Skeletal Muscle AMP-activated Protein Kinase Research Articles

Leaf extracts of Euclea natalensis A.D.C ameliorate biochemical abnormalities in high-fat-low streptozotocin-induced diabetic rats through modulation of the AMPK-GLUT4 pathway

ABSTRACT Euclea natalensis is a commonly used plant in traditional African medicine for treating several diseases, including diabetes. The objective of the present study was to evaluate the antidiabetic effects of the leaf extracts of Euclea natalensis and elucidate some of its mechanisms of action. High-fat diet – low Streptozotocin-induced diabetic Sprague Dawley rats were orally treated with the methanol and dichloromethane extracts (100 and 200 mg/kg bw), and their fasting blood glucose levels, body weights, and food and water intake were measured every seven days for 21 days. Biomarkers of oxidative stress, lipid profile, gluconeogenic enzyme activities, glucose transporter 4 (GLUT 4) protein expression, and AMP-activated protein kinase (AMPK) gene expression were also determined. The 200 mg/kg bw of methanol extracts significantly reduced fasting plasma glucose, glycated hemoglobin (HbAc1), triglycerides, and low-density lipoprotein levels. The extract increased insulin sensitivity and hepatic glycogen synthesis, decreased hepatic glucose output, and increased the activities of antioxidant enzymes in diabetic animals after 21 days of treatment. The methanol extract also significantly upregulated the relative expression of GLUT 4 protein via the promoted expression of skeletal muscle and hepatic AMPK. Histological investigation showed ameliorated pancreatic degeneration and expedited regeneration of islets of Langerhans in diabetic rats treated with the methanol extract compared with the dichloromethane extract. The findings demonstrated that Euclea natalensis has beneficial effects on the biochemical and cell abnormalities of type 2 diabetes, partly via the modulation of the AMPK – GLUT 4 pathway.

Acute, next-generation AMPK activation initiates a disease-resistant gene expression program in dystrophic skeletal muscle.

Duchenne muscular dystrophy (DMD) is a life-limiting neuromuscular disorder characterized by muscle weakness and wasting. Previous proof-of-concept studies demonstrate that the dystrophic phenotype can be mitigated with the pharmacological stimulation of AMP-activated protein kinase (AMPK). However, first-generation AMPK activators have failed to translate from bench to bedside due to either their lack of potency or toxic, off-target effects. The identification of safe and efficacious molecules that stimulate AMPK in dystrophic muscle is of particular importance as it may broaden the therapeutic landscape for DMD patients regardless of their specific dystrophin mutation. Here, we demonstrate that a single dose of the next generation, orally-bioactive AMPK agonist MK-8722 (MK) to mdx mice evoked skeletal muscle AMPK and extensive downstream stimulation within 12h post-treatment. Specifically, MK elicited a gene expression profile indicative of a more disease-resistant slow, oxidative phenotype including increased peroxisome proliferator-activated receptor ɣ coactivator-1⍺ activity and utrophin levels. In addition, we observed augmented autophagy signaling downstream of AMPK, as well as elevations in critical autophagic genes such as Map1lc3 and Sqstm1 subsequent to the myonuclear accumulation of the master regulator of the autophagy gene program, transcription factor EB. Lastly, we show that pharmacological AMPK stimulation normalizes the expression of myogenic regulatory factors and amends activated muscle stem cell content in mdx muscle. Our results indicate that AMPK activation via MK enhances disease-mitigating mechanisms in dystrophic muscle and prefaces further investigation on the chronic effects of novel small molecule AMPK agonists.

Greater Phosphorylation of AMPK and Multiple AMPK Substrates in the Skeletal Muscle of 24-Month-Old Calorie Restricted Compared to Ad-Libitum Fed Male Rats.

