDK2. This study provides a physiologically relevant function for THR phosphorylation, which permits nutritional deprivation and TH to use a common mechanism for acute suppression of the HPT axis.

Intestinal Population in Host with Metabolic Syndrome during Administration of Chitosan and Its Derivatives

Chitosan and its derivatives can alleviate metabolic syndrome by different regulation mechanisms, phosphorylation of AMPK (AMP-activated kinase) and Akt (also known as protein kinase B), suppression of PPAR-γ (peroxisome proliferator-activated receptor-γ) and SREBP-1c (sterol regulatory element–binding proteins), and translocation of GLUT4 (glucose transporter-4), and also the downregulation of fatty-acid-transport proteins, fatty-acid-binding proteins, fatty acid synthetase (FAS), acetyl-CoA carboxylase (acetyl coenzyme A carboxylase), and HMG-CoA reductase (hydroxy methylglutaryl coenzyme A reductase). The improved microbial profiles in the gastrointestinal tract were positively correlated with the improved glucose and lipid profiles in hosts with metabolic syndrome. Hence, this review will summarize the current literature illustrating positive correlations between the alleviated conditions in metabolic syndrome hosts and the normalized gut microbiota in hosts with metabolic syndrome after treatment with chitosan and its derivatives, implying that the possibility of chitosan and its derivatives to serve as therapeutic application will be consolidated. Chitosan has been shown to modulate cardiometabolic symptoms (e.g., lipid and glycemic levels, blood pressure) as well as gut microbiota. However, the literature that summarizes the relationship between such metabolic modulation of chitosan and prebiotic-like effects is limited. This review will discuss the connection among their structures, biological properties, and prebiotic effects for the treatment of metabolic syndrome. Our hope is that future researchers will consider the prebiotic effects as significant contributors to the mitigation of metabolic syndrome.

Differences in intrinsic aerobic capacity alters sensitivity to ischemia-reperfusion injury but not cardioprotective capacity by ischemic preconditioning in rats.

IntroductionAerobic capacity is a strong predictor of cardiovascular mortality. Whether aerobic capacity influences myocardial ischemia and reperfusion (IR) injury is unknown.PurposeTo investigate the impact of intrinsic differences in aerobic capacity and the cardioprotective potential on IR injury.MethodsWe studied hearts from rats developed by selective breeding for high (HCR) or low (LCR) capacity for treadmill running. The rats were randomized to: (1) control, (2) local ischemic preconditioning (IPC) or (3) remote ischemic preconditioning (RIC) followed by 30 minutes of ischemia and 120 minutes of reperfusion in an isolated perfused heart model. The primary endpoint was infarct size. Secondary endpoints included uptake of labelled glucose, content of selected mitochondrial proteins in skeletal and cardiac muscle, and activation of AMP-activated kinase (AMPK).ResultsAt baseline, running distance was 203±7 m in LCR vs 1905±51 m in HCR rats (p<0.01). Infarct size was significantly lower in LCR than in HCR controls (49±5% vs 68±5%, p = 0.04). IPC reduced infarct size by 47% in LCR (p<0.01) and by 31% in HCR rats (p = 0.01). RIC did not modulate infarct size (LCR: 52±5, p>0.99; HCR: 69±6%, p>0.99, respectively). Phosphorylaion of AMPK did not differ between LCR and HCR controls. IPC did not modulate cardiac phosphorylation of AMPK. Glucose uptake during reperfusion was similar in LCR and HCR rats. IPC increased glucose uptake during reperfusion in LCR animals (p = 0.02). Mitochondrial protein content in skeletal muscle was lower in LCR than in HCR (0.77±0.10 arbitrary units (AU) vs 1.09±0.07 AU, p = 0.02), but not in cardiac muscle.ConclusionAerobic capacity is associated with altered myocardial sensitivity to IR injury, but the cardioprotective effect of IPC is not. Glucose uptake, AMPK activation immediately prior to ischemia and basal mitochondrial protein content in the heart seem to be of minor importance as underlying mechanisms for the cardioprotective effects.

Water Extract of Lotus Leaf Alleviates Dexamethasone-Induced Muscle Atrophy via Regulating Protein Metabolism-Related Pathways in Mice.

