AMP-activated Protein Kinase Activity In Muscle Research Articles

CaMKK2 is not involved in contraction-stimulated AMPK activation and glucose uptake in skeletal muscle

ObjectiveThe AMP-activated protein kinase (AMPK) gets activated in response to energetic stress such as contractions and plays a vital role in regulating various metabolic processes such as insulin-independent glucose uptake in skeletal muscle. The main upstream kinase that activates AMPK through phosphorylation of α-AMPK Thr172 in skeletal muscle is LKB1, however some studies have suggested that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) acts as an alternative kinase to activate AMPK. We aimed to establish whether CaMKK2 is involved in activation of AMPK and promotion of glucose uptake following contractions in skeletal muscle. MethodsA recently developed CaMKK2 inhibitor (SGC-CAMKK2-1) alongside a structurally related but inactive compound (SGC-CAMKK2-1N), as well as CaMKK2 knock-out (KO) mice were used. In vitro kinase inhibition selectivity and efficacy assays, as well as cellular inhibition efficacy analyses of CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) were performed. Phosphorylation and activity of AMPK following contractions (ex vivo) in mouse skeletal muscles treated with/without CaMKK inhibitors or isolated from wild-type (WT)/CaMKK2 KO mice were assessed. Camkk2 mRNA in mouse tissues was measured by qPCR. CaMKK2 protein expression was assessed by immunoblotting with or without prior enrichment of calmodulin-binding proteins from skeletal muscle extracts, as well as by mass spectrometry-based proteomics of mouse skeletal muscle and C2C12 myotubes. ResultsSTO-609 and SGC-CAMKK2-1 were equally potent and effective in inhibiting CaMKK2 in cell-free and cell-based assays, but SGC-CAMKK2-1 was much more selective. Contraction-stimulated phosphorylation and activation of AMPK were not affected with CaMKK inhibitors or in CaMKK2 null muscles. Contraction-stimulated glucose uptake was comparable between WT and CaMKK2 KO muscle. Both CaMKK inhibitors (STO-609 and SGC-CAMKK2-1) and the inactive compound (SGC-CAMKK2-1N) significantly inhibited contraction-stimulated glucose uptake. SGC-CAMKK2-1 also inhibited glucose uptake induced by a pharmacological AMPK activator or insulin. Relatively low levels of Camkk2 mRNA were detected in mouse skeletal muscle, but neither CaMKK2 protein nor its derived peptides were detectable in mouse skeletal muscle tissue. ConclusionsWe demonstrate that pharmacological inhibition or genetic loss of CaMKK2 does not affect contraction-stimulated AMPK phosphorylation and activation, as well as glucose uptake in skeletal muscle. Previously observed inhibitory effect of STO-609 on AMPK activity and glucose uptake is likely due to off-target effects. CaMKK2 protein is either absent from adult murine skeletal muscle or below the detection limit of currently available methods.

AICAR promotes endothelium-independent vasorelaxation by activating AMP-activated protein kinase via increased ZMP and decreased ATP/ADP ratio in aortic smooth muscle.

AICAR, an adenosine analog, has been shown to exhibit vascular protective effects through activation of AMP-activated protein kinase (AMPK). However, it remains unclear as to whether adenosine kinase-mediated ZMP formation or adenosine receptor activation contributes to AICAR-mediated AMPK activation and/or vasorelaxant response in vascular smooth muscle. In the present study using endothelium-denuded rat aortic ring preparations, isometric tension measurements revealed that exposure to 1mM AICAR for 30min resulted in inhibition of phenylephrine (1μM)-induced smooth muscle contractility by ∼35%. Importantly, this vasorelaxant response by AICAR was prevented after pretreatment of aortic rings with an AMPK inhibitor (compound C, 40µM) and adenosine kinase inhibitor (5-iodotubercidin, 1µM), but not with an adenosine receptor blocker (8-sulfophenyltheophylline, 100µM). Immunoblot analysis of respective aortic tissues showed that AMPK activation seen during vasorelaxant response by AICAR was abolished by compound C and 5-iodotubercidin, but not by 8-sulfophenyltheophylline, suggesting ZMP involvement in AMPK activation. Furthermore, LC-MS/MS MRM analysis revealed that exposure of aortic smooth muscle cells to 1mM AICAR for 30min enhanced ZMP level to 2014.9±179.4picomoles/mg protein (vs. control value of 8.5±0.6; p<0.01), which was accompanied by a significant decrease in ATP/ADP ratio (1.08±0.02 vs. 2.08±0.06; p<0.01). Together, the present findings demonstrate that AICAR-mediated ZMP elevation and the resultant AMPK activation in vascular smooth muscle contribute to vasorelaxation.