AMP-activated protein kinase (AMPK), a highly conserved, heterotrimeric serine/threonine kinase with critical sensory and regulatory functions, is proposed to induce antiaging actions of caloric restriction (CR). Although earlier studies assessed CR's effects on AMPK in rodent skeletal muscle, the scope of these studies was narrow with a limited focus on older animals. This study's purpose was to fill important knowledge gaps related to CR's influence on AMPK in skeletal muscle of older animals. Therefore, using epitrochlearis muscles from 24-month-old ad-libitum fed (AL) and CR (consuming 65% of AL intake for 8 weeks), male Fischer-344 × Brown Norway F1 rats, we determined: (a) AMPK Thr172 phosphorylation (a key regulatory site) by immunoblot; (b) AMPKα1 and AMPKα2 activity (representing the 2 catalytic α-subunits of AMPK), and AMPKγ3 activity (representing AMPK complexes that include the skeletal muscle-selective regulatory γ3 subunit) using enzymatic assays; (c) phosphorylation of multiple protein substrates that are linked to CR-related effects (acetyl-CoA carboxylase [ACC], that regulates lipid oxidation; Beclin-1 and ULK1 that are autophagy regulatory proteins; Raptor, mTORC1 complex protein that regulates autophagy; TBC1D1 and TBC1D4 that regulate glucose uptake) by immunoblot; and (d) ATP and AMP concentrations (key AMPK regulators) by mass spectrometry. The results revealed significant CR-associated increases in the phosphorylation of AMPKThr172 and 4 AMPK substrates (ACC, Beclin-1, TBC1D1, and TBC1D4), without significant diet-related differences in ATP or AMP concentration or AMPKα1-, AMPKα2-, or AMPKγ3-associated activity. The enhanced phosphorylation of multiple AMPK substrates provides novel mechanistic insights linking AMPK to functionally important consequences of CR.

Structure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator.

The AMP-activated protein kinase (AMPK) αβγ heterotrimer is a primary cellular energy sensor and central regulator of energy homeostasis. Activating skeletal muscle AMPK with small molecule drugs improves glucose uptake and provides an opportunity for new strategies to treat type 2 diabetes and insulin resistance, with recent genetic and pharmacological studies indicating the α2β2γ1 isoform combination as the heterotrimer complex primarily responsible. With the goal of developing α2β2-specific activators, here we perform structure/function analysis of the 2-hydroxybiphenyl group of SC4, an activator with tendency for α2-selectivity that is also capable of potently activating β2 complexes. Substitution of the LHS 2-hydroxyphenyl group with polar-substituted cyclohexene-based probes resulted in two AMPK agonists, MSG010 and MSG011, which did not display α2-selectivity when screened against a panel of AMPK complexes. By radiolabel kinase assay, MSG010 and MSG011 activated α2β2γ1 AMPK with one order of magnitude greater potency than the pan AMPK activator MK-8722. A crystal structure of MSG011 complexed to AMPK α2β1γ1 revealed a similar binding mode to SC4 and the potential importance of an interaction between the SC4 2-hydroxyl group and α2-Lys31 for directing α2-selectivity. MSG011 induced robust AMPK signalling in mouse primary hepatocytes and commonly used cell lines, and in most cases this occurred in the absence of changes in phosphorylation of the kinase activation loop residue α-Thr172, a classical marker of AMP-induced AMPK activity. These findings will guide future design of α2β2-selective AMPK activators, that we hypothesise may avoid off-target complications associated with indiscriminate activation of AMPK throughout the body.

AMP-activated protein kinase activation in skeletal muscle modulates exercise-induced uncoupled protein 1 expression in brown adipocyte in mouse model.