Muscle atrophy is an abnormal condition characterized by loss of skeletal muscle mass and function and is primarily caused by injury, malnutrition, various diseases, and aging. Leaf of lotus (Nelumbo nucifera Gaertn), which has been used for medicinal purposes, contains various active ingredients, including polyphenols, and is reported to exert an antioxidant effect. In this study, we investigated the effect of water extract of lotus leaf (LL) on muscle atrophy and the underlying molecular mechanisms of action. Amounts of 100, 200, or 300 mg/kg/day LL were administered to dexamethasone (DEX)-induced muscle atrophy mice for 4 weeks. Micro-computed tomography (CT) analysis revealed that the intake of LL significantly increased calf muscle volume, surface area, and density in DEX-induced muscle atrophy mice. Administration of LL recovered moving distance, grip strength, ATP production, and body weight, which were decreased by DEX. In addition, muscle damage caused by DEX was also improved by LL. LL reduced the protein catabolic pathway by suppressing gene expression of muscle atrophy F-Box (MAFbx; atrogin-1), muscle RING finger 1 (MuRF1), and forkhead box O (FoxO)3a, as well as phosphorylation of AMP-activated kinase (AMPK). The AKT-mammalian target of the rapamycin (mTOR) signal pathway, which is important for muscle protein synthesis, was increased in LL-administered groups. The HPLC analysis and pharmacological test revealed that quercetin 3-O-beta-glucuronide (Q3G) is a major active component in LL. Thus, Q3G decreased the gene expression of atrogin-1 and MuRF1 and phosphorylation of AMPK. This compound also increased phosphorylation levels of mTOR and its upstream enzyme AKT in DEX-treated C2C12 cells. We identified that LL improves muscle wasting through regulation of muscle protein metabolism in DEX-induced muscle atrophy mice. Q3G is predicted to be one of the major active phenolic components in LL. Therefore, we propose LL as a supplement or therapeutic agent to prevent or treat muscle wasting, such as sarcopenia.

Dysregulated lipid metabolism in hepatocellular carcinoma cancer stem cells.

According to the stem cell theory for cancer, hepatocellular carcinomas are sustained by a group of cancer stem cells (CSCs) which are responsible for resistance to chemotherapy. In the present study we aimed to examine lipid metabolism in cancer stem cells induced by long-term treatment with sorafenib and its relationship with acquisition of a CSC-like phenotype. Two cell lines (HepG2SF1 and Huh7SF1) were generated by incubation with a step-wise increase of sorafenib concentrations for 10 months. These cell lines displayed stem-like characteristics like increase in the expression of ABCB1A, Nanog and Oct4 as well as an E-cadherin/N-cadherin switch. HepG2SF1 and Huh7SF1 cells showed intracellular accumulation of neutral lipids, assessed by flow cytometry and confocal microscopy. The exam of lipid metabolism revealed that HepG2SF1 and Huh7SF1 cells increased the expression of the enzymes involved in de novo lipid synthesis ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN) and that of the fatty acid transporter CD36. In addition, these CSC-like cells had enhanced expression of the lipogenic transcription factor SREBP1c. Analysis of the key metabolic sensor AMP-activated kinase (AMPK) demonstrated that both AMPK phosphorylation and levels were decreased in the CSC-like cells compared to their parental cells. Interestingly, transfection of HepG2SF1 and Huh7SF1 cells with AMPK, restored the levels of the lipogenic enzymes and SREBP1c and decreased the intracellular lipid accumulation. Furthermore, AMPK transfection decreased the stemness markers and inhibited the E-cadherin/N-cadherin switch. Targeting AMPK and lipid metabolism of hepatocellular cancer stem cells is a promising strategy to face stemness and chemotherapy resistance.

AMPK signaling mediates synphilin-1-induced hyperphagia and obesity in Drosophila.