Transthyretin contributes to insulin resistance and diminishes exercise-induced insulin sensitivity in obese mice by inhibiting AMPK activity in skeletal muscle.

Exercise improves obesity-induced insulin resistance and metabolic disorders via mechanisms that remain unclear. Here, we show that the levels of the hepatokine transthyretin (TTR) in circulation are elevated in insulin-resistant individuals including high-fat diet (HFD)-induced obese mice, db/db mice, and patients with metabolic syndrome. Liver Ttr mRNA and circulating TTR levels were reduced in mice by treadmill training, as was the TTR levels in quadriceps femoris muscle; however, AMP-activated protein kinase (AMPK) signaling activity was enhanced. Transgenic overexpression of TTR or injection of purified TTR triggered insulin resistance in mice fed on regular chow (RC). Furthermore, TTR overexpression reduced the beneficial effects of exercise on insulin sensitivity in HFD-fed mice. TTR was internalized by muscle cells via the membrane receptor Grp78 and the internalization into the quadriceps femoris was reduced by treadmill training. The TTR/Grp78 combination in C2C12 cells was increased, whereas the AMPK activity of C2C12 cells was decreased as the TTR concentration rose. In addition, Grp78 silencing prevented the TTR internalization and reversed its inhibitory effect on AMPK activity in C2C12 cells. Our study suggests that elevated circulating TTR may contribute to insulin resistance and counteract the exercise-induced insulin sensitivity improvement; the TTR suppression might be an adaptive response to exercise through enhancing AMPK activity in skeletal muscles.NEW & NOTEWORTHY Exercise improves obesity-induced insulin resistance via mechanisms that remain unclear. The novel findings of the study are that circulating TTR (a hepatokine) level is decreased by exercise, and the elevated circulating TTR, as was the elevated transthyretin internalization mediated by Grp78, counteracts the exercise-induced insulin sensitivity by downregulating AMPK activity in skeletal muscle of obese mice. These data suggest that TTR suppression might be an adaptive response to exercise through the crosstalk between liver and muscle.

Goat Milk Improves Glucose Homeostasis via Enhancement of Hepatic and Skeletal Muscle AMP-Activated Protein Kinase Activation and Modulation of Gut Microbiota in Streptozocin-Induced Diabetic Rats.

Previously, the metabolic benefits of goat milk consumption in high-fat diet-fed rats are demonstrated. However, the effects are only reported in one animal model and the involvement of gut microbiota is not investigated. The aim of this study is to investigate the effects of goat milk consumption on glucose homeostasis and gut microbiota in streptozocin (STZ)-induced diabetic rats. STZ-induced diabetic rats are fed with three dosages of goat milk: 2.5, 5, and 10g kg-1 . Parameters related to glucose homeostasis, hepatic and skeletal muscle AMP-activated protein kinase (AMPK) activation, and gut microbiota are investigated. The dose of 10g kg-1 exerts more metabolic benefits. Goat milk consumption improves fasting glucose levels, glucose tolerance, insulin sensitivity, and promotes hepatic and skeletal muscle AMPK activation in STZ-injected diabetic rats. Goat milk modulates gut microbiota, increases the relative abundance of Lactobacillus, and augments levels of propionic and butyric acids. This study demonstrates the metabolic benefits of goat milk consumption in STZ-induced diabetic rats, which is consistent with the previous observations in high-fat diet-induced diabetic rats. Furthermore, this study elucidates the modulation of gut microbiota by goat milk, which likely mediates the metabolic effects of goat milk.

Skeletal muscle AMPK is not activated during 2h of moderate intensity exercise at ∼65% in endurance trained men.

