ORCID Profile
0000-0001-6139-8268
Current Organisations
Københavns Universitet
,
IT University of Copenhagen
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Publisher: Wiley
Date: 16-10-2014
DOI: 10.1002/OBY.20615
Abstract: In obesity, immune cells infiltrate adipose tissue. Skeletal muscle is the major tissue of insulin‐dependent glucose disposal, and indices of muscle inflammation arise during obesity, but whether and which immune cells increase in muscle remain unclear. Immune cell presence in quadriceps muscle of wild type mice fed high‐fat diet (HFD) was studied for 3 days to 10 weeks, in CCL2‐KO mice fed HFD for 1 week, and in human muscle. Leukocyte presence was assessed by gene expression of lineage markers, cyto/chemokines and receptors immunohistochemistry and flow cytometry. After 1 week HFD, concomitantly with glucose intolerance, muscle gene expression of Ly6b, Emr1 (F4/80) , Tnf, Ccl2 , and Ccr2 rose, as did pro‐ and anti‐inflammatory markers Itgax (CD11c) and Mgl2 . CD11c + proinflammatory macrophages in muscle increased by 76%. After 10 weeks HFD, macrophages in muscle increased by 47%. Quadriceps from CCL2‐KO mice on HFD did not gain macrophages and maintained insulin sensitivity. Muscle of obese, glucose‐intolerant humans showed elevated CD68 (macrophage marker) and ITGAX, correlating with poor glucose disposal and adiposity. Mouse and human skeletal muscles gain a distinct population of inflammatory macrophages upon HFD or obesity, linked to insulin resistance in humans and CCL2 availability in mice.
Publisher: Cold Spring Harbor Laboratory
Date: 17-01-2019
DOI: 10.1101/522805
Abstract: Reactive oxygen species (ROS) act as intracellular compartmentalized second messengers mediating metabolic stress-adaptation. In skeletal muscle fibers, ROS have been suggested to stimulate glucose transporter 4 (GLUT4)-dependent glucose transport during artificially evoked contraction ex vivo but whether myocellular ROS production is stimulated by in vivo exercise to control metabolism is unclear. Here, we combined exercise in humans and mice with fluorescent dyes, genetically-encoded biosensors, and NADPH oxidase 2 (NOX2) loss-of-function models to demonstrate that NOX2 is the main source of cytosolic ROS during moderate-intensity exercise in skeletal muscle. Furthermore, two NOX2 loss-of-function mouse models lacking either p47phox or Rac1 presented striking phenotypic similarities, including greatly reduced exercise-stimulated glucose uptake and GLUT4 translocation. These findings indicate that NOX2 is a major myocellular ROS source regulating glucose transport capacity during moderate-intensity exercise.
Publisher: Cold Spring Harbor Laboratory
Date: 04-11-2021
DOI: 10.1101/2021.11.03.467058
Abstract: Metabolic dysfunction and insulin resistance are emerging as hallmarks of cancer and cachexia, and impair cancer prognosis. Yet, the molecular mechanisms underlying impaired metabolic regulation is not fully understood. To elucidate the mechanisms behind cancer-induced insulin resistance in muscle, we isolated extensor digitorum longus (EDL) and soleus muscles from Lewis Lung Carcinoma tumor-bearing mice. Three weeks after tumor inoculation, muscles were isolated and stimulated with or without a submaximal dose of insulin (1.5 nM). Glucose transport was measured using 2-[ 3 H]Deoxy-Glucose and intramyocellular signaling was investigated using immunoblotting. In soleus muscles from tumor-bearing mice, insulin-stimulated glucose transport was abrogated concomitantly with abolished insulin-induced TBC1D4 and GSK3 phosphorylation. In EDL, glucose transport and TBC1D4 phosphorylation were not impaired in muscles from tumor-bearing mice, while AMPK signaling was elevated. Anabolic insulin signaling via phosphorylation of the mTORC1 targets, p70S6K thr389 and ribosomal-S6 ser235, were decreased by cancer in soleus muscle while increased or unaffected in EDL. In contrast, the mTOR substrate, pULK1 ser757, was reduced in both soleus and EDL by cancer. Hence, cancer causes considerable changes in skeletal muscle insulin signaling that is dependent of muscle-type, which could contribute to metabolic dysregulation in cancer. Thus, skeletal muscle could be a target for managing metabolism in cancer. Cancer abrogates insulin-stimulated glucose transport selectively in oxidative soleus muscle Multiple TBC1D4 phosphorylation sites are reduced in cancer-associated muscle insulin resistance Cancer leads to increased AMPK signaling in the glycolytic EDL muscle Cancer alters anabolic insulin signaling in soleus and EDL muscle
Publisher: Springer Science and Business Media LLC
Date: 06-08-2020
DOI: 10.1038/S41598-020-69936-6
Abstract: Cellular self-organization is the fundamental driving force behind the complex architectures of native tissue. Yet, attempts at replicating native tissue architectures in vitro often involve complex micro-fabrication methods and materials. While impressive progress has been made within engineered models of striated muscle, the wide adaptation of these models is held back by the need for specific tools and knowhow. In this report, we show that C2C12 myoblasts spontaneously organize into highly aligned myotube tissues on the mm to cm scale, when cultured on sufficiently soft yet fully isotropic gelatin hydrogel substrates. Interestingly, we only observed this phenomenon for hydrogels with Young’s modulus of 6 kPa and below. For slightly more rigid compositions, only local micrometer-scale myotube organization was observed, similar to that seen in conventional polystyrene dishes. The hydrogel-supported myotubes could be cultured for multiple weeks and matured into highly contractile phenotypes with notable upregulation of myosin heavy chain, as compared to myotubes developed in conventional petri dishes. The procedure for casting the ultra-soft gelatin hydrogels is straight forward and compatible with standardized laboratory tools. It may thus serve as a simple, yet versatile, approach to generating skeletal muscle tissue of improved physiological relevance for applied and basic research.
Publisher: Elsevier BV
Date: 2019
DOI: 10.1016/J.CELLSIG.2018.10.014
Abstract: Botulinum toxin A (botox) is a toxin used for spasticity treatment and cosmetic purposes. Botox blocks the excitation of skeletal muscle fibers by preventing the release of acetylcholine from motor nerves, a process termed chemical denervation. Surgical denervation is associated with increased expression of the canonical insulin-activated kinase Akt, lower expression of glucose handling proteins GLUT4 and hexokinase II (HKII) and insulin resistant glucose uptake, but it is not known if botox has a similar effect. To test this, we performed a time-course study using supra-maximal insulin-stimulation in mouse soleus ex vivo. No effect was observed in the glucose transport responsiveness at day 1, 7 and 21 after intramuscular botox injection, despite lower expression of GLUT4, HKII and expression and phosphorylation of TBC1D4. Akt protein expression and phosphorylation of the upstream kinase Akt were increased by botox treatment at day 21. In a follow-up study, botox decreased submaximal insulin-stimulated glucose transport. The marked alterations of insulin signaling, GLUT4 and HKII and submaximal insulin-stimulated glucose transport are a potential concern with botox treatment which merit further investigation in human muscle. Furthermore, the botox-induced chemical denervation model may be a less invasive alternative to surgical denervation.
Publisher: Cold Spring Harbor Laboratory
Date: 07-02-2019
DOI: 10.1101/543736
Abstract: Skeletal muscle glucose uptake is essential for maintaining whole-body glucose homeostasis and accounts for the majority of glucose disposal in response to insulin. The group I p21-activated kinase (PAK) isoforms PAK1 and PAK2 are activated in response to insulin in skeletal muscle. Interestingly, PAK1/2 signalling is impaired in insulin-resistant mouse and human skeletal muscle and PAK1 has been suggested to be required for insulin-stimulated GLUT4 translocation. However, the relative contribution of PAK1 and PAK2 to insulin-stimulated glucose uptake in mature skeletal muscle is unresolved. The aim of the present investigation was to determine the requirement for PAK1 and PAK2 in whole-body glucose homeostasis and insulin-stimulated glucose uptake in skeletal muscle. Glucose uptake was measured in isolated skeletal muscle incubated with a pharmacological inhibitor (IPA-3) of group I PAKs and in muscle from whole-body PAK1 knockout (KO), muscle-specific PAK2 (m)KO and double whole-body PAK1 and muscle-specific PAK2 knockout mice. The whole-body respiratory exchange ratio was largely unaffected by lack of PAK1 and/or PAK2. Whole-body glucose tolerance was mildly impaired in PAK2 mKO, but not PAK1 KO mice. IPA-3 partially reduced (−20%) insulin-stimulated glucose uptake in mouse soleus muscle. In contrast to a previous study of GLUT4 translocation in PAK1 KO mice, PAK1 KO muscles displayed normal insulin-stimulated glucose uptake in vivo and in isolated muscle. On the contrary, glucose uptake was slightly reduced in response to insulin in glycolytic extensor digitorum longus muscle lacking PAK2, alone (−18%) or in combination with PAK1 KO (−12%). Insulin-stimulated glucose uptake partly relies on PAK2, but not PAK1, in mouse skeletal muscle. Thus, the present study challenges that group I PAKs, and especially PAK1, are major regulators of whole-body glucose homeostasis and insulin-stimulated glucose uptake in skeletal muscle.
Publisher: Elsevier BV
Date: 09-2020
Publisher: Wiley
Date: 05-2015
DOI: 10.1113/JP270414
Publisher: Proceedings of the National Academy of Sciences
Date: 31-08-2021
Abstract: The function of tissues is controlled by complex biological processes, in which transcriptional mechanisms play a fundamental role. The transcriptional coactivator PGC-1α is a prototypical regulator of adaptive responses evoked by environmental and internal cues (e.g. exercise-adaptation in skeletal muscle). Here, we found that PGC-1α-RNA–binding is central to assemble multiprotein complexes controlling gene transcription. Interestingly, PGC-1α-RNA–binding also regulates chromatin recruitment in transcriptionally active liquid-like nuclear condensates. Collectively, our findings unravel mechanistic insights into the regulation of complex transcriptional networks by PGC-1α, which we demonstrate to be essential to enhance the expression of key genes controlling skeletal muscle oxidative metabolism.