Expression of synphilin-1 in neurons induces hyperphagia and obesity in a Drosophila model. However, the molecular pathways underlying synphilin-1-linked obesity remain unclear. Here, Drosophila models and genetic tools were used to study the synphilin-1-linked pathways in energy balance by combining molecular biology and pharmacological approaches. We found that expression of human synphilin-1 in flies increased AMP-activated kinase (AMPK) phosphorylation at Thr172 compared with that in non-transgenic flies. Knockdown of AMPK reduced AMPK phosphorylation and food intake in non-transgenic flies, and further suppressed synphilin-1-induced AMPK phosphorylation, hyperphagia, fat storage and body weight gain in transgenic flies. Expression of constitutively activated AMPK significantly increased food intake and body weight gain in non-transgenic flies, but it did not alter food intake in the synphilin-1 transgenic flies. In contrast, expression of dominant-negative AMPK reduced food intake in both non-transgenic and synphilin-1 transgenic flies. Treatment with STO-609 also suppressed synphilin-1-induced AMPK phosphorylation, hyperphagia and body weight gain. These results demonstrate that the AMPK signaling pathway plays a critical role in synphilin-1-induced hyperphagia and obesity. These findings provide new insights into the mechanisms of synphilin-1-controlled energy homeostasis.

P738The restoration effect of metformin on the beta-adrenergic signaling, amp-activated kinase and akt-kinase in the myocardium of diabetic rats

Abstract Background The type 2 diabetic mellitus (DM2) induces the myocardial dysfunctions, the impairment of beta-adrenergic regulations and the alterations of energy homeostasis and apoptotic processes in the myocardium are the main causes of diabetic cardiomyopathy. Metformin (MF) is a first-line drug for management of DM2. However, MF effects on the adrenergic signaling, the cell metabolism and survival in the cardiomyocytes are still poorly understood. Purpose To study MF effects on the beta-adrenergic signaling in myocardial membranes and on the expression and activity of beta1- and beta3-adrenergic receptors (ARs), AMP-activated kinase (AMPK), Akt-kinase and the pro- (Bax) and anti-apoptotic (Bcl-2) proteins in the myocardium of diabetic rats. Methods In male rats, DM2 was induced by high-fat/high-carbohydrate diet (three months) and by a single treatment with low-dose streptozotocin. Diabetic rats were treated with MF at two doses, 100 and 200 mg/kg/day, during 28 days. The stimulating effects of beta-AR agonists on adenylyl cyclase (AC) activity in the cardiac membranes were studied by radioisotope method. The gene expression was estimated by qPCR, and the protein amount and their activity were studied by Western blotting. Four groups of animals, such as control (C, n=12), DM2 (D, n=12), and DM2 treated with MF at the doses 100 (D100, n=9) and 200 mg/kg/day (D200, n=9) were investigated. Results MF restores the stimulating AC effect of beta1-agonists, the beta1-AR gene expression and the beta1/beta3-ARs ratio, all reduced in DM2, and restoration effect of MF in Groups D100 and D200 was similar. Meanwhile, in Group D200, but not in Group D100, the AC effect of beta3-agonists and the expression of beta3-AR, both increased in DM2, returns to the control values. In diabetic myocardium, MF restores the AMPK and Akt-kinase activities, reduced in DM2, and it is illustrated by the increased phosphorylation of AMPK by Thr172 and Akt-kinase by Ser473 and Thr308. Despite the fact that MF at a dose of 200 mg/kg/day to a greater extent, as compared to a dose of 100 mg/kg/day, increases the “stimulating” Thr172-phosphorylation, but at the same time it also enhances the “inhibiting” Ser491-phosphorylation of AMPK, decreasing the resulting stimulation of AMPK. In Groups D100 and D200 the Bax/Bcl-2 ratio, significantly increased in DM2, is reduced. This data indicates the weakening of apoptosis in cardiomyocytes in DM2. Conclusion The four-week MF treatment improves the myocardial beta-adrenergic signaling, normalizing the expression and activity of beta1- and beta3-ARs, restores the activity of AMPK and Akt-kinase, the effector components of the signaling cascades responsible for cell survival and metabolism, and normalizes the expression of apoptotic factors. Thus, the cardioprotective MF effect is largely due to the improved myocardial signaling, including the beta1-AR, AMPK and Akt-kinase signaling pathways. Acknowledgement/Funding This work was supported by the Russian Science Foundation (No. 17-75-30052) and partially by the Government Assignment (AAAA-A18-118012290427-7).