Key points AMP‐activated protein kinase (AMPK) is considered a major regulator of skeletal muscle metabolism during exercise.However, we previously showed that, although AMPK activity increases by 8–10‐fold during ∼120 min of exercise at ∼65% V˙O2peak in untrained individuals, there is no increase in these individuals after only 10 days of exercise training (longitudinal study).In a cross‐sectional study, we show that there is also a lack of activation of skeletal muscle AMPK during 120 min of cycling exercise at 65% V˙O2peak in endurance‐trained individuals.These findings indicate that AMPK is not an important regulator of exercise metabolism during 120 min of exercise at 65% V˙O2peak in endurance trained men.It is important that more energy is directed towards examining other potential regulators of exercise metabolism. AMP‐activated protein kinase (AMPK) is considered a major regulator of skeletal muscle metabolism during exercise. Indeed, AMPK is activated during exercise and activation of AMPK by 5‐aminoimidazole‐4‐carboxyamide‐ribonucleoside (AICAR) increases skeletal muscle glucose uptake and fat oxidation. However, we have previously shown that, although AMPK activity increases by 8–10‐fold during ∼120 min of exercise at ∼65% V˙O2peak in untrained individuals, there is no increase in these individuals after only 10 days of exercise training (longitudinal study). In a cross‐sectional study, we examined whether there is also a lack of activation of skeletal muscle AMPK during 120 min of cycling exercise at 65% V˙O2peak in endurance‐trained individuals. Eleven untrained (UT; V˙O2peak = 37.9 ± 5.6 ml.kg−1 min−1) and seven endurance trained (ET; V˙O2peak = 61.8 ± 2.2 ml.kg−1 min−1) males completed 120 min of cycling exercise at 66 ± 4% V˙O2peak (UT: 100 ± 21 W; ET: 190 ± 15 W). Muscle biopsies were obtained at rest and following 30 and 120 min of exercise. Muscle glycogen was significantly (P < 0.05) higher before exercise in ET and decreased similarly during exercise in the ET and UT individuals. Exercise significantly increased calculated skeletal muscle free AMP content and more so in the UT individuals. Exercise significantly (P < 0.05) increased skeletal muscle AMPK α2 activity (4‐fold), AMPK αThr172 phosphorylation (2‐fold) and ACCβ Ser222 phosphorylation (2‐fold) in the UT individuals but not in the ET individuals. These findings indicate that AMPK is not an important regulator of exercise metabolism during 120 min of exercise at 65% V˙O2peak in endurance trained men.

Treatment With Treprostinil and Metformin Normalizes Hyperglycemia and Improves Cardiac Function in Pulmonary Hypertension Associated With Heart Failure With Preserved Ejection Fraction.

Pulmonary hypertension (PH) due to left heart disease (group 2), especially in the setting of heart failure with preserved ejection fraction (HFpEF), is the most common cause of PH worldwide; however, at present, there is no proven effective therapy available for its treatment. PH-HFpEF is associated with insulin resistance and features of metabolic syndrome. The stable prostacyclin analog, treprostinil, is an effective and widely used Food and Drug Administration-approved drug for the treatment of pulmonary arterial hypertension. While the effect of treprostinil on metabolic syndrome is unknown, a recent study suggests that the prostacyclin analog beraprost can improve glucose intolerance and insulin sensitivity. We sought to evaluate the effectiveness of treprostinil in the treatment of metabolic syndrome-associated PH-HFpEF. Approach and Results: Treprostinil treatment was given to mice with mild metabolic syndrome-associated PH-HFpEF induced by high-fat diet and to SU5416/obese ZSF1 rats, a model created by the treatment of rats with a more profound metabolic syndrome due to double leptin receptor defect (obese ZSF1) with a vascular endothelial growth factor receptor blocker SU5416. In high-fat diet-exposed mice, chronic treatment with treprostinil reduced hyperglycemia and pulmonary hypertension. In SU5416/Obese ZSF1 rats, treprostinil improved hyperglycemia with similar efficacy to that of metformin (a first-line drug for type 2 diabetes mellitus); the glucose-lowering effect of treprostinil was further potentiated by the combined treatment with metformin. Early treatment with treprostinil in SU5416/Obese ZSF1 rats lowered pulmonary pressures, and a late treatment with treprostinil together with metformin improved pulmonary artery acceleration time to ejection time ratio and tricuspid annular plane systolic excursion with AMPK (AMP-activated protein kinase) activation in skeletal muscle and the right ventricle. Our data suggest a potential use of treprostinil as an early treatment for mild metabolic syndrome-associated PH-HFpEF and that combined treatment with treprostinil and metformin may improve hyperglycemia and cardiac function in a more severe disease.