Aerobic exercise is an effective intervention in preventing obesity and is also an important factor associated with thermogenesis. There is an increasing interest in the factors and mechanisms induced by aerobic exercise that can influence the metabolism and thermogenic activity in an individual. Recent studies suggest that exercise induced circulating factors (known as ‘exerkines’), which are able to modulate activation of brown adipose tissue (BAT) and browning of white adipose tissue. However, the underlying molecular mechanisms associated with the effect of exercise‐induced peripheral factors on BAT activation remain poorly understood. Furthermore, the role of exercise training in BAT activation is still debatable. Hence, the purpose of our study is to assess whether exercise training affects the expression of uncoupled protein 1 (UCP1) in brown adipocytes via release of different blood factors. Four weeks of exercise training significantly decreased the body weight gain and fat mass gain. Furthermore, trained mice exhibit higher levels of energy expenditure and UCP1 expression than untrained mice. Surprisingly, treatment with serum from exercise‐trained mice increased the expression of UCP1 in differentiated brown adipocytes. To gain a better understanding of these mechanisms, we analysed the conditioned media obtained after treating the C2C12 myotubes with an AMP‐activated protein kinase (AMPK) activator (AICAR; 5‐aminoimidazole‐4‐carboxamide ribonucleotide), which leads to an increased expression of UCP1 when added to brown adipocytes. Our observations suggest the possibility of aerobic exercise‐induced BAT activation via activation of AMPK in skeletal muscles.Key points Exercise promotes thermogenesis by activating uncoupling protein 1 (UCP1), which leads to a decrease in the body weight gain and body fat content. However, little is known about the role of exerkines in modulating UCP1 expression and subsequent brown adipose tissue (BAT) activation.Four weeks of voluntary wheel‐running exercise reduces body weight and fat content.Exercise induces the increase in AMP‐activated protein kinase (AMPK) and slow‐type muscle fibre marker genes in skeletal muscles and promotes UCP1 expression in white and brown adipose tissues.Incubation of brown adipocytes with serum isolated from exercise‐trained mice significantly increased their UCP1 gene and protein levels; moreover, conditioned media of AMPK‐activator‐treated C2C12 myotubes induces increased UCP1 expression in brown adipocytes.These results show that aerobic exercise‐induced skeletal muscle AMPK has a significant effect on UCP1 expression in BAT.

Disrupting AMPK-Glycogen Binding in Mice Increases Carbohydrate Utilization and Reduces Exercise Capacity.

The AMP-activated protein kinase (AMPK) is a central regulator of cellular energy balance and metabolism and binds glycogen, the primary storage form of glucose in liver and skeletal muscle. The effects of disrupting whole-body AMPK-glycogen interactions on exercise capacity and substrate utilization during exercise in vivo remain unknown. We used male whole-body AMPK double knock-in (DKI) mice with chronic disruption of AMPK-glycogen binding to determine the effects of DKI mutation on exercise capacity, patterns of whole-body substrate utilization, and tissue metabolism during exercise. Maximal treadmill running speed and whole-body energy utilization during submaximal running were determined in wild type (WT) and DKI mice. Liver and skeletal muscle glycogen and skeletal muscle AMPK α and β2 subunit content and signaling were assessed in rested and maximally exercised WT and DKI mice. Despite a reduced maximal running speed and exercise time, DKI mice utilized similar absolute amounts of liver and skeletal muscle glycogen compared to WT. DKI skeletal muscle displayed reduced AMPK α and β2 content versus WT, but intact relative AMPK phosphorylation and downstream signaling at rest and following exercise. During submaximal running, DKI mice displayed an increased respiratory exchange ratio, indicative of greater reliance on carbohydrate-based fuels. In summary, whole-body disruption of AMPK-glycogen interactions reduces maximal running capacity and skeletal muscle AMPK α and β2 content and is associated with increased skeletal muscle glycogen utilization. These findings highlight potential unappreciated roles for AMPK in regulating tissue glycogen dynamics and expand AMPK’s known roles in exercise and metabolism.

Skeletal Muscle‐Specific Deletion of AMPKβ1β2 Modulates Age‐Induced Neuromuscular Junction Remodeling in Fast Glycolytic Muscles