170-OR: Tamoxifen Treatment Causes Insulin Resistance in Obese Mice

Breast cancer survivors treated with the selective estrogen receptor modulator tamoxifen have an elevated risk for developing type 2 diabetes (T2D) compared with cancer-free individuals, especially in the context of obesity. Half of T2D diagnoses in breast cancer survivors are attributable to the cancer therapy itself. The underlying mechanisms of tamoxifen-induced T2D are unknown. We hypothesized that the detrimental impact of tamoxifen on insulin sensitivity was mediated by changes in the liver and skeletal muscle, and was exacerbated by high fat diet (HFD). Ovariectomized female mice supplemented with estradiol were randomized to low fat diet (LFD), LFD + tamoxifen, HFD, or HFD + tamoxifen. In general, our preclinical model recapitulated the observations from epidemiological studies on breast cancer survivors. HFD + tamoxifen mice had greater body fat percentage and fasting glucose (P &amp;lt; 0.02 for the HFD-Tamoxifen interaction). The HFD + tamoxifen mice developed insulin resistance determined by HOMA-IR (P &amp;lt; 0.01) and tended towards impaired glucose tolerance measured by glucose AUC from a glucose tolerance test (P = 0.1). In the liver of HFD-fed mice, tamoxifen resulted in greater accumulation of triacylglycerol (P = 0.01) which correlated positively with HOMA-IR (R2= 0.4; P &amp;lt; 0.001). Gastrocnemius citrate synthase activity was elevated with tamoxifen treatment independent of diet, as was phosphorylation of AMP-activated kinase (P &amp;lt; 0.05). HFD + tamoxifen mice had lower content of muscle mitochondrial complex II (P = 0.05) and complex V (P = 0.1). These data suggest that the potential mechanisms underlying tamoxifen-induced T2D is ectopic hepatic fat accumulation and lowered skeletal muscle mitochondrial content. Importantly, this study reveals a difference in response to tamoxifen between lean and obese females. Future studies will explore interventions that can be combined with tamoxifen to prevent hepatosteatosis and protect skeletal muscle mitochondria with the goal of reducing T2D in breast cancer survivors. Disclosure R.L. Scalzo: None. S.E. Hull: None. J.E.B. Reusch: Board Member; Self; American Diabetes Association. Other Relationship; Self; Merck &amp; Co., Inc. P. MacLean: Advisory Panel; Self; Habit LLC. E. Wellberg: None. Funding Susan G. Komen Foundation; National Institutes of Health

Hesperetin is a potent bioactivator that activates SIRT1-AMPK signaling pathway in HepG2 cells.

Sirtuin 1 (SIRT1) is a deacetylase enzyme that plays crucial roles in controlling many cellular processes and its downregulation has been implicated in different metabolic disorders. Recently, several polyphenols have been considered as the effective therapeutic approaches that appear to influence SIRT1. The main goal of this study was to evaluate the effect of hesperetin, a citrus polyphenolic flavonoid, on SIRT1 and AMP-activated kinase (AMPK). HepG2 cells were treated with hesperetin in the presence or absence of EX-527, a SIRT1 specific inhibitor, for 24h. Resveratrol was used as a positive control. SIRT1 gene expression, protein level, and activity were measured by RT-PCR, Western blotting, and fluorometric assay, respectively. AMPK phosphorylation was also determined by Western blotting. Our results indicated a significant increase in SIRT1 protein level and activity as well as an induction of AMPK phosphorylation by hesperetin. These effects of hesperetin were abolished by EX-527. Furthermore, hesperetin reversed the EX-527 inhibitory effects on SIRT1 protein expression and AMPK phosphorylation. These findings suggest that hesperetin can be a novel SIRT1 activator, even stronger than resveratrol. Therefore, the current study may introduce hesperetin as a new strategy aimed at upregulation SIRT1-AMPK pathway resulting in various cellular processes regulation.

β-Hydroxybutyrate, a ketone body, reduces the cytotoxic effect of cisplatin via activation of HDAC5 in human renal cortical epithelial cells