It's well and truly time to stop stating that AMPK regulates glucose uptake and fat oxidation during exercise.

None for Perspective.

Goat Milk Consumption Ameliorates Abnormalities in Glucose Metabolism and Enhances Hepatic and Skeletal Muscle AMP-Activated Protein Kinase Activation in Rats Fed with High-Fat Diets.

Diabetes endangers health and causes serious economic impediment. The aim of this study is to identify the effects of goat milk consumption on glucose metabolism of rats with high-fat (HF) diet. Sixty male Sprague Dawley rats are divided into five groups and fed with different diets for 24 weeks: goat-milk-based HF diet (GHF group; goat milk powder+HF diet), cow-milk-based HF diet (CHF group; cow milk powder+HF diet), HF diet, HF diet plus acarbose (HF+A group; acarbose+HF diet), and chow diet (CD group). Fasting glucose in GHF-fed rats are lower than HF-fed rats on weeks 16 and 20. GHF-fed rats display improved insulin sensitivity in oral glucose and insulin tolerance tests. Compared with HF-fed rats, glycated hemoglobin and triglycerides in GHF-fed rats are lower and high-density lipoprotein level is higher. AMP-activated protein kinase activation (AMPK) in the liver and skeletal muscle is higher in GHF rats than HF rats. Phosphoenolpyruvate carboxykinase and glucose 6-phosphatase protein levels in the liver are lower and hexokinase 2 protein level in the skeletal muscle is higher in GHF rats compared with HF rats. Goat milk consumption can ameliorate abnormalities in glucose metabolism, and AMPK pathway in the liver and skeletal muscle plays an important role in the process.

SCD1 regulates the AMPK/SIRT1 pathway and histone acetylation through changes in adenine nucleotide metabolism in skeletal muscle.

Stearoyl-CoA desaturase (SCD) is a rate-limiting enzyme that catalyzes the synthesis of monounsaturated fatty acids. It plays an important role in regulating skeletal muscle metabolism. Lack of the SCD1 gene increases the rate of fatty acid β-oxidation through activation of the AMP-activated protein kinase (AMPK) pathway and the upregulation of genes that are related to fatty acid oxidation. The mechanism of AMPK activation under conditions of SCD1 deficiency has been unclear. In the present study, we found that the ablation/inhibition of SCD1 led to AMPK activation in skeletal muscle through an increase in AMP levels whereas muscle-specific SCD1 overexpression decreased both AMPK phosphorylation and the adenosine monophosphate/adenosine triphosphate (AMP/ATP) ratio. Changes in AMPK phosphorylation that were caused by SCD1 down- and upregulation affected NAD+ levels following changes in NAD+ -dependent deacetylase sirtuin-1 (SIRT1) activity and histone 3 (H3K9) acetylation and methylation status. Moreover, mice with muscle-targeted overexpression of SCD1 were more susceptible to high-fat diet-induced lipid accumulation and the development of insulin resistance compared with wild-type mice. These data show that SCD1 is involved in nucleotide (ATP and NAD+ ) metabolism and suggest that the SCD1-dependent regulation of muscle steatosis and insulin sensitivity are mediated by cooperation between AMPK- and SIRT1-regulated pathways. Altogether, the present study reveals a novel mechanism that links SCD1 with the maintenance of metabolic homeostasis and insulin sensitivity in skeletal muscle.