Publisher: American Physiological Society
Date: 08-2009
DOI: 10.1152/AJPENDO.90610.2008
Abstract: Conventional (c) protein kinase C (PKC) activity has been shown to increase with skeletal muscle contraction, and numerous studies using primarily pharmacological inhibitors have implicated cPKCs in contraction-stimulated glucose uptake. Here, to confirm that cPKC activity is required for contraction-stimulated glucose uptake in mouse muscles, contraction-stimulated glucose uptake ex vivo was first evaluated in the presence of three commonly used cPKC inhibitors (calphostin C, Gö-6976, and Gö-6983) in incubated mouse soleus and extensor digitorum longus (EDL) muscles. All potently inhibited contraction-stimulated glucose uptake by 50–100%, whereas both Gö compounds, but not calphostin C, inhibited insulin-stimulated glucose uptake modestly. AMP-activated protein kinase (AMPK) and eukaryotic elongation factor 2 phosphorylation was unaffected by the blockers. PKCα was estimated to account for ∼97% of total cPKC protein expression in skeletal muscle. However, in muscles from PKCα knockout (KO) mice, neither contraction- nor phorbol ester-stimulated glucose uptake ex vivo differed compared with the wild type. Furthermore, the effects of calphostin C and Gö-6983 on contraction-induced glucose uptake were similar in muscles lacking PKCα and in the wild type. It can be concluded that PKCα, representing ∼97% of cPKC in skeletal muscle, is not required for contraction-stimulated glucose uptake. Thus the effect of the PKC blockers on glucose uptake is either nonspecific working on other parts of contraction-induced signaling or the remaining cPKC isoforms are sufficient for stimulating glucose uptake during contractions.
Publisher: Wiley
Date: 23-02-2022
Publisher: American Society for Clinical Investigation
Date: 02-2005
Publisher: Elsevier BV
Date: 11-2015
Publisher: Elsevier BV
Date: 10-2014
Publisher: Springer Science and Business Media LLC
Date: 10-01-2022
DOI: 10.1038/S41467-021-27540-W
Abstract: Activation of the sympathetic nervous system causes pronounced metabolic changes that are mediated by multiple adrenergic receptor subtypes. Systemic treatment with β 2- adrenergic receptor agonists results in multiple beneficial metabolic effects, including improved glucose homeostasis. To elucidate the underlying cellular and molecular mechanisms, we chronically treated wild-type mice and several newly developed mutant mouse strains with clenbuterol, a selective β 2 -adrenergic receptor agonist. Clenbuterol administration caused pronounced improvements in glucose homeostasis and prevented the metabolic deficits in mouse models of β-cell dysfunction and insulin resistance. Studies with skeletal muscle-specific mutant mice demonstrated that these metabolic improvements required activation of skeletal muscle β 2 -adrenergic receptors and the stimulatory G protein, G s . Unbiased transcriptomic and metabolomic analyses showed that chronic β 2 -adrenergic receptor stimulation caused metabolic reprogramming of skeletal muscle characterized by enhanced glucose utilization. These findings strongly suggest that agents targeting skeletal muscle metabolism by modulating β 2 -adrenergic receptor-dependent signaling pathways may prove beneficial as antidiabetic drugs.
Publisher: Wiley
Date: 17-09-2020
DOI: 10.1113/JP280294
Publisher: American Physiological Society
Date: 07-2007
DOI: 10.1152/AJPENDO.00693.2006
Abstract: Previous studies have proposed that caffeine-induced activation of glucose transport in skeletal muscle is independent of AMP-activated protein kinase (AMPK) because α-AMPK Thr172 phosphorylation was not increased by caffeine. However, our previous studies, as well as the present, show that AMPK phosphorylation measured in whole muscle lysate is not a good indicator of AMPK activation in rodent skeletal muscle. In lysates from incubated rat soleus muscle, a predominant model in previous caffeine-studies, both acetyl-CoA carboxylase-β (ACCβ) Ser221 and immunoprecipitated α 1 -AMPK activity increased with caffeine incubation, without changes in AMPK phosphorylation or immunoprecipitated α 2 -AMPK activity. This pattern was also observed in mouse soleus muscle, where only ACCβ and α 1 -AMPK phosphorylation were increased following caffeine treatment. Preincubation with the selective CaMKK inhibitor STO-609 (5 μM), the CaM-competitive inhibitor KN-93 (10 μM), or the SR Ca 2+ release blocking agent dantrolene (10 μM) all inhibited ACCβ phosphorylation and α 1 -AMPK phosphorylation, suggesting that SR Ca 2+ release may work through a CaMKK-AMPK pathway. Caffeine-stimulated 2-deoxyglucose (2DG) uptake reflected the AMPK activation pattern, being increased with caffeine and inhibited by STO-609, KN-93, or dantrolene. The inhibition of 2DG uptake is likely causally linked to AMPK activation, since muscle-specific expression of a kinase-dead AMPK construct greatly reduced caffeine-stimulated 2DG uptake in mouse soleus. We conclude that a SR Ca 2+ -activated CaMKK may control α 1 -AMPK activation and be necessary for caffeine-stimulated glucose uptake in mouse soleus muscle.
Publisher: American Physiological Society
Date: 10-2016
DOI: 10.1152/AJPENDO.00237.2016
Abstract: AMP-activated protein kinase (AMPK) plays erse roles and coordinates complex metabolic pathways for maintenance of energy homeostasis. This could be explained by the fact that AMPK exists as multiple heterotrimer complexes comprising a catalytic α-subunit (α1 and α2) and regulatory β (β1 and β2)- and γ (γ1, γ2, γ3)-subunits, which are uniquely distributed across different cell types. There has been keen interest in developing specific and isoform-selective AMPK-activating drugs for therapeutic use and also as research tools. Moreover, establishing ways of enhancing cellular AMPK activity would be beneficial for both purposes. Here, we investigated if a recently described potent AMPK activator called 991, in combination with the commonly used activator 5-aminoimidazole-4-carboxamide riboside or contraction, further enhances AMPK activity and glucose transport in mouse skeletal muscle ex vivo. Given that the γ3-subunit is exclusively expressed in skeletal muscle and has been implicated in contraction-induced glucose transport, we measured the activity of AMPKγ3 as well as ubiquitously expressed γ1-containing complexes. We initially validated the specificity of the antibodies for the assessment of isoform-specific AMPK activity using AMPK-deficient mouse models. We observed that a low dose of 991 (5 μM) stimulated a modest or negligible activity of both γ1- and γ3-containing AMPK complexes. Strikingly, dual treatment with 991 and 5-aminoimidazole-4-carboxamide riboside or 991 and contraction profoundly enhanced AMPKγ1/γ3 complex activation and glucose transport compared with any of the single treatments. The study demonstrates the utility of a dual activator approach to achieve a greater activation of AMPK and downstream physiological responses in various cell types, including skeletal muscle.
Publisher: Wiley
Date: 16-06-2016
DOI: 10.1113/JP272039
Publisher: American Chemical Society (ACS)
Date: 04-01-2023
Publisher: Elsevier BV
Date: 09-2023
Publisher: Wiley
Date: 14-03-2019
DOI: 10.1113/EP087545
Publisher: Public Library of Science (PLoS)
Date: 28-07-2016
Publisher: Wiley
Date: 03-2012
Publisher: Wiley
Date: 04-2011
DOI: 10.1002/JCB.23053
Abstract: AMP-activated protein kinase (AMPK) is a serine/threonine kinase that regulates cellular and whole body energy homeostasis. In adipose tissue, activation of AMPK has been demonstrated in response to a variety of extracellular stimuli. However, the upstream kinase that activates AMPK in adipocytes remains elusive. Previous studies have identified LKB1 as a major AMPK kinase in muscle, liver, and other tissues. In certain cell types, Ca(2+) /calmodulin-dependent protein kinase kinase β (CaMKKβ) has been shown to activate AMPK in response to increases of intracellular Ca(2+) levels. Our aim was to investigate if LKB1 and/or CaMKK function as AMPK kinases in adipocytes. We used adipose tissue and isolated adipocytes from mice in which the expression of LKB1 was reduced to 10-20% of that of wild-type (LKB1 hypomorphic mice). We show that adipocytes from LKB1 hypomorphic mice display a 40% decrease in basal AMPK activity and a decrease of AMPK activity in the presence of the AMPK activator phenformin. We also demonstrate that stimulation of 3T3L1 adipocytes with intracellular [Ca(2+) ]-raising agents results in an activation of the AMPK pathway. The inhibition of CaMKK isoforms, particularly CaMKKβ, by the inhibitor STO-609 or by siRNAs, blocked Ca(2+) -, but not phenformin-, AICAR-, or forskolin-induced activation of AMPK, indicating that CaMKK activated AMPK in response to Ca(2+) . Collectively, we show that LKB1 is required to maintain normal AMPK-signaling in non-stimulated adipocytes and in the presence of phenformin. In addition, we demonstrate the existence of a Ca(2+) /CaMKK signaling pathway that can also regulate the activity of AMPK in adipocytes.
Publisher: Elsevier BV
Date: 10-2022
DOI: 10.1016/J.CMET.2022.07.003
Abstract: Exercise induces signaling networks to improve muscle function and confer health benefits. To identify ergent and common signaling networks during and after different exercise modalities, we performed a phosphoproteomic analysis of human skeletal muscle from a cross-over intervention of endurance, sprint, and resistance exercise. This identified 5,486 phosphosites regulated during or after at least one type of exercise modality and only 420 core phosphosites common to all exercise. One of these core phosphosites was S67 on the uncharacterized protein C18ORF25, which we validated as an AMPK substrate. Mice lacking C18ORF25 have reduced skeletal muscle fiber size, exercise capacity, and muscle contractile function, and this was associated with reduced phosphorylation of contractile and Ca 2+ handling proteins. Expression of C18ORF25 S66/67D phospho-mimetic reversed the decreased muscle force production. This work defines the ergent and canonical exercise phosphoproteome across different modalities and identifies C18ORF25 as a regulator of exercise signaling and muscle function.
Publisher: Elsevier BV
Date: 05-2019
DOI: 10.1016/J.CELLSIG.2019.02.001
Abstract: AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis, acting as a sensor of energy and nutrient status. As such, AMPK is considered a promising drug target for treatment of medical conditions particularly associated with metabolic dysfunctions. To better understand the downstream effectors and physiological consequences of AMPK activation, we have employed a chemical genetic screen in mouse primary hepatocytes in an attempt to identify novel AMPK targets. Treatment of hepatocytes with a potent and specific AMPK activator 991 resulted in identification of 65 proteins phosphorylated upon AMPK activation, which are involved in a variety of cellular processes such as lipid/glycogen metabolism, vesicle trafficking, and cytoskeleton organisation. Further characterisation and validation using mass spectrometry followed by immunoblotting analysis with phosphorylation site-specific antibodies identified AMPK-dependent phosphorylation of Gapex-5 (also known as GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1)) on Ser902 in hepatocytes and starch-binding domain 1 (STBD1) on Ser175 in multiple cells/tissues. As new promising roles of AMPK as a key metabolic regulator continue to emerge, the substrates we identified could provide new mechanistic and therapeutic insights into AMPK-activating drugs in the liver.