The neuromuscular junction (NMJ) is the electrochemical signaling apparatus between an ⍺-motoneuron and the skeletal muscle fibers that it innervates. AMP-activated protein kinase (AMPK) regulates neuromuscular phenotype. However, the precise role of this kinase at the NMJ has yet to be elucidated. The purpose of the current study was to examine the role of skeletal muscle AMPK on NMJ biology across the lifespan. We utilized 3- and 12-month-old (mo) skeletal muscle-specific AMPKβ1β2 knock out (mKO, n = 10) and wild-type (WT, n = 10) mice, as well as 22 mo WT (n = 3) animals. The extensor digitorum longus (EDL) and soleus (SOL) muscles were examined to represent fast glycolytic and slow oxidative tissues, respectively. Utilizing confocal microscopy, we acquired 20-25 NMJ images per muscle to conduct 3D NMJ morphology analyses. Contralateral EDL and SOL muscles were also collected for immunofluorescence staining and muscle innervation determination. Quantitative real-time PCR was employed to examine genes critical for NMJ maintenance and remodeling in tibialis anterior (TA) muscles. Presynaptic axonal blebbing and sprouting was observed more frequently (p < 0.05) in the EDL and SOL muscles of aged WT animals relative to their younger counterparts. Additionally, the proportion of dysmorphic axons was greater (p < 0.05) in mKO animals compared with age-matched WT mice. As expected, there was a significant main effect of age on the total number of fragmented and ectopic NMJs in both genotypes. mKO animals displayed a greater (p < 0.05) number of dysmorphic NMJs than their age-matched WT littermates, which suggests that AMPK is required for the development and maintenance of NMJ structure. Indeed, the number of NMJs positive for postsynaptic abnormalities in the EDL muscles of 12 mo mKO animals was significantly greater than 22 mo WT mice. Fiber type grouping was significantly higher in EDL and SOL muscles of 22 mo WT animals relative to their younger counterparts. Statistical trends (p = 0.09) were also observed towards greater fiber type grouping and increased muscle fibers co-expressing multiple myosin heavy chain isoforms in only the SOL muscles of mKO animals relative to their age-matched WT controls, which suggests that AMPK elicits muscle-specific denervation/reinnervation cycling. mRNA expression of the acetylcholine receptor gamma subunit was significantly greater in mKO mice relative to WT animals. Interestingly, additional NMJ transcripts, such as agrin, rapsyn, docking protein 7, and fibroblast growth factor binding protein 1, were significantly lower in mKO animals relative to their WT littermates. Collectively, these data suggest that the absence of skeletal muscle AMPK accelerates age-associated changes at the NMJ in more glycolytic muscle fibers, potentially due to a greater ability for reinnervation in oxidative muscles. These results reveal a novel role for AMPK in the maintenance and remodeling of the NMJ during aging.

Acute exercise stimulates AMPK and corrects some disease characteristics in the skeletal muscle of myotonic dystrophy type 1 mice

Myotonic Dystrophy type I (DM1) is the most prevalent adult form of muscular dystrophy and is characterized by myotonia, skeletal muscle weakness and wasting. The disease pathogenesis is characterized by a CTG microsatellite repeat expansion, leading to the dysregulation of pre-messenger RNA splicing of numerous muscle genes. There is currently no cure for this disorder, but recent evidence indicate that exercise is a safe and modestly effective therapy. AMP-activated protein kinase (AMPK) is critical to the acute responses and chronic adaptations to physical activity at the cellular and molecular level in the healthy condition, as well as in various disease states. However, the AMPK-signaling response to exercise in DM1 remains largely unknown. Therefore, the purpose of this study is to examine whether a single bout of exercise 1) activates AMPK and its downstream signalling network in DM1 skeletal muscle, and 2) modulates the DM1 molecular signature. Wild-type (WT) and HSA-LR (DM1) mice ran on a motor-driven treadmill until the inability to continue exercise was objectively determined, and the molecular response to physical activity at various timepoints post-exercise was examined using Western blotting, immunofluorescence microscopy, and qPCR techniques. WT mice ran for 937.6 ± 193.8 meters, which was significantly greater than DM1 mice at 573.3 ± 181.4meters. In the skeletal muscle of WT mice, AMPK activation status was significantly augmented immediately after exercise. This coincided with an increase (p < 0.05) in peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) transcript levels, as well as significant elevations in unc-51-like kinase 1 (ULK1) and calcium/calmodulin-dependent protein kinase type 2β (CAMKIIβ) activation. In DM1 mice, acute exercise also significantly stimulated AMPK, however, PGC-1α, ULK1 and CAMKIIβ were unaffected. In resting muscle, several markers related to autophagy downstream of AMPK, for example ULK1 activation status, p62 protein level, and the lipidated form of microtubule-associated protein 1-light chain 3, were significantly elevated in DM1 mice compared to their WT counterparts. These normalized after exercise. Acute physical activity did not impact either the prevalence of toxic myonuclear foci formed by the CTG microsatellite repeat or the proportion of mis-spliced muscle-specific chloride channel in DM1 mice. Collectively, these results indicate that while exercise-induced AMPK phosphorylation was preserved in the skeletal muscle of DM1 mice, markers of upstream and downstream AMPK signalling were attenuated in response to acute physical activity. Nonetheless, our data also suggest that exercise-evoked AMPK activation normalizes perturbations in the autophagy pathway observed in DM1 muscle. Thus, skeletal muscle AMPK stimulation after a single bout of exercise is very likely necessary but insufficient to elicit beneficial structural and functional adaptations in DM1.