Adenosine and adenosine-5\u2032-monophosphate ingestion ameliorates abnormal glucose metabolism in mice fed a high-fat diet

BackgroundWe have previously reported that ingestion of adenosine (ADN) and adenosine-5′-monophosphate (AMP) improves abnormal glucose metabolism in the stroke-prone spontaneously hypertensive rat model of non-obesity-associated insulin resistance. In this study, we investigated the effect of ADN and AMP ingestion on glucose metabolism in mice with high-fat diet-induced obesity.MethodsSeven-week-old C57BL/6 J mice were administered distilled water (as a control), 10 mg/L ADN, or 13 mg/L AMP via their drinking water for 14 or 25 weeks, during which they were fed a high-fat diet. Oral glucose tolerance test (OGTT) was conducted on 21-week-old mice fasted for 16 h. Insulin tolerance test (ITT) was performed on 22-week-old mice fasted for 3 h. Blood and muscle were collected for further analysis of serum parameters, gene and protein expression levels, respectively.ResultsGlucose metabolism in the ADN and AMP groups was significantly improved compared with the control. OGTT and ITT showed that ADN and AMP groups lower than control group. Furthermore, phosphorylation of AMP-activated protein kinase (AMPK) and mRNA levels of genes involved in lipid oxidation were enhanced in the skeletal muscle of ADN- and AMP-treated mice.ConclusionThese results indicate that ingestion of ADN or AMP induces activation of AMPK in skeletal muscle and mitigates insulin resistance in mice with high-fat diet-induced diabetes.

PO-104 The shifted balance of high-energy phosphates drives the AMPK dephosphorylation and reduced slow myosin expression during unloading

Objective Mechanical unloading of postural muscles results in both muscle atrophy and a slow to fast fibre type transition. The cause of such changes is a reduction in slow-type MyHC isoform expression and an increase in fast-type MyHC isoform expression. It is believed that calcineurin/NFAT signalling pathway and AMP-activated protein kinase (AMPK) are involved in the regulation of slow-type MyHC isoform. Previously we showed that AMPK phosphorylation is significantly decreased in rat soleus at the early stage of mechanical unloading. We found, that stimulation of AMPK activity at the early stage of gravitational unloading prevents a decline of slow MyHC expression. We assumed that a decrease in AMPK activity in rat soleus at the early stage of gravitational unloading could be associated with changes in the ratio of intracellular high-energy phosphates (ATP/ADP). The aim of the study was to investigate the impact of high-energy phosphates ratio changes on AMPK activity and slow-type MyHC isoform expression in rat soleus muscle at the early stages of gravitational unloading.&#x0D; Methods To verify the hypothesis, we used administration of β-guanidinopropionic acid (βGPA), before (6 day) and during 24-h hindlimb suspension. The content of phospho-AMPK, phospho-ACC, phospho-PKD, HDAC4/5 in rat soleus was determined by Western-blotting. The amount of MyHCI(β) pre-mRNA and mRNA was evaluated by RT-PCR. &#x0D; Results After 24-h HS we observed a decrease (p&lt;0.05) in phospho-AMPK content vs. control group, but in HS+ βGPA group didn’t differ from the control. After 24-h unloading we found a significant increase in the content of nuclear HDAC4 in the HS group, but in the HS+ βGPA group the content of nuclear HDAC4 didn’t differ from the control group. 24-h unloading resulted in a decrease in MyHCI(β) pre-mRNA and mRNA expression vs. the control group. The expression level of MyHCI(β) pre-mRNA and mRNA in HS+ βGPA group didn’t differ from the control.&#x0D; Conclusions Thus βGPA administration prevents a decline in AMPK phosphorylation. Therefore, we can conclude that at the early stage of gravitational unloading an accumulation of high-energy phosphates (ATP, ADP and creatine phosphate) takes place and leads to reduced AMPK activity and a slow to fast myosin fibre type transition. The study was supported by Russian Science Foundation grant # 18-15-00107.

PL - 032 Physiological triggers involved in reduced slow myosin expression in disused postural muscle

PL - 032 Physiological triggers involved in reduced slow myosin expression in disused postural muscle

Role of Angptl4/Fiaf in exercise-induced skeletal muscle AMPK activation.