Publisher: American Diabetes Association
Date: 09-2020
DOI: 10.2337/DB20-0062
Abstract: Women with polycystic ovary syndrome (PCOS) have been shown to be less insulin sensitive compared with control (CON) women, independent of BMI. Training is associated with molecular adaptations in skeletal muscle, improving glucose uptake and metabolism in both healthy in iduals and patients with type 2 diabetes. In the current study, lean hyperandrogenic women with PCOS (n = 9) and healthy CON women (n = 9) completed 14 weeks of controlled and supervised exercise training. In CON, the training intervention increased whole-body insulin action by 26% and insulin-stimulated leg glucose uptake by 53% together with increased insulin-stimulated leg blood flow and a more oxidative muscle fiber type distribution. In PCOS, no such changes were found, despite similar training intensity and improvements in VO2max. In skeletal muscle of CON but not PCOS, training increased GLUT4 and HKII mRNA and protein expressions. These data suggest that the impaired increase in whole-body insulin action in women with PCOS with training is caused by an impaired ability to upregulate key glucose-handling proteins for insulin-stimulated glucose uptake in skeletal muscle and insulin-stimulated leg blood flow. Still, other important benefits of exercise training appeared in women with PCOS, including an improvement of the hyperandrogenic state.
Publisher: Elsevier BV
Date: 06-2019
Publisher: Cold Spring Harbor Laboratory
Date: 30-01-2020
DOI: 10.1101/2020.01.29.925024
Abstract: Muscle contraction stimulates skeletal muscle glucose transport. Since it occurs independently of insulin, it is an important alternative pathway to increase glucose uptake in insulin-resistant states, but the intracellular signalling mechanisms are not fully understood. Muscle contraction activates group I p21-activated kinases (PAKs) in mouse and human skeletal muscle. PAK1 and PAK2 are downstream targets of Rac1, which is a key regulator of contraction-stimulated glucose transport. Thus, PAK1 and PAK2 could be downstream effectors of Rac1 in contraction-stimulated glucose transport. The current study aimed to test the hypothesis that PAK1 and/or PAK2 regulate contraction-induced glucose transport. Glucose transport was measured in isolated soleus and extensor digitorum longus (EDL) mouse skeletal muscle incubated either in the presence or absence of a pharmacological inhibitor (IPA-3) of group I PAKs or originating from whole-body PAK1 knockout (KO), muscle-specific PAK2 (m)KO or double whole-body PAK1 and muscle-specific PAK2 knockout mice. IPA-3 attenuated (−22%) the increase in muscle glucose transport in response to electrically-stimulated contraction. PAK1 was dispensable for contraction-stimulated glucose uptake in both soleus and EDL muscle. Lack of PAK2, either alone (−13%) or in combination with PAK1 (−14%), reduced contraction-stimulated glucose transport compared to control littermates in EDL, but not soleus muscle. Contraction-stimulated glucose transport in isolated glycolytic mouse EDL muscle is partly dependent on PAK2, but not PAK1.
Publisher: Wiley
Date: 11-06-2017
DOI: 10.1113/JP274203
Publisher: Elsevier BV
Date: 11-2018
Publisher: Cold Spring Harbor Laboratory
Date: 09-10-2023
Publisher: Cold Spring Harbor Laboratory
Date: 28-05-2019
DOI: 10.1101/652479
Abstract: Immobilization of skeletal muscle in a stretched position is associated with marked protection against disuse atrophy. Some intramyocellular changes in known proteins and post-translational modifications were previously linked to this phenomenon but there are likely many presently unknown proteins and post-translational modifications that contribute to this beneficial effect. To identify novel proteins and phosphorylation events involved in stretch-induced reduction of disuse atrophy, we conducted a global unbiased screen of the changes occurring in skeletal muscle in control vs. 1 day and 1 week stretched cast-immobilized mouse tibialis anterior muscle, using quantitative tandem mass spectrometry on HILIC-fractionated muscle peptides with follow-up studies in transgenic mice and humans. Our mass spectrometry analyses detected 11714 phosphopeptides and 2081 proteins, of which 53 phosphopeptides and 5 proteins, 125 phosphopeptides and 43 proteins were deregulated after 1D and 7D of stretched immobilization, respectively. The sarcomere and muscle tendinous junction-associated putative multi-adaptor protein Xin was among the most highly upregulated proteins both in terms of phosphorylation and protein expression and was confirmed to increase with stretch but not disuse atrophy in mice and to increase and decrease with exercise and cast immobilization, respectively, in humans. Xin -/- mice were partially protected against disuse but not denervation atrophy in both stretched and flexed immobilized muscles compared to WT. This study identified Xin as a novel protein involved in disuse atrophy and also provides a resource to guide future hypothesis-driven investigations into uncovering critical factors in the protection against disuse atrophy.
Publisher: Wiley
Date: 29-01-2004
Publisher: American Diabetes Association
Date: 14-03-2013
DOI: 10.2337/DB12-0491
Abstract: In skeletal muscle, the actin cytoskeleton-regulating GTPase, Rac1, is necessary for insulin-dependent GLUT4 translocation. Muscle contraction increases glucose transport and represents an alternative signaling pathway to insulin. Whether Rac1 is activated by muscle contraction and regulates contraction-induced glucose uptake is unknown. Therefore, we studied the effects of in vivo exercise and ex vivo muscle contractions on Rac1 signaling and its regulatory role in glucose uptake in mice and humans. Muscle Rac1-GTP binding was increased after exercise in mice (∼60–100%) and humans (∼40%), and this activation was AMP-activated protein kinase independent. Rac1 inhibition reduced contraction-stimulated glucose uptake in mouse muscle by 55% in soleus and by 20–58% in extensor digitorum longus (EDL P & 0.01). In agreement, the contraction-stimulated increment in glucose uptake was decreased by 27% (P = 0.1) and 40% (P & 0.05) in soleus and EDL muscles, respectively, of muscle-specific inducible Rac1 knockout mice. Furthermore, depolymerization of the actin cytoskeleton decreased contraction-stimulated glucose uptake by 100% and 62% (P & 0.01) in soleus and EDL muscles, respectively. These are the first data to show that Rac1 is activated during muscle contraction in murine and human skeletal muscle and suggest that Rac1 and possibly the actin cytoskeleton are novel regulators of contraction-stimulated glucose uptake.
Publisher: Cold Spring Harbor Laboratory
Date: 30-10-2023
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1748-1716.2009.01979.X
Abstract: In skeletal muscle, the contraction-activated heterotrimeric 5'-AMP-activated protein kinase (AMPK) protein is proposed to regulate the balance between anabolic and catabolic processes by increasing substrate uptake and turnover in addition to regulating the transcription of proteins involved in mitochondrial biogenesis and other aspects of promoting an oxidative muscle phenotype. Here, the current knowledge on the expression of AMPK subunits in human quadriceps muscle and evidence from rodent studies suggesting distinct AMPK subunit expression pattern in different muscle types is reviewed. Then, the intensity and time dependence of AMPK activation in human quadriceps and rodent muscle are evaluated. Subsequently, a major part of this review critically examines the evidence supporting a necessary and/or sufficient role of AMPK in a broad spectrum of skeletal muscle contraction-relevant processes. These include glucose uptake, glycogen synthesis, post-exercise insulin sensitivity, fatty acid (FA) uptake, intramuscular triacylglyceride hydrolysis, FA oxidation, suppression of protein synthesis, proteolysis, autophagy and transcriptional regulation of genes relevant to promoting an oxidative phenotype.
Publisher: Springer Science and Business Media LLC
Date: 14-10-2017
Abstract: Skeletal muscle extracts glucose from the blood to maintain demand for carbohydrates as an energy source during exercise. Such uptake involves complex molecular signalling processes that are distinct from those activated by insulin. Exercise-stimulated glucose uptake is preserved in insulin-resistant muscle, emphasizing exercise as a therapeutic cornerstone among patients with metabolic diseases such as diabetes mellitus. Exercise increases uptake of glucose by up to 50-fold through the simultaneous stimulation of three key steps: delivery, transport across the muscle membrane and intracellular flux through metabolic processes (glycolysis and glucose oxidation). The available data suggest that no single signal transduction pathway can fully account for the regulation of any of these key steps, owing to redundancy in the signalling pathways that mediate glucose uptake to ensure maintenance of muscle energy supply during physical activity. Here, we review the molecular mechanisms that regulate the movement of glucose from the capillary bed into the muscle cell and discuss what is known about their integrated regulation during exercise. Novel developments within the field of mass spectrometry-based proteomics indicate that the known regulators of glucose uptake are only the tip of the iceberg. Consequently, many exciting discoveries clearly lie ahead.
Publisher: Cold Spring Harbor Laboratory
Date: 22-09-2022
DOI: 10.1101/2022.09.19.508621
Abstract: Microtubules serve as tracks for long-range intracellular trafficking of glucose transporter 4 (GLUT4), but the role of this process in skeletal muscle and insulin resistance is unclear. Here, we used fixed and live-cell imaging to study microtubule-based GLUT4 trafficking in human and mouse muscle fibers and L6 rat muscle cells. We found GLUT4 localized along and on the microtubules in mouse and human muscle fibers. Pharmacological microtubule disruption using Nocodazole (Noco) prevented long-range GLUT4 trafficking and depleted GLUT4-enriched structures at microtubule nucleation sites in a fully reversible manner. Using a perfused muscle-on-a-chip system to enable real-time glucose uptake measurements in isolated mouse skeletal muscle fibers, we observed that Noco maximally disrupted the microtubule network after 5 min without affecting insulin-stimulated glucose uptake. In contrast, a 2h Noco treatment markedly decreased insulin responsiveness of glucose uptake. Insulin resistance in mouse muscle fibers induced either in vitro by C2 ceramides or in vivo by diet-induced obesity, impaired microtubule-based GLUT4 trafficking. In L6 muscle cells, pharmacological activation of the microtubule motor protein kinesin-1 increased basal and insulin-stimulated GLUT4 translocation, whereas shRNA-mediated knockdown of the kinesin-1 protein encoding gene Kif5B reduced insulin-stimulated GLUT4 translocation. Thus, in adult skeletal muscle fibers, the microtubule network is essential for intramyocellular GLUT4 movement, likely functioning to maintain an insulin-responsive cell-surface recruitable GLUT4 pool via kinesin-1 mediated trafficking.