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.

CARM1 Regulates AMPK Signaling in Skeletal Muscle.

SummaryCoactivator-associated arginine methyltransferase 1 (CARM1) is an emerging mediator of skeletal muscle plasticity. We employed genetic, physiologic, and pharmacologic approaches to determine whether CARM1 regulates the master neuromuscular phenotypic modifier AMP-activated protein kinase (AMPK). CARM1 skeletal muscle-specific knockout (mKO) mice displayed reduced muscle mass and dysregulated autophagic and atrophic processes downstream of AMPK. We observed altered interactions between CARM1 and AMPK and its network, including forkhead box protein O1, during muscle disuse. CARM1 methylated AMPK during the early stages of muscle inactivity, whereas CARM1 mKO mitigated progression of denervation-induced atrophy and was accompanied by attenuated phosphorylation of AMPK targets such as unc-51 like autophagy-activating kinase 1Ser555. Lower acetyl-coenzyme A corboxylaseSer79 phosphorylation, as well as reduced peroxisome proliferator-activated receptor-γ coactivator-1α, was also observed in mKO animals following acute administration of the direct AMPK activator MK-8722. Our study suggests that targeting CARM1-AMPK interplay may have broad impacts on neuromuscular health and disease.

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.

Contraction‐regulated mTORC1 and protein synthesis: Influence of AMPK and glycogen

AMP-activated protein kinase (AMPK)-dependent Raptor Ser792 phosphorylation does not influence mechanistic target of rapamycin complex 1 (mTORC1)-S6K1 activation by intense muscle contraction. α2 -AMPK activity-deficient mice have lower contraction-stimulated protein synthesis. Increasing glycogen activates mTORC1-S6K1. Normalizing muscle glycogen content rescues reduced protein synthesis in AMPK-deficient mice. The mechansitic target of rapamycin complex 1 (mTORC1)-S6K1 signalling pathway regulates muscle growth-related protein synthesis and is antagonized by AMP-activated protein kinase (AMPK) in multiple cell types. Resistance exercise stimulates skeletal muscle mTORC1-S6K1 and AMPK signalling and post-contraction protein synthesis. Glycogen inhibits AMPK and has been proposed as a pro-anabolic stimulus. The present study aimed to investigate how muscle mTORC1-S6K1 signalling and protein synthesis respond to resistance exercise-mimicking contraction in the absence of AMPK and with glycogen manipulation. Resistance exercise-mimicking unilateral in situ contraction of musculus quadriceps femoris in anaesthetized wild-type and dominant negative α2 AMPK kinase dead transgenic (KD-AMPK) mice, measuring muscle mTORC1 and AMPK signalling immediately (0h) and 4h post-contraction, and protein-synthesis at 4h. Muscle glycogen manipulation by 5day oral gavage of the glycogen phosphorylase inhibitor CP316819 and sucrose (80gL-1 ) in the drinking water prior to in situ contraction. The mTORC1-S6K1 and AMPK signalling axes were coactivated immediately post-contraction, despite potent AMPK-dependent Ser792 phosphorylation on the mTORC1subunit raptor. KD-AMPK muscles displayed normal mTORC1-S6K1 activation at 0h and 4h post-exercise, although there was impaired contraction-stimulated protein synthesis 4h post-contraction. Pharmacological/dietary elevation of muscle glycogen content augmented contraction-stimulated mTORC1-S6K1-S6 signalling and rescued the reduced protein synthesis-response in KD-AMPK to wild-type levels. mTORC-S6K1 signalling is not influenced by α2 -AMPK during or after intense muscle contraction. Elevated glycogen augments mTORC1-S6K1 signalling. α2 -AMPK-deficient KD-AMPK mice display impaired contraction-induced muscle protein synthesis, which can be rescued by normalizing muscle glycogen content.

