Skeletal muscle glucose transporter 4 (GLUT4) translocation to the plasma membrane determines glucose uptake in response to insulin and exercise and is disrupted in insulin resistance, making its experimental measurement critical. Confocal light microscopy is widely used for this purpose because of its ability to provide quantitative, high-resolution spatial information from small tissue amounts. However, conventional immunofluorescence colocalization microscopy lacks sensitivity and specificity in the detection of GLUT4 translocation. We validated the use of exofacial epitope-specific GLUT4 antibodies to quantify sarcolemmal GLUT4 translocation in fixed, nonpermeabilized adult human and rodent muscle fibers. Across human, mouse, and rat muscles, these antibodies sensitively detected stimulus-induced GLUT4 translocation, and labeling was abolished in muscle-specific GLUT4-knockout muscle, confirming specificity. Importantly, this study includes the first unambiguous visualization of endogenous GLUT4 translocation in intact human skeletal muscle fibers after insulin stimulation and exercise. In TBC1D4-knockout rats, insulin-stimulated GLUT4 translocation was absent despite wild-type-level GLUT4 expression, confirming an essential role for TBC1D4 in this process. Thus, exofacial GLUT4 antibodies provide a straightforward, sensitive, and specific approach to quantify endogenous GLUT4 translocation in fixed adult skeletal muscle. ARTICLE HIGHLIGHTS:Reliable quantification of glucose transporter 4 (GLUT4) translocation in intact skeletal muscle is essential for understanding insulin and exercise responses but remains technically challenging. We aimed to test whether exofacial GLUT4 antibodies can specifically detect sarcolemmal GLUT4 translocation in fixed, nonpermeabilized muscle fibers from humans and rodents. GLUT4 translocation in response to insulin, AMPK activation, and exercise was detectable in human and rodent muscles. Insulin-stimulated translocation correlated with 2-deoxyglucose uptake and was abolished in TBC1D4-knockout muscle. Exofacial GLUT4 antibodies enable straightforward, specific quantification of endogenous GLUT4 translocation in rodent and human muscles in healthy and insulin-resistant states.

Human skeletal muscle is a major regulator of whole-body metabolic homeostasis, yet mechanistic insight into human muscle plasticity is limited by the lack of in vitro models with adult-like metabolic and functional maturity. Here, we develop a workflow for generating bioengineered human skeletal muscle tissues that undergo coordinated structural, molecular, and functional maturation and stabilize in an adult-like state by day 21. Time-resolved RNA-seq and protein profiling reveal consolidation of contractile programs alongside progressive metabolic maturation, including increased mitochondrial electron transport chain content, mature mitochondrial network organization, and upregulation of glucose- and glycogen-handling proteins as well as muscle-enriched AMPK isoforms. Functionally, the tissues develop physiological force-frequency behavior, post-tetanic potentiation, and reproducible fatigue responses that are exacerbated by hypoxia and glucose withdrawal. Exercise-like chronic stimulation increases force and endurance with hypertrophy-like remodeling, and these adaptations reverse with detraining. The model also captures pharmacological responsiveness. Beta-adrenergic stimulation activates canonical signaling, increases force, limits disuse-related decline, and improves endurance in a glucose-dependent manner. Under physiological insulin and IGF-1 conditions, tissues show robust insulin-stimulated glucose uptake and glycogen synthesis, with punctate glucose transporter 4 (GLUT4) localization. Finally, knockdown of muscle glycogen synthase (GYS1) preserves peak tetanic force but impairs endurance and force recovery under fuel stress, indicating that glycogen metabolism is a key determinant of human muscle resilience. ### Competing Interest Statement CHO, MKH, CTAL, TW, ZL, PJ, PB, BFH, ET, JH, ZW, CSW, AB, CP, and JRK were employed at Novo Nordisk A/S while the experimental work took place. JFPW is a shareholder and external consultant for Novo Nordisk Inc. Danish Diabetes Academy, https://ror.org/05xbkrn90, 17SA0031406 Novo Nordisk Foundation, NNF 082659, NF 0085866