Publisher: Wiley
Date: 02-2022
DOI: 10.14814/PHY2.15183
Publisher: Elsevier BV
Date: 2022
Publisher: Cold Spring Harbor Laboratory
Date: 06-02-2019
DOI: 10.1101/542514
Abstract: Objective: Reactive oxygen species (ROS) have been proposed as signaling molecules mediating exercise training adaptation, but the ROS source has remained unclear. This study aimed to investigate the requirement for NADPH oxidase (NOX)2-dependent redox changes induced by acute and long-term high-intensity interval training (HIIT) in skeletal muscle in a mouse model lacking functional NOX2 complex due to deficient p47phox (Ncf1) subunit expression (ncf1* mutation). Methods: HIIT was investigated after an acute bout of exercise and after a chronic intervention (3x week for 6 weeks) in wildtype (WT) vs. NOX2 activity-deficient (ncf1*) mice. NOX2 activation during HIIT was measured using a genetically-encoded biosensor. Immunoblotting and single-fiber staining were performed to measure classical exercise-training responsive endpoints in skeletal muscle. Results: A single bout of HIIT increased NOX2 activity measured using electroporated p47roGFP oxidation immediately after exercise but not 1h after exercise. After a 6-week of HIIT regime, improvements in maximal running capacity and some muscle training-markers responded less to HIIT in the ncf1* mice compared to WT, including superoxide dismutase (SOD)2, catalase, hexokinase II (HK II), pyruvate dehydrogenase (PDH) and protein markers of mitochondrial oxidative phosphorylation complexes. Strikingly, HIIT-training increased mitochondrial network area and decreased fragmentation in WT mice only. Conclusion: This study provided evidence that HIIT exercise activates NOX2 complex in skeletal muscle and that the presence of functional NOX2 is required for specific skeletal muscle adaptations to HIIT relating to antioxidant defense, glucose metabolism, and mitochondria.
Publisher: American Physiological Society
Date: 2022
DOI: 10.1152/AJPENDO.00389.2021
Abstract: The mTORC2 readout p-NDRG Thr346 is a novel exercise-responsive protein in human skeletal muscle. β2-AR and G S signaling are not sufficient to induce mTORC2 signaling in adult muscle. In vivo, but not ex vivo, contraction induced p-NDRG Thr346, which indicates requirement of a systemic factor for exercise-induced mTORC2 activation.
Publisher: Elsevier BV
Date: 10-2011
DOI: 10.1016/J.CELLSIG.2011.05.022
Abstract: Small Rho family GTPases are important regulators of cellular traffic. Emerging evidence now implicates Rac1 and Rac-dependent actin reorganisation in insulin-induced recruitment of glucose transporter-4 (GLUT4) to the cell surface of muscle cells and mature skeletal muscle. This review summarises the current thinking on the regulation of Rac1 by insulin, the role of Rac-dependent cortical actin remodelling in GLUT4 traffic, and the impact of Rac1 towards insulin resistance in skeletal muscle.
Publisher: The Endocrine Society
Date: 10-06-2016
DOI: 10.1210/EN.2016-1220
Abstract: Exercise has a potent insulin-sensitivity enhancing effect on skeletal muscle, but the intracellular mechanisms that mediate this effect are not well understood. In muscle, Ras-related C3 botulinum toxin substrate 1 (Rac1) regulates both insulin- and contraction-stimulated glucose transport and is dysregulated in insulin resistant muscle. However, whether Rac1 is involved in mediating enhanced insulin sensitivity after an acute bout of exercise is unresolved. To address this question, we investigated after exercise whole-body (insulin tolerance test) as well as muscle (insulin-stimulated 2-deoxyglucose transport in isolated soleus muscle) insulin sensitivity in inducible muscle-specific Rac1 knockout (mKO) and wild-type (WT) littermate mice. Previous exercise enhanced whole-body insulin sensitivity by 40% in WT mice and rescued the insulin intolerance in Rac1 mKO mice by improving whole-body insulin sensitivity by 230%. In agreement, previous exercise significantly improved insulin sensitivity by 20% in WT and by 40% in Rac1 mKO soleus muscles. These findings suggest that muscle Rac1 is dispensable for the insulin sensitizing effect of exercise. Moreover, insulin resistance in Rac1 mKO mice can be completely normalized by previous exercise explaining why insulin resistant patients can increase insulin action with exercise despite dysfunctional Rac1 activity in muscle.
Publisher: American Physiological Society
Date: 03-2011
Publisher: Cold Spring Harbor Laboratory
Date: 12-04-2023
DOI: 10.1101/2023.04.10.535538
Abstract: Pre-clinical models suggest a causative nexus between mitochondrial oxidative stress and insulin resistance. However, the translational and pathophysiological significance of this mechanism in humans remains unclear. Herein, we employed an invasive in vivo mechanistic approach in humans to manipulate mitochondrial redox state while assessing insulin action. To this end, we combined intravenous infusion of a lipid overload with intake of a mitochondria-targeted antioxidant (mtAO) in conjunction with insulin cl studies. During lipid overload, insulin-stimulated muscle glucose uptake, as determined by the femoral arteriovenous balance technique, was increased by mtAO. At the muscle molecular level, mtAO did not affect canonical insulin signaling but augmented insulin-stimulated GLUT4 translocation while decreasing the mitochondrial oxidative burden under lipid oversupply. Ex vivo studies revealed that mtAO ameliorated features of mitochondrial bioenergetics, including diminished mitochondrial H 2 O 2 emission, in muscle fibers exposed to high intracellular lipid levels. These findings provide translational and mechanistic evidence implicating mitochondrial oxidants in the development of lipid-induced muscle insulin resistance in humans.
Publisher: Cold Spring Harbor Laboratory
Date: 07-06-2022
DOI: 10.1101/2022.06.07.495096
Abstract: Metabolic dysfunction and cancer cachexia are associated with poor cancer prognosis, yet the molecular mechanisms causing cancer-induced metabolic dysfunction and cachexia remain to be defined. A key link between metabolic- and muscle mass-regulation is adenosine monophosphate-activated protein kinase (AMPK). As AMPK could be a potential treatment, it is important to determine the function for AMPK in cancer-associated metabolic dysfunction and cachexia. Here we determined the function of AMPK in cancer-associated metabolic dysfunction, insulin resistance, and cachexia. In vastus lateralis muscle biopsies from pre-cachectic and cachectic patients with Non-Small-Cell Lung Carcinoma (NSCLC), AMPK signaling and expression were examined by immunoblotting. To investigate the role of muscle AMPK, male mice overexpressing a dominant-negative AMPKα2 (kinase-dead) specifically in striated muscle (mAMPK-KD) were inoculated with Lewis Lung Carcinoma (LLC) cells. In a subsequent cohort, male LLC-tumor-bearing mice were treated with/without 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) to activate AMPK for 13 days. Littermate mice were used as control. Metabolic phenotyping of mice was performed via indirect calorimetry, body composition analyses, glucose- and insulin tolerance tests, tissue-specific 2-deoxy- glucose (2-DG) uptake, and immunoblotting. In muscle from patients with NSCLC, we found increased expression of AMPK subunits α1, α2, β2, γ1, and γ3 ranging from +27% to +79% compared to healthy control subjects. AMPK subunit expression correlated with indices of cachexia, including cross sectional area and weight loss. Tumor-bearing mAMPK-KD mice presented increased fat loss as well as glucose and insulin intolerance. LLC in mAMPK-KD mice displayed lower insulin-stimulated 2-DG uptake in skeletal muscle (quadriceps −35%, soleus −49%, EDL −48%) and the heart (−29%) compared to non-tumor-bearing mice. In skeletal muscle, mAMPK-KD abrogated the tumor-induced increase in phosphorylation of TBC1D4 thr642 . Additionally, protein expression of TBC1D4 (+26%), pyruvate dehydrogenase (PDH, +94%), and PDH-kinases (PDKs, +45% to +100%), and glycogen synthase (+48%) were increased in skeletal muscle of tumor-bearing mice in an AMPK-dependent manner. Lastly, chronic AICAR treatment elevated hexokinase-II protein expression and normalized phosphorylation of p70S6K thr389 (mTORC1 substrate) and ACC ser212 (AMPK substrate) and rescued the cancer-induced insulin intolerance. Upregulated protein expression of AMPK subunits observed in skeletal muscle of (pre)cachectic patients with non-small-cell lung carcinoma. This seemed protective inferred by AMPK-deficient tumor-bearing mice being highly prone to developing metabolic dysfunction, which included the AMPK-dependent regulation of several proteins involved in glucose metabolism. These observations highlight the potential for targeting AMPK to counter cancer-associated metabolic dysfunction and cachexia.
Publisher: American Physiological Society
Date: 12-2011
DOI: 10.1152/AJPREGU.00066.2011
Abstract: Exercise-induced phosphorylation of FXYD1 is a potential important regulator of Na + -K + -pump activity. It was investigated whether skeletal muscle contractions induce phosphorylation of FXYD1 and whether protein kinase Cα (PKCα) activity is a prerequisite for this possible mechanism. In part 1, human muscle biopsies were obtained at rest, after 30 s of high-intensity exercise (166 ± 31% of V̇o 2max ) and after a subsequent 20 min of moderate-intensity exercise (79 ± 8% of V̇o 2max ). In general, FXYD1 phosphorylation was increased compared with rest both after 30 s ( P 0.05) and 20 min ( P 0.001), and more so after 20 min compared with 30 s ( P 0.05). Specifically, FXYD1 ser63, ser68, and combined ser68 and thr69 phosphorylation were 26–45% higher ( P 0.05) after 20 min of exercise than at rest. In part 2, FXYD1 phosphorylation was investigated in electrically stimulated soleus and EDL muscles from PKCα knockout (KO) and wild-type (WT) mice. Contractile activity caused FXYD1 ser68 phosphorylation to be increased ( P 0.001) in WT soleus muscles but to be reduced ( P 0.001) in WT extensor digitorum longus. In contrast, contractile activity did not affect FXYD1 ser68 phosphorylation in the KO mice. In conclusion, exercise induces FXYD1 phosphorylation at multiple sites in human skeletal muscle. In mouse muscles, contraction-induced changes in FXYD1 ser68 phosphorylation are fiber-type specific and dependent on PKCα activity.
Publisher: American Physiological Society
Date: 05-2007
DOI: 10.1152/AJPENDO.00456.2006
Abstract: The Ca 2+ /calmodulin (CaM) competitive inhibitor KN-93 has previously been used to evaluate 5′-AMP-activated protein kinase (AMPK)-independent Ca 2+ -signaling to contraction-stimulated glucose uptake in muscle during intense electrical stimulation ex vivo. With the use of low-intensity tetanic contraction of mouse soleus and extensor digitorum longus (EDL) muscles ex vivo, this study demonstrates that KN-93 can potently inhibit AMPK phosphorylation and activity after 2 min but not 10 min of contraction while strongly inhibiting contraction-stimulated 2-deoxyglucose uptake at both the 2- and 10-min time points. These data suggest inhibition of Ca 2+ /CaM-dependent signaling events upstream of AMPK, the most likely candidate being the novel AMPK kinase CaM-dependent protein kinase kinase (CaMKK). CaMKK protein expression was detected in mouse skeletal muscle. Similar to KN-93, the CaMKK inhibitor STO-609 strongly reduced AMPK phosphorylation and activity at 2 min and less potently at 10 min. Pretreatment with STO-609 inhibited contraction-stimulated glucose uptake at 2 min in soleus, but not EDL, and in both muscles after 10 min. Neither KN-93 nor STO-609 inhibited 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside-stimulated glucose uptake, AMPK phosphorylation, or recombinant LKB1 activity, suggestive of an LKB1-independent effect. Finally, neither KN-93 nor STO-609 had effects on the reductions in glucose uptake seen in mice overexpressing a kinase-dead AMPK construct, indicating that the effects of KN-93 and STO-609 on glucose uptake require inhibition of AMPK activity. We propose that CaMKKs act in mouse skeletal muscle regulating AMPK phosphorylation and glucose uptake at the onset of mild tetanic contraction and that an intensity- and/or time-dependent switch occurs in the relative importance of AMPKKs during contraction.