Investigating the Function of Co‐activator‐associated Arginine Methyltransferase 1 (CARM1) During Denervation‐induced Skeletal Muscle Atrophy

CARM1 is a type I protein arginine methyltransferase (PRMT) that forms asymmetric dimethylarginines (ADMA) by transferring monomethyl groups to arginine residues on target proteins. We previously observed elevated CARM1 expression and methyltransferase activity in response to neurogenic muscle disuse. Transient knockdown of CARM1 in muscle also represses the progression of muscle wasting and the expression of atrophy‐related genes following denervation. Notably, CARM1 regulates intracellular signaling molecules that are important for atrophy such as AMP‐activated protein kinase (AMPK) and downstream unc‐51 like autophagy activating kinase 1 (ULK1), forkhead box class O (FOXO), and transcription factor EB (TFEB) in non‐muscle tissues. The purpose of this study was to more comprehensively elucidate the function of CARM1 during neurogenic muscle disuse. We employed the Cre/loxP system to generate skeletal muscle‐specific CARM1 knockout (mKO) mice. Following unilateral sectioning of the sciatic nerve, mKO and wild type (WT) mice were subjected to 3 and 7 days of denervation. The contralateral limb served as an internal control. Hematoxylin and eosin staining analyses were performed to assess cross‐sectional area (CSA) in the extensor digitorum longus (EDL) muscle. We also employed Western blot and immunoprecipitation analyses to evaluate protein expression and protein interaction levels, respectively, in the non‐denervated and denervated tibialis anterior muscles. Our CSA analyses revealed that the absence of CARM1 mitigated denervation‐induced atrophy in the EDL muscle after 7 days of disuse (p &lt; 0.05). Compared to WT animals, phosphorylation levels of AMPK and ULK1 were lower (p &lt; 0.05) in mKO mice following 3 days of neurogenic muscle disuse. We also report that CARM1 interacts with AMPK, FOXO1, and TFEB in skeletal muscle. Relative to the contralateral, non‐denervated control (CON) limb, the interactions between CARM1‐AMPK and CARM1‐FOXO1 increased (p &lt; 0.05) after 3 days of denervation in WT mice. When compared to the CON limb, the CARM1‐TFEB interaction was lower (p &lt; 0.05) in WT mice following 7 days of neurogenic muscle disuse. In comparison to WT animals, the methylation of AMPK was significantly blunted in mKO mice after 3 days of denervation, suggesting that CARM1 methyltransferase activity regulates AMPK signaling. Collectively, these results suggest that CARM1 interacts with intracellular signaling molecules that are integral for muscle wasting. Importantly, targeting the interplay between CARM1‐AMPK, CARM1‐FOXO1, and CARM1‐TFEB may provide a novel therapeutic strategy for mitigating neurogenic skeletal muscle atrophy.Support or Funding InformationNatural Sciences and Engineering Research Council of CanadaCanada Research Chairs

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.

Mechanisms of exercise-induced survival motor neuron expression in the skeletal muscle of spinal muscular atrophy-like mice.