Angiopoietin-like protein 4 (Angptl4)/fasting-induced adipose factor (Fiaf) expression levels are increased by exercise in skeletal muscle. We have previously shown that Angptl4 regulates food intake and energy expenditure via modulation of hypothalamic AMP-activated protein kinase (AMPK) activity. AMPK is an important signaling molecule that integrates skeletal muscle metabolism during exercise. Therefore, we investigated the involvement of Angptl4 in exercise-induced AMPK activation in skeletal muscle. Angptl4 protein and mRNA expression levels were significantly increased in the gastrocnemius and soleus muscles of mice following a 50-min running bout. Treatment of C2C12 myotubes with Angptl4 increased phosphorylation of AMPK and acetyl-CoA carboxylase (ACC), which were markers of AMPK activation, and the mitochondrial maximum respiratory capacity. Treadmill exercise increased AMPK and ACC phosphorylation in the gastrocnemius of normal mice; this phosphorylation increase was attenuated in mice lacking Angptl4. Endurance to swimming and hanging was also reduced in Angptl4 knockout mice. Taken together, our current data demonstrate that exercise-induced upregulation of skeletal muscle Angptl4 is critical for AMPK activation and exercise tolerance. These findings unveil a new role for skeletal muscle Angptl4 in exercise physiology. NEW & NOTEWORTHY 1) Angiopoietin-like protein 4 (Angptl4) treatment activates AMP-activated protein kinase (AMPK) signaling in skeletal muscle cells. 2) Angptl4 increases the maximum mitochondrial oxidative capacity through AMPK activation in skeletal muscle cells. 3) Lack of Angptl4 mitigates exercise-induced skeletal muscle AMPK activation. 4) Angptl4-deficient mice show a lower endurance to exercise.

Taurine supplementation induces long-term beneficial effects on glucose homeostasis in ob/ob mice.

The sulfur-containing amino acid, taurine (Tau), regulates glucose and lipid homeostasis under normal, pre- and diabetic conditions. Here, we aimed to verify whether Tau supplementation exerts its beneficial effects against obesity, hyperglycemia and alterations in islet functions, in leptin-deficient obese (ob/ob), over a long period of treatment. From weaning until 12months of age, female ob/ob mice received, or not, 5% Tau in drinking water (obTau group). After this period, a reduction in hypertriglyceridemia and an improvement in glucose tolerance and insulin sensitivity were observed in obTau mice. In addition, the daily metabolic flexibility was restored in obTau mice. In the gastrocnemius muscle of obTau mice, the activation of AMP-activated protein kinase (AMPK) was increased, while total AMPK protein content was reduced. Finally, isolated islets from obTau mice expressed high amounts of pyruvate carboxylase (PC) protein and lower glucose-induced insulin secretion. Taking these evidences together Tau supplementation had long-term positive actions on glucose tolerance and insulin sensitivity, associated with a reduction in glucose-stimulated insulin secretion, in ob/ob mice. The improvement in insulin actions in obTau mice was due, at least in part, to increased activation of AMPK in skeletal muscle, while the increased content of the PC enzyme in pancreatic islets may help to preserve glucose responsiveness in obTau islets, possibly contributing to islet cell survive.

Regulation of energy metabolism during social interactions in rainbow trout: a role for AMP-activated protein kinase.

Rainbow trout (Oncorhynchus mykiss) confined in pairs form social hierarchies in which subordinate fish typically experience fasting and high circulating cortisol levels, resulting in low growth rates. The present study investigated the role of AMP-activated protein kinase (AMPK) in mediating metabolic adjustments associated with social status in rainbow trout. After 3 days of social interaction, liver AMPK activity was significantly higher in subordinate than dominant or sham (fish handled in the same fashion as paired fish but held individually) trout. Elevated liver AMPK activity in subordinate fish likely reflected a significantly higher ratio of phosphorylated AMPK (phospho-AMPK) to total AMPK protein, which was accompanied by significantly higher AMPKα1 relative mRNA abundance. Liver ATP and creatine phosphate concentrations in subordinate fish also were elevated, perhaps as a result of AMPK activity. Sham fish that were fasted for 3 days exhibited effects parallel to those of subordinate fish, suggesting that low food intake was an important trigger of elevated AMPK activity in subordinate fish. Effects on white muscle appeared to be influenced by the physical activity associated with social interaction. Overall, muscle AMPK activity was significantly higher in dominant and subordinate than sham fish. The ratio of phospho-AMPK to total AMPK protein in muscle was highest in subordinate fish, while muscle AMPKα1 relative mRNA abundance was elevated by social dominance. Muscle ATP and creatine phosphate concentrations were high in dominant and subordinate fish at 6 h of interaction and decreased significantly thereafter. Collectively, the findings of the present study support a role for AMPK in mediating liver and white muscle metabolic adjustments associated with social hierarchy formation in rainbow trout.