Publisher: Canadian Science Publishing
Date: 10-2007
DOI: 10.1139/H07-079
Abstract: The 5′-AMP-activated protein kinase (AMPK) functions as an intracellular fuel sensor that affects metabolism and gene expression. AMPK is activated in skeletal muscle in response to exercise and is therefore believed to be an important signalling molecule in regulating adaptation of skeletal muscle to exercise training. This review first focuses on mechanisms regulating AMPK activity during muscle contraction. We then discuss the role of AMPK in regulating expression of genes encoding various enzymes in muscle in the basal state and in relation to exercise training. Although decreased AMPK activity in muscle causes reduced protein expression of mitochondrial enzymes in the basal state, AMPK does not appear to be indispensable for exercise-training induced increase in mitochondrial enzyme expression.
Publisher: eLife Sciences Publications, Ltd
Date: 31-03-2023
Publisher: American Diabetes Association
Date: 07-2006
DOI: 10.2337/DB06-0175
Abstract: AMP-activated protein kinase (AMPK) is a heterotrimeric protein that regulates glucose transport mediated by cellular stress or pharmacological agonists such as 5-aminoimidazole-4-carboxamide 1 β-d-ribonucleoside (AICAR). AS160, a Rab GTPase-activating protein, provides a mechanism linking AMPK signaling to glucose uptake. We show that AICAR increases AMPK, acetyl-CoA carboxylase, and AS160 phosphorylation by insulin-independent mechanisms in isolated skeletal muscle. Recombinant AMPK heterotrimeric complexes (α1β1γ1 and α2β2γ1) phosphorylate AS160 in a cell-free assay. In mice deficient in AMPK signaling (α2 AMPK knockout [KO], α2 AMPK kinase dead [KD], and γ3 AMPK KO), AICAR effects on AS160 phosphorylation were severely blunted, highlighting that complexes containing α2 and γ3 are necessary for AICAR-stimulated AS160 phosphorylation in intact skeletal muscle. Contraction-mediated AS160 phosphorylation was also impaired in α2 AMPK KO and KD but not γ3 AMPK KO mice. Our results implicate AS160 as a downstream target of AMPK.
Publisher: Proceedings of the National Academy of Sciences
Date: 26-06-2023
Abstract: The molecular events governing skeletal muscle glucose uptake have pharmacological potential for managing insulin resistance in conditions such as obesity, diabetes, and cancer. With no current pharmacological treatments to target skeletal muscle insulin sensitivity, there is an unmet need to identify the molecular mechanisms that control insulin sensitivity in skeletal muscle. Here, the Rho guanine dissociation inhibitor α (RhoGDIα) is identified as a point of control in the regulation of insulin sensitivity. In skeletal muscle cells, RhoGDIα interacted with, and thereby inhibited, the Rho GTPase Rac1. In response to insulin, RhoGDIα was phosphorylated at S101 and Rac1 dissociated from RhoGDIα to facilitate skeletal muscle GLUT4 translocation. Accordingly, siRNA-mediated RhoGDIα depletion increased Rac1 activity and elevated GLUT4 translocation. Consistent with RhoGDIα’s inhibitory effect, rAAV-mediated RhoGDIα overexpression in mouse muscle decreased insulin-stimulated glucose uptake and was detrimental to whole-body glucose tolerance. Aligning with RhoGDIα’s negative role in insulin sensitivity, RhoGDIα protein content was elevated in skeletal muscle from insulin-resistant patients with type 2 diabetes. These data identify RhoGDIα as a clinically relevant controller of skeletal muscle insulin sensitivity and whole-body glucose homeostasis, mechanistically by modulating Rac1 activity.
Publisher: Wiley
Date: 04-2015
DOI: 10.14814/PHY2.13798
Publisher: Wiley
Date: 29-09-2014
DOI: 10.1113/EXPPHYSIOL.2014.081679
Abstract: In many cell types, Ca(2+) signals to increase the movement and surface membrane insertion of vesicles. In skeletal muscle, Ca(2+) is predominantly released from the sarcoplasmic reticulum (SR) to initiate contraction. Sarcoplasmic reticulum Ca(2+) release is widely believed to be a direct feedforward regulator of the translocation of glucose transporter 4 to the cell surface to facilitate transmembrane glucose transport. This review summarizes the evidence supporting the Ca(2+) feedforward model and its proposed signalling links to regulation of glucose transport in skeletal muscle and other cell types. The literature is contrasted against our recent findings suggesting that SR Ca(2+) release is neither essential nor adequate to stimulate glucose transport in muscle. Instead, feedback signals through AMPK and mechanical stress are likely to account for most of contraction-stimulated glucose transport. A revised working model is proposed, in which muscle glucose transport during contraction is not directly regulated by SR Ca(2+) release but rather responds exclusively to feedback signals activated secondary to cross-bridge cycling and tension development.
Publisher: Portland Press Ltd.
Date: 17-04-2015
DOI: 10.1042/BJ20141142
Abstract: PT-1 activates AMP-activated protein kinase (AMPK)-γ1- but not-γ3-containing complexes in mouse muscle, but PT-1 activates all three γ isoforms in HEK293 cells. PT-1 activates AMPK not by direct binding to α subunits, but by inhibiting the respiratory chain and increasing cellular AMP.
Publisher: Elsevier BV
Date: 04-2020
DOI: 10.1016/J.METABOL.2020.154169
Abstract: Redirecting glucose from skeletal muscle and adipose tissue, likely benefits the tumor's energy demand to support tumor growth, as cancer patients with type 2 diabetes have 30% increased mortality rates. The aim of this study was to elucidate tissue-specific contributions and molecular mechanisms underlying cancer-induced metabolic perturbations. Glucose uptake in skeletal muscle and white adipose tissue (WAT), as well as hepatic glucose production, were determined in control and Lewis lung carcinoma (LLC) tumor-bearing C57BL/6 mice using isotopic tracers. Skeletal muscle microvascular perfusion was analyzed via a real-time contrast-enhanced ultrasound technique. Finally, the role of fatty acid turnover on glycemic control was determined by treating tumor-bearing insulin-resistant mice with nicotinic acid or etomoxir. LLC tumor-bearing mice displayed reduced insulin-induced blood-glucose-lowering and glucose intolerance, which was restored by etomoxir or nicotinic acid. Insulin-stimulated glucose uptake was 30-40% reduced in skeletal muscle and WAT of mice carrying large tumors. Despite compromised glucose uptake, tumor-bearing mice displayed upregulated insulin-stimulated phosphorylation of TBC1D4 Cancer can result in marked perturbations on at least six metabolically essential functions i) insulin's blood-glucose-lowering effect, ii) glucose tolerance, iii) skeletal muscle and WAT insulin-stimulated glucose uptake, iv) intramyocellular insulin signaling, v) muscle microvascular perfusion, and vi) basal hepatic glucose production in mice. The mechanism causing cancer-induced insulin resistance may relate to fatty acid metabolism.
Publisher: Springer Science and Business Media LLC
Date: 11-10-2019
DOI: 10.1038/S41467-019-12523-9
Abstract: Reactive oxygen species (ROS) act as intracellular compartmentalized second messengers, mediating metabolic stress-adaptation. In skeletal muscle fibers, ROS have been suggested to stimulate glucose transporter 4 (GLUT4)-dependent glucose transport during artificially evoked contraction ex vivo, but whether myocellular ROS production is stimulated by in vivo exercise to control metabolism is unclear. Here, we combined exercise in humans and mice with fluorescent dyes, genetically-encoded biosensors, and NADPH oxidase 2 (NOX2) loss-of-function models to demonstrate that NOX2 is the main source of cytosolic ROS during moderate-intensity exercise in skeletal muscle. Furthermore, two NOX2 loss-of-function mouse models lacking either p47phox or Rac1 presented striking phenotypic similarities, including greatly reduced exercise-stimulated glucose uptake and GLUT4 translocation. These findings indicate that NOX2 is a major myocellular ROS source, regulating glucose transport capacity during moderate-intensity exercise.
Publisher: Frontiers Media SA
Date: 2012
Publisher: American Diabetes Association
Date: 06-08-2010
DOI: 10.2337/DB09-1716
Abstract: The induction of obesity, dyslipidemia, and insulin resistance by high-fat diet in rodents can be prevented by n-3 long-chain polyunsaturated fatty acids (LC-PUFAs). We tested a hypothesis whether AMP-activated protein kinase (AMPK) has a role in the beneficial effects of n-3 LC-PUFAs. Mice with a whole-body deletion of the α2 catalytic subunit of AMPK (AMPKα2−/−) and their wild-type littermates were fed on either a low-fat chow, or a corn oil-based high-fat diet (cHF), or a cHF diet with 15% lipids replaced by n-3 LC-PUFA concentrate (cHF+F). Feeding a cHF diet induced obesity, dyslipidemia, hepatic steatosis, and whole-body insulin resistance in mice of both genotypes. Although cHF+F feeding increased hepatic AMPKα2 activity, the body weight gain, dyslipidemia, and the accumulation of hepatic triglycerides were prevented by the cHF+F diet to a similar degree in both AMPKα2−/− and wild-type mice in ad libitum-fed state. However, preservation of hepatic insulin sensitivity by n-3 LC-PUFAs required functional AMPKα2 and correlated with the induction of adiponectin and reduction in liver diacylglycerol content. Under hyperinsulinemic-euglycemic conditions, AMPKα2 was essential for preserving low levels of both hepatic and plasma triglycerides, as well as plasma free fatty acids, in response to the n-3 LC-PUFA treatment. Our results show that n-3 LC-PUFAs prevent hepatic insulin resistance in an AMPKα2-dependent manner and support the role of adiponectin and hepatic diacylglycerols in the regulation of insulin sensitivity. AMPKα2 is also essential for hypolipidemic and antisteatotic effects of n-3 LC-PUFA under insulin-stimulated conditions.