Obesity and Type 2 Diabetes are major contributors to cardiac hypertrophy and dysfunction, yet the molecular mechanisms driving early myocardial alterations remain incompletely understood. Evidence from rodent models and end-stage human disease suggests that cytoskeletal remodeling and oxidative stress may contribute to early increases in cardiomyocyte stiffness and hypertrophy. Whether these processes are involved at earlier disease stages in translationally relevant large-animal models remains unclear. Heart tissue from male Göttingen minipigs subjected to a 13-month intervention with a standard control diet, high-fat-fructose-cholesterol diet, or high-fat-fructose-cholesterol with streptozotocin-induced diabetes was analyzed. Untargeted proteomics was performed on left atrium and left ventricle tissue, followed by pathway enrichment analyses to identify diet- and chamber-specific proteomic alterations. Global proteomic analyses revealed that anatomical region represented the dominant source of variance, with 200–300 proteins significantly regulated across dietary interventions. Pathway enrichment analyses highlighted alterations in protein and macronutrient metabolism, mitochondrial function, and extracellular matrix organization. Correlation and Hallmark analyses further linked ventricular remodeling to glucose-associated and mitochondrial pathways, while atrial remodeling was more closely associated with metabolic and nutrient-sensing pathways. Among 75 microtubule and 53 redox-related proteins examined, 16 and 14, respectively, were significantly altered in a chamber- and intervention-dependent manner. Early cardiac hypertrophy associated with obesity and/or diabetes is accompanied by extensive proteomic remodeling, characterized by distinct atrial and ventricular profiles. However, the relatively modest changes in microtubule and redox-related proteins suggest that these are unlikely to be primary drivers of early myocardial remodeling.

The intracellular redox state is crucial for insulin responses in peripheral tissues. Despite the longstanding belief that insulin signaling increases hydrogen peroxide (H2O2) production leading to reversible oxidation of cysteine thiols, evidence is inconsistent and rarely involves human tissues. In this study, we systematically investigated insulin-dependent changes in subcellular H2O2 levels and reversible cysteine modifications across mouse and human skeletal muscle models. Utilizing advanced redox tools—including genetically encoded H2O2 sensors and non-reducing immunoblotting—we consistently observed no increase in subcellular H2O2 levels following insulin stimulation. Instead, stoichiometric cysteine proteome analyses revealed a selective pro-reductive shift in cysteine modifications affecting insulin transduction related proteins, including Cys179 on GSK3β and Cys416 on Ras and Rab Interactor 2 (RIN2). Our findings challenge the prevailing notion that insulin promotes H2O2 generation in skeletal muscle and suggest that an insulin-stimulated pro-reductive shift modulates certain aspects of insulin signal transduction.

Exercise not only regulates energy expenditure but also appetite, yet the underpinnings remain unclear. We describe that increased energy intake is a defense against energy loss that depends on initial running distance and operates independently of diet and age. Running caused a rapid circadian-dependent fat remodeling leading to a decline in circulating leptin accompanied by the activation of hypothalamic neurons. We discovered that the activation of the β3-adrenergic receptor drives running-induced fat loss and the lower leptin triggers energy compensation by upregulating Neuropeptide Y . Once energy compensation is achieved, running is associated with molecular changes in hypothalamic signaling related to appetite and functional adaptations, such as enhanced sensitivity to hunger and satiety signals and increased responsiveness to appetite suppression induced by α-Melanocyte-Stimulating Hormone. The increased food intake persisted without fat rebound beyond running in both lean and obese young mice, uncovering a new homeostatic balance in young mice. ### Competing Interest Statement Claus Brandt and Diana Samodova-Sommer are currently working at Novo Nordisk. The other authors declare no competing interests. All authors gave their approval for the current version to be published. Novo Nordisk Foundation, 0059436, NNF20SA0064340, ID 0074481, NNF18CC0034900, NNF19SA0059305 Lundbeck Foundation, R380-2021-1300, R436-2023-1225 TrygFonden, ID 101390, ID 20045, ID 125132, ID 177225

SummaryCancer cachexia is a metabolic condition affecting up to 80% of patients with cancer. Cachexia is mediated by reduced muscle and fat mass and impaired function, and it lowers survival for patients. With no approved drugs to treat cachexia, preclinical efforts focus on understanding the molecular mechanisms underlying this condition to reveal treatment targets. Housing laboratory mice at ambient temperature imposes cold stress, leading to induced thermogenic activity and consequent whole-body metabolic adaptations. Yet, the impact of housing temperature inin vivopreclinical cachexia remains unknown. We found that thermoneutral (TN) housing in C26 carcinoma-bearing (C26) mice affected lean and fat mass, but not muscle weight or force. TN housing improved glucose tolerance in C26 mice, while enhancing circulating abundance of FGF21 and IL-6. Thermogenic tissues, especially brown adipose tissue, exhibited housing temperature-dependent molecular responses to cancer in oxygen consumption, ATP levels and SERCA ATPase activity, which are all crucial for cancer-induced whole-body metabolic adaptations. We conclude that molecular and systemic adaptations to cancer in mice critically depend on housing temperature, which should be considered in the design and interpretation of preclinical cancer studies.