Activation of Skeletal Muscle AMPK Promotes Glucose Disposal and Glucose Lowering in Non-human Primates and Mice

The AMP-activated protein kinase (AMPK) is a potential therapeutic target for metabolic diseases based on its reported actions in the liver and skeletal muscle. We evaluated two distinct direct activators of AMPK: a non-selective activator of all AMPK complexes, PF-739, and an activator selective for AMPK β1-containing complexes, PF-249. In cells and animals, both compounds were effective at activating AMPK in hepatocytes, but only PF-739 was capable of activating AMPK in skeletal muscle. In diabetic mice, PF-739, but not PF-249, caused a rapid lowering of plasma glucose levels that was diminished in the absence of skeletal muscle, but not liver, AMPK heterotrimers and was the result of an increase in systemic glucose disposal with no impact on hepatic glucose production. Studies of PF-739 in cynomolgus monkeys confirmed translation of the glucose lowering and established activation of AMPK in skeletal muscle as a potential therapeutic approach to treat diabetic patients.

Skeletal muscle PI3K p110β regulates expression of AMP-activated protein kinase

Skeletal muscle metabolic homeostasis is maintained through numerous biochemical and physiological processes. Two principal molecular regulators of skeletal muscle metabolism include AMP-activated protein kinase (AMPK) and phosphatidylinositol 3-kinase (PI3K); however, PI3K exists as multiple isoforms, and specific metabolic actions of each isoform have not yet been fully elucidated in skeletal muscle. Given this lack of knowledge, we performed a series of experiments to define the extent to which PI3K p110β mediated expression and (or) activation of AMPK in skeletal muscle. To determine the effect of p110β inhibition on AMPK expression and phosphorylation in cultured cells, C2C12 myoblasts were treated with a pharmacological inhibitor of p110β (TGX-221), siRNA against p110β, or overexpression of kinase-dead p110β. Expression and phosphorylation of AMPK were unaffected in myoblasts treated with TGX-221 or expressing kinase-dead p110β. However, expressions of total and phosphorylated AMPK at T172 were reduced in myoblasts treated with p110β siRNA. When normalized to expression of total AMPK, phosphorylation of AMPK S485/491 was elevated in p110β-deficient myoblasts. Similar results were observed in tibialis anterior muscle from mice with conditional deletion of p110β (p110β-mKO mice). Analysis of AMPK transcript expression revealed decreased expression of Prkaa2 in p110β-deficient myoblasts and in p110β-mKO muscle. Loss of p110β had no effect on oligomycin-stimulated phosphorylation of AMPK or phosphorylated Acetyl-CoA carboxylase (ACC), although oligomycin-induced AMPK and ACC phosphorylation were increased in p110β-deficient myoblasts compared to oligomycin-stimulated control myoblasts when normalized to levels of total AMPK or ACC. Overall, these results suggest that p110β positively regulates expression of AMPK in cultured myoblasts and in skeletal muscle in vivo; moreover, despite the reduced abundance of AMPK in p110β-deficient myoblasts, loss of p110β does not appear to impair AMPK activation following stimulus. These findings thus reveal a novel role for p110β in mediating skeletal muscle metabolic signaling.

Histone acetyltransferase inhibitors antagonize AMP-activated protein kinase in postmortem glycolysis.