Publisher: American Diabetes Association
Date: 18-10-2010
DOI: 10.2337/DB10-0698
Abstract: We have previously shown that overnight fasted women have higher insulin-stimulated whole body and leg glucose uptake despite a higher intramyocellular triacylglycerol concentration than men. Women also express higher muscle mRNA levels of proteins related to lipid metabolism than men. We therefore hypothesized that women would be less prone to lipid-induced insulin resistance. Insulin sensitivity of whole-body and leg glucose disposal was studied in 16 young well-matched healthy men and women infused with intralipid or saline for 7 h. Muscle biopsies were obtained before and during a euglycemic-hyperinsulinemic cl (1.42 mU · kg−1 · min−1). Intralipid infusion reduced whole-body glucose infusion rate by 26% in women and 38% in men (P & 0.05), and insulin-stimulated leg glucose uptake was reduced significantly less in women (45%) than men (60%) after intralipid infusion. Hepatic glucose production was decreased during the cl similarly in women and men irrespective of intralipid infusion. Intralipid did not impair insulin or AMPK signaling in muscle and subcutaneous fat, did not cause accumulation of muscle lipid intermediates, and did not impair insulin-stimulated glycogen synthase activity in muscle or increase plasma concentrations of inflammatory cytokines. In vitro glucose transport in giant sarcolemmal vesicles was not decreased by acute exposure to fatty acids. Leg lactate release was increased and respiratory exchange ratio was decreased by intralipid. Intralipid infusion causes less insulin resistance of muscle glucose uptake in women than in men. This insulin resistance is not due to decreased canonical insulin signaling, accumulation of lipid intermediates, inflammation, or direct inhibition of GLUT activity. Rather, a higher leg lactate release and lower glucose oxidation with intralipid infusion may suggest a metabolic feedback regulation of glucose metabolism.
Publisher: American Society for Clinical Investigation
Date: 11-2004
DOI: 10.1172/JCI21480
Publisher: Wiley
Date: 27-08-2009
Publisher: American Physiological Society
Date: 10-2009
DOI: 10.1152/AJPENDO.90653.2008
Abstract: Some studies suggest that the 5′-AMP-activated protein kinase (AMPK) is important in regulating muscle glucose uptake in response to intense electrically stimulated contractions. However, it is unknown whether AMPK regulates muscle glucose uptake during in vivo exercise. We studied this in male and female mice overexpressing kinase-dead AMPKα2 (AMPK-KD) in skeletal and heart muscles. Wild-type and AMPK-KD mice were exercised at the same absolute intensity and the same relative intensity (30 and 70% of in idual maximal running speed) to correct for reduced exercise capacity of the AMPK-KD mouse. Muscle glucose clearance was measured using 2-deoxy-[ 3 H]glucose as tracer. In wild-type mice, glucose clearance was increased at 30 and 70% of maximal running speed by 40 and 350% in the quadriceps muscle and by 120 and 380% in gastrocnemius muscle, respectively. Glucose clearance was not lower in AMPK-KD muscles compared with wild-type regardless of whether animals were exercised at the same relative or the same absolute intensity. In agreement, surface membrane content of the glucose transporter GLUT4 was increased similarly in AMPK-KD and wild-type muscle in response to running. We also measured signaling of alternative exercise-sensitive pathways that might be compensatorily increased in AMPK-KD muscles. However, increases in phosphorylation of CaMKII, Trisk95, p38 MAPK, and ERK1/2 were not higher in AMPK-KD than in WT muscle. Collectively, these findings suggest that AMPKα2 signaling is not essential in regulating glucose uptake in mouse skeletal muscle during treadmill exercise and that other mechanisms play a central role.
Publisher: MDPI AG
Date: 28-03-2020
DOI: 10.3390/IJMS21072344
Abstract: The small molecule kinase inhibitor SBI-0206965 was originally described as a specific inhibitor of ULK1/2. More recently, it was reported to effectively inhibit AMPK and several studies now report its use as an AMPK inhibitor. Currently, we investigated the specificity of SBI-0206965 in incubated mouse skeletal muscle, measuring the effect on analog 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)-stimulated AMPK-dependent glucose transport and insulin-stimulated AMPK-independent glucose uptake. Pre-treatment with 10 µM SBI-0206965 for 50 min potently suppressed AICAR-stimulated glucose transport in both the extensor digitorum longus (EDL) and soleus muscle. This was despite only a modest lowering of AICAR-stimulated AMPK activation measured as ACC2 Ser212, while ULK1/2 Ser555 phosphorylation was prevented. Insulin-stimulated glucose transport was also potently inhibited by SBI-0206965 in soleus. No major changes were observed on insulin-stimulated cell signaling. No general effect of SBI-0206965 on intracellular membrane morphology was observed by transmission electron microscopy. As insulin is known to neither activate AMPK nor require AMPK to stimulate glucose transport, and insulin inhibits ULK1/2 activity, these data strongly suggest that SBI-0206965 has a non-specific off-target inhibitory effect on muscle glucose transport. Thus, SBI-0206965 is not a specific inhibitor of the AMPK/ULK-signaling axis in skeletal muscle, and data generated with this inhibitor must be interpreted with caution.
Publisher: American Physiological Society
Date: 07-2018
DOI: 10.1152/AJPENDO.00392.2017
Abstract: Studies in skeletal muscle cell cultures suggest that the cortical actin cytoskeleton is a major requirement for insulin-stimulated glucose transport, implicating the β-actin isoform, which in many cell types is the main actin isoform. However, it is not clear that β-actin plays such a role in mature skeletal muscle. Neither dependency of glucose transport on β-actin nor actin reorganization upon glucose transport have been tested in mature muscle. To investigate the role of β-actin in fully differentiated muscle, we performed a detailed characterization of wild type and muscle-specific β-actin knockout (KO) mice. The effects of the β-actin KO were subtle however, we confirmed the previously reported decline in running performance of β-actin KO mice compared with wild type during repeated maximal running tests. We also found insulin-stimulated glucose transport into incubated muscles reduced in soleus but not in extensor digitorum longus muscle of young adult mice. Contraction-stimulated glucose transport trended toward the same pattern, but the glucose transport phenotype disappeared in soleus muscles from mature adult mice. No genotype-related differences were found in body composition or glucose tolerance or by indirect calorimetry measurements. To evaluate β-actin mobility in mature muscle, we electroporated green fluorescent protein (GFP)-β-actin into flexor digitorum brevis muscle fibers and measured fluorescence recovery after photobleaching. GFP-β-actin showed limited unstimulated mobility and no changes after insulin stimulation. In conclusion, β-actin is not required for glucose transport regulation in mature mouse muscle under the majority of the tested conditions. Thus, our work reveals fundamental differences in the role of the cortical β-actin cytoskeleton in mature muscle compared with cell culture.
Publisher: American Diabetes Association
Date: 04-2019
DOI: 10.2337/DB18-0796
Abstract: Skeletal muscle (SKM) insulin resistance plays a central role in the pathogenesis of type 2 diabetes. Because G-protein–coupled receptors (GPCRs) represent excellent drug targets, we hypothesized that activation of specific functional classes of SKM GPCRs might lead to improved glucose homeostasis in type 2 diabetes. At present, little is known about the in vivo metabolic roles of the various distinct GPCR signaling pathways operative in SKM. In this study, we tested the hypothesis that selective activation of SKM Gq signaling can improve SKM glucose uptake and whole-body glucose homeostasis under physiological and pathophysiological conditions. Studies with transgenic mice expressing a Gq-linked designer GPCR selectively in SKM cells demonstrated that receptor-mediated activation of SKM Gq signaling greatly promoted glucose uptake into SKM and significantly improved glucose homeostasis in obese, glucose-intolerant mice. These beneficial metabolic effects required the activity of SKM AMPK. In contrast, obese mutant mice that lacked both Gαq and Gα11 selectively in SKM showed severe deficits in glucose homeostasis. Moreover, GPCR-mediated activation of Gq signaling also stimulated glucose uptake in primary human SKM cells. Taken together, these findings strongly suggest that agents capable of enhancing SKM Gq signaling may prove useful as novel antidiabetic drugs.
Publisher: Wiley
Date: 19-05-2021
DOI: 10.1113/JP281187
Abstract: Tamoxifen‐inducible skeletal muscle‐specific AXIN1 knockout (AXIN1 imKO) in mouse does not affect whole‐body energy substrate metabolism. AXIN1 imKO does not affect AICAR or insulin‐stimulated glucose uptake in adult skeletal muscle. AXIN1 imKO does not affect adult skeletal muscle AMPK or mTORC1 signalling during AICAR/insulin/amino acid incubation, contraction and exercise. During exercise, α2/β2/γ3AMPK and AMP/ATP ratio show greater increases in AXIN1 imKO than wild‐type in gastrocnemius muscle. AXIN1 is a scaffold protein known to interact with proteins in signal transduction pathways regulating cellular development and function. Recently, AXIN1 was proposed to assemble a protein complex essential to catabolic‐anabolic transition by coordinating AMPK activation and inactivation of mTORC1 and to regulate glucose uptake‐stimulation by both AMPK and insulin. To investigate whether AXIN1 is permissive for adult skeletal muscle function, a phenotypic in vivo and ex vivo characterization of tamoxifen‐inducible skeletal muscle‐specific AXIN1 knockout (AXIN1 imKO) mice was conducted. AXIN1 imKO did not influence AMPK/mTORC1 signalling or glucose uptake stimulation at rest or in response to different exercise/contraction protocols, pharmacological AMPK activation, insulin or amino acids stimulation. The only genotypic difference observed was in exercising gastrocnemius muscle, where AXIN1 imKO displayed elevated α2/β2/γ3 AMPK activity and AMP/ATP ratio compared to wild‐type mice. Our work shows that AXIN1 imKO generally does not affect skeletal muscle AMPK/mTORC1 signalling and glucose metabolism, probably due to functional redundancy of its homologue AXIN2.