Long-term exercise training elicits tremendous health benefits; however, the molecular understanding is incomplete and identifying therapeutic targets has been challenging. Rho GTPases are among the most regulated groups of proteins after exercise in human skeletal muscle, yet, unexplored candidates for mediating the effects of exercise training.We found that the Rho GTPase Rac1 was activated acutely after multiple exercise modalities in human skeletal muscle. Loss of Rac1 specifically in muscle attenuated contraction-induced muscle protein synthesis, diminished improvements in running capacity, and prevented muscle hypertrophy after exercise training in mice. Additionally, Ncf1∗ mice revealed that Rac1 regulated glycogen resynthesis via a NOX2-dependent mechanism. Molecularly, Rac1 was required for contraction-induced p38MAPK signaling towards HSP27, MNK1, and CREB phosphorylation. In vivo muscle-targeted overexpression of a hyperactive Rac1-mutant elevated reactive oxidant species production during exercise but did not affect muscle mass. Using mass spectrometry-based proteomics, we found that loss or gain of Rac1 muscle protein affected pathways related to cytoskeleton organization, muscle adaptation, and large ribosomal subunits. Thus, skeletal muscle Rac1 mediates both molecular and functional adaptation to exercise training.

The kinases AMPK, and mTOR as part of either mTORC1 or mTORC2, are major orchestrators of cellular growth and metabolism. Phosphorylation of mTOR Ser1261 is reportedly stimulated by both insulin and AMPK activation and a regulator of both mTORC1 and mTORC2 activity. Intrigued by the possibilities that Ser1261 might be a convergence point between insulin and AMPK signaling in skeletal muscle, we investigated the regulation and function of this site using a combination of human exercise, transgenic mouse, and cell culture models. Ser1261 phosphorylation on mTOR did not respond to insulin in any of our tested models, but instead responded acutely to contractile activity in human and mouse muscle in an AMPK activity-dependent manner. Contraction-stimulated mTOR Ser1261 phosphorylation in mice was decreased by Raptor muscle knockout (mKO) and increased by Raptor muscle overexpression, yet was not affected by Rictor mKO, suggesting most of Ser1261 phosphorylation occurs within mTORC1 in skeletal muscle. In accordance, HEK293 cells mTOR Ser1261Ala mutation strongly impaired phosphorylation of mTORC1 substrates but not mTORC2 substrates. However, neither mTORC1 nor mTORC2-dependent phosphorylations were affected in muscle-specific kinase-dead AMPK mice with no detectable mTOR Ser1261 phosphorylation in skeletal muscle. Thus, mTOR Ser1261 is an exercise but not insulin-responsive AMPK-dependent phosphosite in human and murine skeletal muscle, playing an unclear role in mTORC1 regulation but clearly not required for mTORC2 activity.

Metabolic flexibility in skeletal muscle is essential for maintaining healthy glucose and lipid metabolism, and its dysfunction is closely linked to metabolic diseases. Exercise enhances metabolic flexibility, making it an important tool for discovering mechanisms that promote metabolic health. Here we show that pantothenate kinase 4 (PanK4) is a new conserved exercise target with high abundance in muscle. Muscle-specific deletion of PanK4 impairs fatty acid oxidation which is related to higher intramuscular acetyl-CoA and malonyl-CoA levels. Elevated acetyl-CoA levels persist regardless of feeding state and are associated with whole-body glucose intolerance, reduced insulin-stimulated glucose uptake in glycolytic muscle, and impaired glucose uptake during exercise. Conversely, increasing PanK4 levels in glycolytic muscle lowers acetyl-CoA and enhances glucose uptake. Our findings highlight PanK4 as an important regulator of acetyl-CoA levels, playing a key role in both muscle lipid and glucose metabolism.

AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1) are crucial kinase signalling hubs that regulate the balance between catabolism and anabolism in skeletal muscle. The scaffold protein AXIN1 has been proposed to regulate the switch between these pathways and be required for GLUT4 translocation in skeletal muscle and adipocyte cell lines. Muscle-specific AXIN1 knockout (KO) mice exhibit no discernable phenotype, possibly due to compensation by AXIN2 upon AXIN1 loss. Thus we generated and characterized muscle-specific inducible AXIN1 and AXIN2 double knockout (dKO) mice. Surprisingly AXIN1/2 dKO mice displayed normal AMPK and mTORC1 signalling and glucose uptake in response to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), insulin and in situ muscle contraction. These findings suggest that AXIN proteins are not essential for the regulation of AMPK and mTORC1 signalling or glucose uptake in skeletal muscle. This study challenges the previously indicated critical roles of AXIN1 in exercise-stimulated AMPK activation and GLUT4-mediated glucose uptake in skeletal muscle. KEY POINTS: Phenotyping of tamoxifen-inducible muscle-specific AXIN1/2 double knockout (dKO) mice. We find no evidence for AXIN-dependent AMPK or mTORC1 regulation in skeletal muscle by insulin, AMPK activation or contraction. Glucose uptake regulation by insulin and AMPK activation is normal in AXIN1/2 dKO mice.