Aimsβ-Hydroxybutyrate (βOHB) is a metabolic intermediate that constitutes about 70% of ketone bodies produced in liver from oxidation of fatty acids released from adipose tissue. A recent study showed that βOHB inhibits HDAC1, 3 and 4 (classes I and IIa) in human embryonic kidney 293 (HEK293) cells. Therefore, βOHB could regulate epigenetics via modulating HDACs. However, little is known about the protective effect of βOHB on renal cells through epigenetics. The aim of this study is to investigate whether βOHB reduces cisplatin-induced nephrotoxicity in human renal cortical epithelial (HRCE) cells by modulating HDACs. Main methodsIn this study, we used human renal cortical epithelial (HRCE) cells. The anti-apoptotic effect of βOHB was evaluated using flow cytometry analysis. The expression of apoptosis-related proteins and HDACs was evaluated by western immunoblot. Key findingsThe results showed that βOHB significantly reduced cisplatin-induced apoptosis in HRCE cells. Furthermore, βOHB significantly reduced cisplatin-induced cleavage of caspase-3, acetylation of histone H3, and phosphorylation of AMP-activated kinase. This anti-apoptotic effect of βOHB was markedly attenuated by an inhibitor of HDAC4/5, and βOHB-mediated suppression of cleavage of caspase3 was significantly blocked by siRNA-induced gene silencing of HDAC5. SignificanceβOHB attenuates cisplatin-induced apoptosis by activation of HDAC5 in HRCE cells, suggesting that βOHB may be a new therapeutic agent for cisplatin nephropathy.

The red pepper's spicy ingredient capsaicin activates AMPK in HepG2 cells through CaMKKβ.

Capsaicin is a natural compound present in chili and red peppers and the responsible of their spicy flavor. It has recently provoked interest because of its antitumoral effects in many cell types although its action mechanism is not clearly understood. As metabolic dysregulation is one of the hallmarks of cancer cells and the key metabolic sensor in the AMP-activated kinase (AMPK), in this study we explored the ability of capsaicin to modulate AMPK activity. We found that capsaicin activated AMPK in HepG2 cells by increasing AMPK phosphorylation and its downstream target ACC. Mechanistically, we determined that capsaicin activated AMPK through the calcium/calmodulin-dependent protein kinase kinase β, CaMKKβ as either the CaMKK inhibitor STO-609 or CaMKK knock down with siRNA abrogated the activation of AMPK. Moreover, capsaicin decreased cell viability, inhibited Akt/mTOR pathway and increased reactive oxygen species (ROS) in HepG2 cells. AMPK activation was involved in the underpinning mechanism of capsaicin-induced cell death.

A novel phosphorylation by AMP-activated kinase regulates RUNX2 from ubiquitination in osteogenesis over adipogenesis

Mesenchymal stem cells (MSCs) function as progenitors to a variety of cell types. The reported association between osteogenic and adipogenic commitment during differentiation is due to the regulation of key transcription factors in the signaling pathways. However, the process of adipogenesis at the expense of osteogenic phenotype during metabolic stress is still unclear. In this study, we showed for the first time that RUNX2 is a novel substrate of AMP-activated kinase (AMPK), which directly phosphorylates at serine 118 residue in the DNA-binding domain of RUNX2. Our results in in vitro MSC lineage differentiation models confirmed that active AMPK and RUNX2-S118 phosphorylation are preferentially associated with osteogenic commitment, whereas the lack of this phosphorylation leads to adipogenesis. This interplay is regulated by the ubiquitination of non-phosphorylated RUNX2-S118, which is evident in the dominant mutant RUNX2-S118D. Pharmacological activation of AMPK by metformin significantly abrogated the loss of RUNX2-S118 phosphorylation and protected from tunicamycin-induced endoplasmic reticulum stress, high glucose-induced in vitro adipogenesis and streptozotocin-induced in vivo bone adiposity and bone phenotype. In conclusion, results from this study demonstrated that RUNX2 is a direct target of AMPK which simplified the outlook towards several complex mechanisms that are currently established concerning cellular metabolism and pathogenesis.

AMP-activated kinase (AMPK) promotes innate immunity and antiviral defense against Zika virus induced ocular infection