Publisher: American Diabetes Association
Date: 28-07-2023
DOI: 10.2337/DB23-0358
Publisher: Elsevier BV
Date: 09-2014
Publisher: Elsevier BV
Date: 03-2009
DOI: 10.1016/J.BBALIP.2008.12.009
Abstract: Enhanced contractile activity increases cardiac long-chain fatty acid (LCFA) uptake via translocation of CD36 to the sarcolemma, similarly to increase in glucose uptake via GLUT4 translocation. AMP-activated protein kinase (AMPK) is assumed to mediate contraction-induced LCFA utilization. However, which catalytic isoform (AMPKalpha1 versus AMPKalpha2) is involved, is unknown. Furthermore, no studies have been performed on the role of LKB1, a kinase with AMPKK activity, on the regulation of cardiac LCFA utilization. Using different mouse models (AMPKalpha2-kinase-dead, AMPKalpha2-knockout and LKB1-knockout mice), we tested whether LKB1 and/or AMPK are required for stimulation of LCFA and glucose utilization upon treatment of cardiomyocytes with compounds (oligomycin/AICAR/dipyridamole) which induce CD36 translocation similar to that seen upon contraction. In AMPKalpha2- kinase-dead cardiomyocytes, the stimulating effects of oligomycin and AICAR on palmitate and deoxyglucose uptake and palmitate oxidation were almost completely lost. Moreover, in AMPKalpha2- and LKB1-knockout cardiomyocytes, oligomycin-induced LCFA and deoxyglucose uptake were completely abolished. However, the stimulatory effect of dipyridamole on palmitate uptake and oxidation was preserved in AMPKalpha2-kinase-dead cardiomyocytes. In conclusion, in the heart there is a signaling axis consisting of LKB1 and AMPKalpha2 which activation results in enhanced LCFA utilization, similarly to enhanced glucose uptake. In addition, an unknown dipyridamole-activated pathway can stimulate cardiac LCFA utilization by activating signaling components downstream of AMPK.
Publisher: Wiley
Date: 20-10-2014
DOI: 10.1113/EXPPHYSIOL.2014.079194
Abstract: Muscle contraction stimulates muscle glucose uptake by facilitating translocation of glucose transporter 4 from intracellular locations to the cell surface, which allows for diffusion of glucose into the myofibres. The intracellular mechanisms regulating this process are not well understood. The GTPase Rac1 has, until recently, been investigated only with regard to its involvement in insulin-stimulated glucose uptake. However, we recently found that Rac1 is activated during muscle contraction and exercise in mice and humans. Remarkably, Rac1 seems to be necessary for exercise and contraction-stimulated glucose uptake in skeletal muscle, because muscle-specific Rac1 knockout mice display reduced ex vivo contraction- and in vivo exercise-stimulated glucose uptake. The molecular mechanism by which Rac1 regulates glucose uptake is presently unknown. However, recent studies link Rac1 to the actin cytoskeleton, the small GTPase RalA and/or free radical production, which have previously been shown to be regulators of glucose uptake in muscle. We propose a model in which Rac1 is activated by contraction- and exercise-induced mechanical stress signals and that Rac1 in conjunction with other signalling regulates glucose uptake during muscle contraction and exercise.
Publisher: Wiley
Date: 15-01-2015
Publisher: Public Library of Science (PLoS)
Date: 07-05-2008
Publisher: eLife Sciences Publications, Ltd
Date: 29-10-2015
DOI: 10.7554/ELIFE.06011
Abstract: Adipose tissue is crucial for the maintenance of energy and metabolic homeostasis and its deregulation can lead to obesity and type II diabetes (T2D). Using gene disruption in the mouse, we discovered a function for a RhoA-specific guanine nucleotide exchange factor PDZ-RhoGEF (Arhgef11) in white adipose tissue biology. While PDZ-RhoGEF was dispensable for a number of RhoA signaling-mediated processes in mouse embryonic fibroblasts, including stress fiber formation and cell migration, it's deletion led to a reduction in their proliferative potential. On a whole organism level, PDZ-RhoGEF deletion resulted in an acute increase in energy expenditure, selectively impaired early adipose tissue development and decreased adiposity in adults. PDZ-RhoGEF-deficient mice were protected from diet-induced obesity and T2D. Mechanistically, PDZ-RhoGEF enhanced insulin/IGF-1 signaling in adipose tissue by controlling ROCK-dependent phosphorylation of the insulin receptor substrate-1 (IRS-1). Our results demonstrate that PDZ-RhoGEF acts as a key determinant of mammalian metabolism and obesity-associated pathologies.
Publisher: Public Library of Science (PLoS)
Date: 08-08-2016
Publisher: eLife Sciences Publications, Ltd
Date: 19-04-2023
DOI: 10.7554/ELIFE.83338
Abstract: Microtubules serve as tracks for long-range intracellular trafficking of glucose transporter 4 (GLUT4), but the role of this process in skeletal muscle and insulin resistance is unclear. Here, we used fixed and live-cell imaging to study microtubule-based GLUT4 trafficking in human and mouse muscle fibers and L6 rat muscle cells. We found GLUT4 localized on the microtubules in mouse and human muscle fibers. Pharmacological microtubule disruption using Nocodazole (Noco) prevented long-range GLUT4 trafficking and depleted GLUT4-enriched structures at microtubule nucleation sites in a fully reversible manner. Using a perifused muscle-on-a-chip system to enable real-time glucose uptake measurements in isolated mouse skeletal muscle fibers, we observed that Noco maximally disrupted the microtubule network after 5 min without affecting insulin-stimulated glucose uptake. In contrast, a 2-hr Noco treatment markedly decreased insulin responsiveness of glucose uptake. Insulin resistance in mouse muscle fibers induced either in vitro by C2 ceramides or in vivo by diet-induced obesity, impaired microtubule-based GLUT4 trafficking. Transient knockdown of the microtubule motor protein kinesin-1 protein KIF5B in L6 muscle cells reduced insulin-stimulated GLUT4 translocation while pharmacological kinesin-1 inhibition in incubated mouse muscles strongly impaired insulin-stimulated glucose uptake. Thus, in adult skeletal muscle fibers, the microtubule network is essential for intramyocellular GLUT4 movement, likely functioning to maintain an insulin-responsive cell surface recruitable GLUT4 pool via kinesin-1-mediated trafficking.
Publisher: Wiley
Date: 27-05-2020
DOI: 10.1113/JP279780
Publisher: Wiley
Date: 23-09-2020
DOI: 10.1113/JP280528
Publisher: American Diabetes Association
Date: 07-04-2017
DOI: 10.2337/DB16-1138
Abstract: Exercise bypasses insulin resistance to increase glucose uptake in skeletal muscle and therefore represents an important alternative to stimulate glucose uptake in insulin-resistant muscle. Both Rac1 and AMPK have been shown to partly regulate contraction-stimulated muscle glucose uptake, but whether those two signaling pathways jointly account for the entire signal to glucose transport is unknown. We therefore studied the ability of contraction and exercise to stimulate glucose transport in isolated muscles with AMPK loss of function combined with either pharmacological inhibition or genetic deletion of Rac1. Muscle-specific knockout (mKO) of Rac1, a kinase-dead α2 AMPK (α2KD), and double knockout (KO) of β1 and β2 AMPK subunits (β1β2 KO) each partially decreased contraction-stimulated glucose transport in mouse soleus and extensor digitorum longus (EDL) muscle. Interestingly, when pharmacological Rac1 inhibition was combined with either AMPK β1β2 KO or α2KD, contraction-stimulated glucose transport was almost completely inhibited. Importantly, α2KD+Rac1 mKO double-transgenic mice also displayed severely impaired contraction-stimulated glucose transport, whereas exercise-stimulated glucose uptake in vivo was only partially reduced by Rac1 mKO with no additive effect of α2KD. It is concluded that Rac1 and AMPK together account for almost the entire ex vivo contraction response in muscle glucose transport, whereas only Rac1, but not α2 AMPK, regulates muscle glucose uptake during submaximal exercise in vivo.
Publisher: Wiley
Date: 06-2020
DOI: 10.14814/PHY2.14460
Publisher: Public Library of Science (PLoS)
Date: 06-05-2013
Publisher: Elsevier BV
Date: 06-2016
DOI: 10.1016/J.CELLSIG.2016.03.005
Abstract: During induction of the autophagosomal degradation process, LC3-I is lipidated to LC3-II and associates to the cargo isolation membrane allowing for autophagosome formation. Lipidation of LC3 results in an increased LC3-II/LC3-I ratio, and this ratio is an often used marker for autophagy in various tissues, including skeletal muscle. From cell studies AMPK has been proposed to be necessary and sufficient for LC3 lipidation. The aim of the present study was to investigate the role of AMPK in regulation of LC3 lipidation as a marker of autophagy in skeletal muscle. We observed an increase in the LC3-II/LC3-I ratio in skeletal muscle of AMPKα2 kinase-dead (KD) (p<0.001) and wild type (WT) (p<0.05) mice after 12h of fasting, which was greater (p<0.05) in AMPKα2 KD mice than in WT. The fasting-induced increase in the LC3-II/LC3-I ratio in both genotypes coincided with an initial decrease (p<0.01) in plasma insulin concentration, a subsequent decrease in muscle mTORC1 signaling and increased (p<0.05) levels of the autophagy-promoting proteins, FoxO3a and ULK1. Furthermore, a higher (p<0.01) LC3-II/LC3-I ratio was observed in old compared to young mice. We were not able to detect any change in LC3 lipidation with either in vivo treadmill exercise or in situ contractions. Collectively, these findings suggest that AMPKα2 is not necessary for induction of LC3 lipidation with fasting and aging. Furthermore, LC3 lipidation is increased in muscle lacking functional AMPKα2 during fasting and aging. Moreover, LC3 lipidation seems not to be a universal response to muscle contraction in mice.
Publisher: Elsevier BV
Date: 02-2014
DOI: 10.1016/J.CELLSIG.2013.11.007
Abstract: Skeletal muscle plays a major role in regulating whole body glucose metabolism. Akt and Rac1 are important regulators of insulin-stimulated glucose uptake in skeletal muscle. However the relative role of each pathway and how they interact are not understood. Here we delineate how Akt and Rac1 pathways signal to increase glucose transport independently of each other and are simultaneously downregulated in insulin resistant muscle. Pharmacological inhibition of Rac1 and Akt signaling was used to determine the contribution of each pathway to insulin-stimulated glucose uptake in mouse muscles. The actin filament-depolymerizing agent LatrunculinB was combined with pharmacological inhibition of Rac1 or Akt, to examine whether either pathway mediates its effect via the actin cytoskeleton. Akt and Rac1 signaling were investigated under each condition, as well as upon Akt2 knockout and in ob/ob mice, to uncover whether Akt and Rac1 signaling are independent and whether they are affected by genetically-induced insulin resistance. While in idual inhibition of Rac1 or Akt partially decreased insulin-stimulated glucose transport by ~40% and ~60%, respectively, their simultaneous inhibition completely blocked insulin-stimulated glucose transport. LatrunculinB plus Akt inhibition blocked insulin-stimulated glucose uptake, while LatrunculinB had no additive effect on Rac1 inhibition. In muscles from severely insulin-resistant ob/ob mice, Rac1 and Akt signaling were severely dysregulated and the increment in response to insulin reduced by 100% and 90%, respectively. These findings suggest that Rac1 and Akt regulate insulin-stimulated glucose uptake via distinct parallel pathways, and that insulin-induced Rac1 and Akt signaling are both dysfunctional in insulin resistant muscle. There may thus be multiple treatment targets for improving insulin sensitivity in muscle.