Decline in mitochondrial function associates with decreased muscle mass and strength in multiple conditions, including sarcopenia and type 2 diabetes. Optimal treatment could include improving mitochondrial function, however, there are limited and equivocal data regarding the molecular cues controlling muscle mitochondrial plasticity. Here we uncover the mitochondrial-mRNA-stabilizing protein SLIRP, in complex with LRPPRC, as a PGC-1α target that regulates mitochondrial structure, respiration, and mitochondrially-encoded-mRNA pools in skeletal muscle. Exercise training effectively counteracted mitochondrial defects induced by loss of LRPPRC/SLIRP, despite sustained low mitochondrially-encoded-mRNA pools, via increased mitoribosome translation capacity. In humans, exercise training robustly increased muscle SLIRP and LRPPRC protein content across exercise modalities and sexes, yet this increase was less prominent in subjects with type 2 diabetes. Our work identifies a mechanism of post-transcriptional mitochondrial regulation in skeletal muscle through mitochondrial mRNA stabilization. It emphasizes exercise as an effective approach to alleviate mitochondrial defects by possibly increasing mitoribosome capacity.

Striated muscle cells, encompassing cardiac myocytes and skeletal muscle fibers, are fundamental to athletic performance, facilitating blood circulation and coordinated movement through contraction. Despite their distinct functional roles, these muscle types exhibit similarities in cytoarchitecture, protein expression, and excitation-contraction coupling. Both muscle types also undergo molecular remodeling in energy metabolism and cell size in response to acute and repeated exercise stimuli to enhance exercise performance. Reactive oxygen species (ROS) produced by NADPH oxidase (NOX) isoforms 2 and 4 have emerged as signaling molecules that regulate exercise adaptations. This review systematically compares NOX2 and NOX4 expression, regulation, and roles in cardiac and skeletal muscle responses across exercise modalities. We highlight the many gaps in our knowledge and opportunities to let future skeletal muscle research into NOX-dependent mechanisms be inspired by cardiac muscle studies and vice versa. Understanding these processes could enhance the development of exercise routines to optimize human performance and health strategies that capitalize on the advantages of physical activity.

Preclinical models suggest mitochondria-derived oxidative stress as an underlying cause of insulin resistance. However, it remains unknown whether this pathophysiological mechanism is conserved in humans. Here, we used an invasive in vivo mechanistic approach to interrogate muscle insulin action while selectively manipulating the mitochondrial redox state in humans. To this end, we conducted insulin clamp studies combining intravenous infusion of a lipid overload with intake of a mitochondria-targeted antioxidant (mitoquinone). Under lipid overload, selective modulation of mitochondrial redox state by mitoquinone enhanced insulin-stimulated glucose uptake in skeletal muscle. Mechanistically, mitoquinone did not affect canonical insulin signaling but augmented insulin-stimulated glucose transporter type 4 (GLUT4) translocation while reducing the mitochondrial oxidative burden under lipid oversupply. Complementary ex vivo studies in human muscle fibers exposed to high intracellular lipid levels revealed that mitoquinone improves features of mitochondrial bioenergetics, including diminished mitochondrial H 2 O 2 emission. These findings provide translational and mechanistic evidence implicating mitochondrial oxidants in the development of lipid-induced muscle insulin resistance in humans.

In the Greenlandic Inuit population, 4% are homozygous carriers of a genetic nonsense TBC1D4 p.Arg684Ter variant leading to loss of the muscle-specific isoform of TBC1D4 and an approximately tenfold increased risk of type 2 diabetes1. Here we show the metabolic consequences of this variant in four female and four male homozygous carriers and matched controls. An extended glucose tolerance test reveals prolonged hyperglycaemia followed by reactive hypoglycaemia in the carriers. Whole-body glucose disposal is impaired during euglycaemic-hyperinsulinaemic clamp conditions and associates with severe insulin resistance in skeletal muscle only. Notably, a marked reduction in muscle glucose transporter GLUT4 and associated proteins is observed. While metabolic regulation during exercise remains normal, the insulin-sensitizing effect of a single exercise bout is compromised. Thus, loss of the muscle-specific isoform of TBC1D4 causes severe skeletal muscle insulin resistance without baseline hyperinsulinaemia. However, physical activity can ameliorate this condition. These observations offer avenues for personalized interventions and targeted preventive strategies. In Greenlandic Inuit, a TBC1D4 loss-of-function mutation increases type 2 diabetes risk by tenfold. Carriers show severe muscle insulin resistance, impaired glucose disposal and reduced muscle GLUT4, yet exercise mitigates these defects, offering potential for personalized lifestyle interventions.