Abstract Purpose We recently reported that Zika Virus infected multiple retinal cell types and caused chorioretinal atrophy in mouse eyes. In this study, we investigated the potential role of AMPK, a master regulator of cellular metabolism, in pathogenesis of ocular ZIKV infection. Methods Primary human RPE cells were infected with ZIKV for various time points and AMPK phosphorylation was detected Western blotting (WB). AMPK activity was modulated by pharmacological activators (AICAR and Metformin) and inhibitors (Compound C) of AMPK. ZIKV replication was assessed by immunofluorscence staining for 4G2 and western blot detection of NS3 proteins. For in vivo studies B6 mice eye were challenged with ZIKV with or without AICAR treatment and disease progression were measured by fundus imaging. Activation of innate immune and antiviral response genes was assessed by qPCR. Results ZIKV challenge upregulated the AMPK phosphorylation in Pr. RPE cells in a time-dependent fashion. Our results indicate that AMPK deficient mouse embryonic fibroblast (MEF) cells were more permissive to ZIKV replication as compared to WT MEFs. AMPK activation using AICAR and Metformin restricted ZIKV replication in Pr. RPE cells and presence of Compound C reversed the effect of AICAR/Metformin. In vivo AICAR treatment ameliorated ZIKV-induced chorioretinal atrophy in mice with increased antiviral responses. ZIKV infection leads to activation of MEK pathways and an MEK pathways inhibitor, U0126, attenuated ZIKV replication. Conclusion Collectively, these findings indicate the AMPK activation evokes antiviral innate responses and restricts ZIKV replication. Hence, targeting AMPK signaling can be explored to develop anti-viral therapy against ZIKV infection.

Carnosol Increases Skeletal Muscle Cell Glucose Uptake via AMPK-Dependent GLUT4 Glucose Transporter Translocation.

Skeletal muscle is a major insulin-target tissue and plays an important role in glucose homeostasis. Insulin action in muscle activates the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway causing the translocation of intracellularly stored GLUT4 glucose transporters to the plasma membrane and increased glucose uptake. Impaired insulin action in muscle results in insulin resistance and type 2 diabetes mellitus (T2DM). Activation of the energy sensor AMP-activated kinase (AMPK) increases muscle glucose uptake and the use of AMPK activators is viewed as an effective strategy to combat insulin resistance. Rosemary extract (RE) has been shown to stimulate muscle AMPK and glucose uptake, but the exact components responsible for these effects are unknown. In the current study, we investigated the effect of carnosol, a RE polyphenol, in L6 rat muscle cells. Carnosol stimulated glucose uptake in L6 myotubes in a dose- and time-dependent manner, did not affect Akt, increased AMPK phosphorylation and plasma membrane GLUT4 levels. The carnosol-stimulated glucose uptake and GLUT4 translocation was significantly reduced by the AMPK inhibitor compound C (CC). Our study is the first to show an AMPK-dependent increase in muscle glucose uptake by carnosol. Carnosol has potential as a glucose homeostasis regulating agent and deserves further study.

Carnosol increases skeletal muscle cell glucose uptake via AMPK‐dependent GLUT 4 glucose transporter translocation

Skeletal muscle is highly important in glucose homeostasis since it is quantitatively a major insulin‐target tissue. Insulin action in muscle cells activates the phosphatidylinositol‐3 kinase (PI3K)/Akt signaling pathway causing the translocation of intracellularly stored GLUT4 glucose transporters to the plasma membrane leading to increased glucose uptake. Impaired insulin action in muscle leads to insulin resistance and type 2 diabetes mellitus (T2DM).AMP‐activated kinase (AMPK) is a cellular energy sensor and its activation increases glucose uptake by skeletal muscle cells. Finding AMPK activators is viewed as an effective approach to combat insulin resistance and T2DM. Rosemary extract (RE) has been shown to increase muscle glucose uptake and AMPK activity but the components responsible for these effects have not been identified yet. In the current study, we investigated the effect of carnosol, a polyphenol found in high concentrations in RE. L6 rat muscle cells were used to measure uptake of [3H]‐2‐deoxy‐D‐glucose and the signaling molecules involved were investigated by immunoblotting. Carnosol stimulated glucose uptake in L6 myotubes in a dose‐ and time‐dependent manner. A response comparable to maximum insulin stimulation (196±9.2 % of control) was seen with 50μM of carnosol (2h) (182±7.8 % of control). Carnosol did not affect Akt phosphorylation while it significantly increased AMPK phosphorylation. Furthermore, the increase in glucose uptake in the presence of carnosol was significantly reduced by the AMPK inhibitor compound C (CC) while it was not affected by the PI3K inhibitor wortmannin. Carnosol increased plasma membrane GLUT4 glucose transporter levels in GLUT4myc overexpressing L6 cells and this response was abolished by the AMPK inhibitor CC. Our study is the first to show a significant increase in muscle glucose uptake by carnosol via a mechanism that involves AMPK.Support or Funding InformationSupported by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant to ET.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.