Publisher: Springer Science and Business Media LLC
Date: 27-05-2015
DOI: 10.1038/JP.2015.31
Abstract: We report significant hypotension and prerenal failure in an extremely preterm infant following two doses of oral sildenafil that warranted discontinuation of the drug and treatment with inotropes. Blood pressure and urine output normalized after 24 h of withdrawal of the oral drug. Sildenafil should be used cautiously in extremely preterm infants early in the neonatal course, where there is limited data on its efficacy and safety.
Publisher: Public Library of Science (PLoS)
Date: 19-03-2012
Publisher: Wiley
Date: 15-01-2016
DOI: 10.1113/JP271405
Publisher: Springer Science and Business Media LLC
Date: 16-07-2018
DOI: 10.1038/S41598-018-28540-5
Abstract: Regular exercise elicits advantageous metabolic adaptations in skeletal muscle, such as improved insulin sensitivity. However, the underpinning molecular mechanisms and the effect of diet on muscle exercise training benefits are unclear. We therefore characterized the skeletal muscle proteome following exercise training (ET) in mice fed chow or high-fat diet (HFD). ET increased exercise performance, lowered body-weight, decreased fat mass and improved muscle insulin action in chow- and HFD-fed mice. At the molecular level, ET regulated 170 muscle proteins in chow-fed mice, but only 29 proteins in HFD-fed mice. HFD per se altered 56 proteins, most of which were regulated in a similar direction by ET. To identify proteins that might have particular health-related bearing on skeletal muscle metabolism, we filtered for differentially regulated proteins in response to ET and HFD. This yielded 15 proteins, including the major urinary protein 1 (MUP1), which was the protein most decreased after HFD, but increased with ET. The ET-induced Mup1 expression was absent in mouse muscle lacking functional AMPK. MUP1 also potentiated insulin-stimulated GLUT4 translocation in cultured muscle cells. Collectively, we provide a resource of ET-regulated proteins in insulin-sensitive and insulin-resistant skeletal muscle. The identification of MUP1 as a diet-, ET- and AMPK-regulated skeletal muscle protein that improves insulin sensitivity in muscle cells demonstrates the usefulness of these data.
Publisher: Wiley
Date: 11-08-2019
DOI: 10.1002/JCSM.12474
Abstract: Skeletal muscle wasting is often associated with insulin resistance. A major regulator of muscle mass is the transforming growth factor β (TGF‐β) superfamily, including activin A, which causes atrophy. TGF‐β superfamily ligands also negatively regulate insulin‐sensitive proteins, but whether this pathway contributes to insulin action remains to be determined. To elucidate if TGF‐β superfamily ligands regulate insulin action, we used an adeno‐associated virus gene editing approach to overexpress an activin A inhibitor, follistatin (Fst288), in mouse muscle of lean and diet‐induced obese mice. We determined basal and insulin‐stimulated 2‐deoxy‐glucose uptake using isotopic tracers in vivo . Furthermore, to evaluate whether circulating Fst and activin A concentrations are associated with obesity, insulin resistance, and weight loss in humans, we analysed serum from morbidly obese subjects before, 1 week, and 1 year after Roux‐en‐Y gastric bypass (RYGB). Fst288 muscle overexpression markedly increased in vivo insulin‐stimulated (but not basal) glucose uptake (+75%, P 0.05) and increased protein expression and intracellular insulin signalling of AKT, TBC1D4, PAK1, pyruvate dehydrogenase‐E1α, and p70S6K, while decreasing TBC1D1 signaling ( P 0.05). Fst288 increased both basal and insulin‐stimulated protein synthesis, but no correlation was observed between the Fst288‐driven hypertrophy and the increase in insulin‐stimulated glucose uptake. Importantly, Fst288 completely normalized muscle glucose uptake in insulin‐resistant diet‐induced obese mice. RYGB surgery doubled circulating Fst and reduced activin A (−24%, P 0.05) concentration 1 week after surgery before any significant weight loss in morbidly obese normoglycemic patients, while major weight loss after 1 year did not further change the concentrations. We here present evidence that Fst is a potent regulator of insulin action in muscle, and in addition to AKT and p70S6K, we identify TBC1D1, TBC1D4, pyruvate dehydrogenase‐E1α, and PAK1 as Fst targets. Circulating Fst more than doubled post‐RYGB surgery, a treatment that markedly improved insulin sensitivity, suggesting a role for Fst in regulating glycaemic control. These findings demonstrate the therapeutic potential of inhibiting TGF‐β superfamily ligands to improve insulin action and Fst's relevance to muscle wasting‐associated insulin‐resistant conditions in mice and humans.
Publisher: Public Library of Science (PLoS)
Date: 08-02-2012
Publisher: Portland Press Ltd.
Date: 05-01-2016
DOI: 10.1042/BJ20150910
Abstract: The γ subunits of heterotrimeric AMPK complexes contain the binding sites for the regulatory adenine nucleotides AMP, ADP and ATP. We addressed whether complexes containing different γ isoforms display different responses to adenine nucleotides by generating cells stably expressing FLAG-tagged versions of the γ1, γ2 or γ3 isoform. When assayed at a physiological ATP concentration (5 mM), γ1- and γ2-containing complexes were allosterically activated almost 10-fold by AMP, with EC50 values one to two orders of magnitude lower than the ATP concentration. By contrast, γ3 complexes were barely activated by AMP under these conditions, although we did observe some activation at lower ATP concentrations. Despite this, all three complexes were activated, due to increased Thr172 phosphorylation, when cells were incubated with mitochondrial inhibitors that increase cellular AMP. With γ1 complexes, activation and Thr172 phosphorylation induced by the upstream kinase LKB1 [liver kinase B1 but not calmodulin-dependent kinase kinase (CaMKKβ)] in cell-free assays was markedly promoted by AMP and, to a smaller extent and less potently, by ADP. However, effects of AMP or ADP on activation and phosphorylation of the γ2 and γ3 complexes were small or insignificant. Binding of AMP or ADP protected all three γ subunit complexes against inactivation by Thr172 dephosphorylation with γ2 complexes, ADP had similar potency to AMP, but with γ1 and γ3 complexes, ADP was less potent than AMP. Thus, AMPK complexes containing different γ subunit isoforms respond differently to changes in AMP, ADP or ATP. These differences may tune the responses of the isoforms to fit their differing physiological roles.
Publisher: American Physiological Society
Date: 10-2021
DOI: 10.1152/AJPENDO.00164.2021
Abstract: Resistance exercise is known to increase c-Myc expression, which is known to stimulate ribosome biogenesis and protein synthesis in a variety of cells. However, whether the increase in c-Myc stimulates ribosome biogenesis and protein synthesis in skeletal muscles remains unknown. We found that c-Myc overexpression is sufficient to stimulate skeletal muscle ribosome biogenesis and protein synthesis without activation of mTORC1.
Publisher: American Diabetes Association
Date: 17-05-2013
DOI: 10.2337/DB12-1148
Abstract: The actin cytoskeleton–regulating GTPase Rac1 is required for insulin-stimulated GLUT4 translocation in cultured muscle cells. However, involvement of Rac1 and its downstream signaling in glucose transport in insulin-sensitive and insulin-resistant mature skeletal muscle has not previously been investigated. We hypothesized that Rac1 and its downstream target, p21-activated kinase (PAK), are regulators of insulin-stimulated glucose uptake in mouse and human skeletal muscle and are dysregulated in insulin-resistant states. Muscle-specific inducible Rac1 knockout (KO) mice and pharmacological inhibition of Rac1 were used to determine whether Rac1 regulates insulin-stimulated glucose transport in mature skeletal muscle. Furthermore, Rac1 and PAK1 expression and signaling were investigated in muscle of insulin-resistant mice and humans. Inhibition and KO of Rac1 decreased insulin-stimulated glucose transport in mouse soleus and extensor digitorum longus muscles ex vivo. Rac1 KO mice showed decreased insulin and glucose tolerance and trended toward higher plasma insulin concentrations after intraperitoneal glucose injection. Rac1 protein expression and insulin-stimulated PAKThr423 phosphorylation were decreased in muscles of high fat–fed mice. In humans, insulin-stimulated PAK activation was decreased in both acute insulin-resistant (intralipid infusion) and chronic insulin-resistant states (obesity and diabetes). These findings show that Rac1 is a regulator of insulin-stimulated glucose uptake and a novel candidate involved in skeletal muscle insulin resistance.
Publisher: Wiley
Date: 31-03-2009
Publisher: Frontiers Media SA
Date: 04-04-2019
Publisher: Wiley
Date: 09-2018
DOI: 10.14814/PHY2.13800
Publisher: Wiley
Date: 10-05-2018
DOI: 10.1113/JP275602
Publisher: American Physiological Society
Date: 11-2007
DOI: 10.1152/AJPENDO.00382.2007
Abstract: 5′-AMP-activated protein kinase (AMPK) was recently suggested to regulate pyruvate dehydrogenase (PDH) activity and thus pyruvate entry into the mitochondrion. We aimed to provide evidence for a direct link between AMPK and PDH in resting and metabolically challenged (exercised) skeletal muscle. Compared with rest, treadmill running increased AMPKα1 activity in α 2 KO mice (90%, P 0.01) and increased AMPKα2 activity in wild-type (WT) mice (110%, P 0.05), leading to increased AMPKα Thr 172 (WT: 40%, α 2 KO: 100%, P 0.01) and ACCβ Ser 227 phosphorylation (WT: 70%, α 2 KO: 210%, P 0.01). Compared with rest, exercise significantly induced PDH-E 1 α site 1 (WT: 20%, α 2 KO: 62%, P 0.01) and site 2 (only α 2 KO: 83%, P 0.01) dephosphorylation and PDH a [∼200% in both genotypes ( P 0.01)]. Compared with WT, PDH dephosphorylation and activation was markedly enhanced in the α 2 KO mice both at rest and during exercise. The increased PDH a activity during exercise was associated with elevated glycolytic flux, and muscles from the α 2 KO mice displayed marked lactate accumulation and deranged energy homeostasis. Whereas mitochondrial DNA content was normal, the expression of several mitochondrial proteins was significantly decreased in muscle of α 2 KO mice. In isolated resting EDL muscles, activation of AMPK signaling by AICAR did not change PDH-E 1 α phosphorylation in either genotype. PDH is activated in mouse skeletal muscle in response to exercise and is independent of AMPKα2 expression. During exercise, α 2 KO muscles display deranged energy homeostasis despite enhanced glycolytic flux and PDH a activity. This may be linked to decreased mitochondrial oxidative capacity.
No related grants have been discovered for Thomas Jensen.