ObjectiveThe purpose of this study was to investigate the influence of AMP-activated protein kinase (AMPK) activation on protein acetylation and glycolysis in postmortem muscle to better understand the mechanism by which AMPK regulates postmortem glycolysis and meat quality.MethodsA total of 32 mice were randomly assigned to four groups and intraperitoneally injected with 5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR, a specific activator of AMPK), AICAR and histone acetyltransferase inhibitor II, or AICAR, Trichostatin A (TSA, an inhibitor of histone deacetylase I and II) and Nicotinamide (NAM, an inhibitor of the Sirt family deacetylases). After mice were euthanized, the Longissimus dorsi muscle was collected at 0 h, 45 min, and 24 h postmortem. AMPK activity, protein acetylation and glycolysis in postmortem muscle were measured.ResultsActivation of AMPK by AICAR significantly increased glycolysis in postmortem muscle. At the same time, it increased the total acetylated proteins in muscle 45 min postmortem. Inhibition of protein acetylation by histone acetyltransferase inhibitors reduced AMPK activation induced increase in the total acetylated proteins and glycolytic rate in muscle early postmortem, while histone deacetylase inhibitors further promoted protein acetylation and glycolysis. Several bands of proteins were detected to be differentially acetylated in muscle with different glycolytic rates.ConclusionProtein acetylation plays an important regulatory role in postmortem glycolysis. As AMPK mediates the effects of pre-slaughter stress on postmortem glycolysis, protein acetylation is likely a mechanism by which antemortem stress influenced postmortem metabolism and meat quality though the exact mechanism is to be elucidated.

ORM Promotes Skeletal Muscle Glycogen Accumulation via CCR5-Activated AMPK Pathway in Mice.

We found previously that acute phase protein orosomucoid reacts to fatigue and activates C-C chemokine receptor type 5 to increase muscle glycogen storage and enhance muscle endurance (Lei et al., 2016). To explore the underlying molecular mechanisms, we investigated the role of AMP-activated protein kinase, a critical fuel sensor in skeletal muscle, in C-C chemokine receptor type 5-mediated orosomucoid action. It was found orosomucoid increased skeletal muscle AMP-activated protein kinase activation in a time- and dose- dependent manner, which was largely prevented by pharmacological blocking or knockout of C-C chemokine receptor type 5. Administration of orosomucoid also significantly increased the de-phosphorylation and activity of muscle glycogen synthase, the rate-limiting enzyme for glycogen synthesis. The effect was largely absent in mice deficient in C-C chemokine receptor type 5−/− or AMP-activated protein kinase α2−/−, the predominant isoform in skeletal muscle. Moreover, deletion of AMP-activated protein kinase α2 abolished the effect of orosomucoid on fatigue and muscle glycogen. These findings indicate that orosomucoid may promote glycogen storage and enhance muscle function through C-C chemokine receptor type 5-mdiated activation of AMP-activated protein kinase, which in turn activates glycogen synthase and increases muscle glycogen.

Exercise reverses age-related vulnerability of the retina to injury by preventing complement-mediated synapse elimination via a BDNF-dependent pathway.

SummaryRetinal ganglion cells (RGCs) become increasingly vulnerable to injury with advancing age. We recently showed that this vulnerability can be strongly modified in mice by exercise. However, the characteristics and underlying mechanisms of retinal protection with exercise remain unknown. Hence, the aim of this study was to investigate cellular changes associated with exercise‐induced protection of aging retinal cells and the role of local and peripheral trophic signalling in mediating these effects. We focussed on two molecules that are thought to play key roles in mediating beneficial effects of exercise: brain‐derived neurotrophic factor (BDNF) and AMP‐activated protein kinase (AMPK). In middle‐aged (12 months old) C57BL/6J mice, we found that exercise protected RGCs against dysfunction and cell loss after an acute injury induced by elevation of intra‐ocular pressure. This was associated with preservation of inner retinal synapses and reduced synaptic complement deposition. Retinal expression of BDNF was not upregulated in response to exercise alone. Rather, exercise maintained BDNF levels in the retina, which were decreased postinjury in nonexercised animals. Confirming a critical role for BDNF, we found that blocking BDNF signalling during exercise by pharmacological means or genetic knock‐down suppressed the functional protection of RGCs afforded by exercise. Protection of RGCs with exercise was independent of activation of AMPK in either retina or skeletal muscle. Our data support a previously unidentified mechanism in which exercise prevents loss of BDNF in the retina after injury and preserves neuronal function and survival by preventing complement‐mediated elimination of synapses.