Key points Spinal muscular atrophy (SMA) is a health‐ and life‐limiting neuromuscular disorder caused by a deficiency in survival motor neuron (SMN) protein.While historically considered a motor neuron disease, current understanding of SMA emphasizes its systemic nature, which requires addressing affected peripheral tissues such as skeletal muscle in particular.Chronic physical activity is beneficial for SMA patients, but the cellular and molecular mechanisms of exercise biology are largely undefined in SMA.After a single bout of exercise, canonical responses such as skeletal muscle AMP‐activated protein kinase (AMPK), p38 mitogen‐activated protein kinase (p38) and peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α) activation were preserved in SMA‐like Smn2B/− animals. Furthermore, molecules involved in SMN transcription were also altered following physical activity. Collectively, these changes were coincident with an increase in full‐length SMN transcription and corrective SMN pre‐mRNA splicing.This study advances understanding of the exercise biology of SMA and highlights the AMPK–p38–PGC‐1α axis as a potential regulator of SMN expression in muscle. Chronic physical activity is safe and effective in spinal muscular atrophy (SMA) patients, but the underlying cellular events that drive physiological adaptations are undefined. We examined the effects of a single bout of exercise on molecular mechanisms associated with adaptive remodelling in the skeletal muscle of Smn2B/− SMA‐like mice. Skeletal muscles were collected from healthy Smn2B/+ mice and Smn2B/− littermates at pre‐ (postnatal day (P) 9), early‐ (P13) and late‐ (P21) symptomatic stages to characterize SMA disease progression. Muscles were also collected from Smn2B/− animals exercised to fatigue on a motorized treadmill. Intracellular signalling and gene expression were examined using western blotting, confocal immunofluorescence microscopy, real‐time quantitative PCR and endpoint PCR assays. Basal skeletal muscle AMP‐activated protein kinase (AMPK) and p38 mitogen‐activated protein kinase (p38) expression and activity were not affected by SMA‐like conditions. Canonical exercise responses such as AMPK, p38 and peroxisome proliferator‐activated receptor γ coactivator‐1α (PGC‐1α) activation were observed following a bout of exercise in Smn2B/− animals. Furthermore, molecules involved in survival motor neuron (SMN) transcription, including protein kinase B (AKT) and extracellular signal‐regulated kinases (ERK)/ETS‐like gene 1 (ELK1), were altered following physical activity. Acute exercise was also able to mitigate aberrant proteolytic signalling in the skeletal muscle of Smn2B/− mice. Collectively, these changes were coincident with an exercise‐evoked increase in full‐length SMN mRNA expression. This study advances our understanding of the exercise biology of SMA and highlights the AMPK–p38–PGC‐1α axis as a potential regulator of SMN expression alongside AKT and ERK/ELK1 signalling.

Effect of electroacupuncture combined with treadmill exercise on body weight and expression of PGC-1α， Irisin and AMPK in skeletal muscle of diet-induced obesity rats

To investigate the effect of electroacupuncture (EA) plus treadmill exercise on the expression of peroxisome proliferator activated receptor γ coactivator 1α(PGC-1α), Irisin, AMP-activated protein kinase (AMPK) in skeletal muscle of diet-induced obesity (DIO) rats, so as to explore its mechanism underlying body reduction promotion. Forty-two male SD rats were divided into normal diet (control, n＝10), high fat diet (model), EA, treadmill exercise and EA plus treadmill exercise (combination) groups (n＝8 in each of the latter 4 groups). The obesity model was established by feeding the rats with high fat diet. EA (2 Hz/15 Hz, 1 mA) was applied to bilateral "Zusanli" (ST36) and "Tianshu" (ST25) for 30 min, 5 times per week for a total of 8 weeks. Rats of the treadmill exercise group were forced to perform exercise on a treadmill (16 m/min) for 30 min, 5 times per week for a total of 8 weeks. Rats in the combination group received the above-mentioned two methods. During the treatment, rats in the control group were fed with normal fodder, rats in other groups were fed with high fat fodder, and their body weight was measured once a week. The expression levels of PGC-1α， fibronectin type Ⅲ domain containing 5 (FNDC5), AMPK mRNA and protein of skeletal muscle were measured by quantitative real-time PCR and Western blot，respectively. After modeling, the body weight was significantly increased (P<0.05), and the expression levels of PGC-1α and FNDC5 mRNA and protein, AMPK mRNA and phosphorylated AMPK (p-AMPK) protein in the skeletal muscle were considerably decreased in the model group relevant to the control group (P<0.05). Following the treatment, the body weight was significantly down-regulated, while the expression levels of PGC-1α and FNDC5 mRNAs and proteins, AMPK mRNA and p-AMPK protein were obviously up-regulated in the EA, treadmill exercise and combination groups relevant to the model group (P<0.05). The therapeutic effect of EA plus treadmill exercise was significantly superior to those of both simple EA and simple treadmill exercise in down-regulating the body weight, as well as in up-regulating the expression of PGC-1α and FNDC5 mRNAs and proteins, AMPK mRNA, and p-AMPK protein (P<0.05). Both EA and treadmill exercise can significantly increase the expression of PGC-1α， FNDC5 and p-AMPK in skeletal muscle of DIO rats, suggesting their efficacy in restoring fatty acid oxidation in skeletal muscle cells and improving mitochondrial function, which may contribute to their function in body reduction. The therapeutic effect of EA plus treadmill exercise is better than that of simple EA and simple treadmill exercise.

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-245 Exercise at the lactate threshold (LT) and above the LT increases phosphorylation of AMPK and Akt in rat skeletal muscle

&#x0D; Objective A single bout of exercise can enhance glucose uptake in skeletal muscle. It is well established that AMP-activated protein kinase (AMPK) activation is required for stimulation of glucose uptake by exercise. After the initial phosphorylation of glucose by hexokinase, glucose is further utilized to mitochondrial oxidation during exercise. The direct or functional interaction between hexokinase and Akt may act to integrate glucose metabolism in working muscle. Hence, AMPK and Akt activation would be cooperatively regulated exercise-induced activation of glucose metabolism. Although exercise at the lactate threshold (LT) and above the LT sharply increase glucose uptake via increasing AMPK activity, whether LT exercise can also increase Akt activity is still unknown. Therefore, we examined the AMPK and Akt activity immediately after several intensities of exercise.&#x0D; Methods Male wistar rats (250-270 g) were randomly assigned to 3 groups: Resting control (sedentary, n=16), Low-intensity exercise (LIE: 10 m/min for 30 min, n=8), LT intensity exercise 1 (LTE1: 17.5 m/min for 30 min, n=8), LT intensity exercise 2 (LTE2: 22.5 m/min for 30 min, n=8), and High-intensity exercise (HE: 27.5 m/min for 30 min, n=8). Immediately after each treadmill exercise, plantaris and soleus muscles were dissected.&#x0D; Results LIE exercise did not changed AMPK phosphorylation site (Thr172), indicator of AMPK activity, and Akt phosphorylation site (Ser473, Thr308), indicator of Akt activity, in these muscles compared with resting control. At and above LTE1 exercise increased the phosphorylation of AMPK in these tissues. At and above LTE2 exercise increased the phosphorylation of Akt in these tissues. Therefore, increasing AMPK and Akt activity immediately after LT exercise possibly involved with regulating glucose metabolism.&#x0D; Conclusions Phosphorylation of AMPK and Akt is increased immediately after at and above LT exercise in rat soleus and plantaris muscle.&#x0D;

METRNL attenuates lipid-induced inflammation and insulin resistance via AMPK or PPAR\u03b4-dependent pathways in skeletal muscle of mice

Physical activity has many beneficial effects on metabolic disorders, such as obesity, insulin resistance, and diabetes. Meteorin-like protein (METRNL), a novel secreted protein homologous to the neurotrophin Metrn, is induced after exercise in the skeletal muscle. Herein, we investigated the effects of METRNL on lipid-mediated inflammation and insulin resistance in skeletal muscle via AMP-activated protein kinase (AMPK) or peroxisome proliferator-activated receptor δ (PPARδ). Treatment with METRNL suppressed inflammatory markers, such as nuclear factor κB (NFκB) nuclear translocation, inhibitory κBα (IκBα) phosphorylation, interleukin-6 (IL-6) expression, and pro-inflammatory cytokines (such as TNFα and MCP-1). METRNL treatment also attenuated the impaired insulin response both in palmitate-treated differentiated C2C12 cells and the skeletal muscle of high-fat diet (HFD)-fed mice. Furthermore, METRNL administration rescued glucose intolerance and reduced HFD-induced body weight gain in mice; however, METRNL did not affect calorie intake. METRNL treatment increased AMPK phosphorylation and PPARδ expression both in differentiated C2C12 cells and mouse skeletal muscle. siRNA-mediated suppression of AMPK and PPARδ abrogated the suppressive effects of METRNL on palmitate-induced inflammation and insulin resistance. Moreover, METRNL augmented the mRNA expression of fatty acid oxidation-associated genes, such as carnitine palmitoyltransferase 1 (CPT1), acyl-CoA oxidase (ACO), and fatty acid binding protein 3 (FABP3). siRNAs for AMPK and PPARδ reversed these changes. In the current study, we report for the first time that METRNL alleviates inflammation and insulin resistance and induces fatty acid oxidation through AMPK or PPARδ-dependent signaling in skeletal muscle.