For three-dimensional (3D) bioprinting to fulfill its promise and enable the automated fabrication of complex tissue-mimicking constructs, there is a need for developing bioinks that are not only printable and biocompatible but also have integrated cell-instructive properties. Toward this goal, we here present a scalable technique for generating nanofiber 3D printing inks with unique tissue-guiding capabilities. Our core methodology relies on tailoring the size and dispersibility of cellulose fibrils through a solvent-controlled partial carboxymethylation. This way, we generate partially negatively charged cellulose nanofibers with diameters of ∼250 nm and lengths spanning tens to hundreds of microns. In this range, the fibers structurally match the size and dimensions of natural collagen fibers making them sufficiently large to orient cells. Yet, they are simultaneously sufficiently thin to be optically transparent. By adjusting fiber concentration, 3D printing inks with excellent shear-thinning properties can be established. In addition, as the fibers are readily dispersible, composite inks with both carbohydrates and extracellular matrix (ECM)-derived proteins can easily be generated. We apply such composite inks for 3D printing cell-laden and cross-linkable structures, as well as tissue-guiding gel substrates. Interestingly, we find that the spatial organization of engineered tissues can be defined by the shear-induced alignment of fibers during the printing procedure. Specifically, we show how myotubes derived from human and murine skeletal myoblasts can be programmed into linear and complex nonlinear architectures on soft printed substrates with intermediate fiber contents. Our nanofibrillated cellulose inks can thus serve as a simple and scalable tool for engineering anisotropic human muscle tissues that mimic native structure and function.

BackgroundMetabolic dysfunction and cachexia are associated with poor cancer prognosis. With no pharmacological treatments, it is crucial to define the molecular mechanisms causing cancer-induced metabolic dysfunction and cachexia. Adenosine monophosphate-activated protein kinase (AMPK) connects metabolic and muscle mass regulation. As AMPK could be a potential treatment target, it is important to determine the function for AMPK in cancer-associated metabolic dysfunction and cachexia. We therefore established AMPK's roles in cancer-associated metabolic dysfunction, insulin resistance and cachexia. MethodsIn vastus lateralis muscle biopsies from n = 26 patients with non-small cell lung cancer (NSCLC), AMPK signalling and protein content were examined by immunoblotting. To determine the role of muscle AMPK, male mice overexpressing a dominant-negative AMPK alpha 2 (kinase-dead [KiDe]) specifically in striated muscle were inoculated with Lewis lung carcinoma (LLC) cells (wild type [WT]: n = 27, WT + LLC: n = 34, mAMPK-KiDe: n = 23, mAMPK-KiDe + LLC: n = 38). Moreover, male LLC-tumour-bearing mice were treated with (n = 10)/without (n = 9) 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to activate AMPK for 13 days. Littermate mice were used as controls. Metabolic phenotyping of mice was performed via indirect calorimetry, body composition analyses, glucose and insulin tolerance tests, tissue-specific 2-[3H]deoxy-d-glucose (2-DG) uptake and immunoblotting. ResultsPatients with NSCLC presented increased muscle protein content of AMPK subunits alpha 1, alpha 2, beta 2, gamma 1 and gamma 3 ranging from +27% to +79% compared with control subjects. In patients with NSCLC, AMPK subunit protein content correlated with weight loss (alpha 1, alpha 2, beta 2 and gamma 1), fat-free mass (alpha 1, beta 2 and gamma 1) and fat mass (alpha 1 and gamma 1). Tumour-bearing mAMPK-KiDe mice presented increased fat loss and glucose and insulin intolerance. LLC in mAMPK-KiDe mice displayed lower insulin-stimulated 2-DG uptake in skeletal muscle (quadriceps: -35%, soleus: -49%, extensor digitorum longus: -48%) and the heart (-29%) than that in non-tumour-bearing mice. In skeletal muscle, mAMPK-KiDe abrogated the tumour-induced increase in insulin-stimulated TBC1D4(thr642) phosphorylation. The protein content of TBC1D4 (+26%), pyruvate dehydrogenase (PDH; +94%), PDH kinases (+45% to +100%) and glycogen synthase (+48%) was increased in skeletal muscle of tumour-bearing mice in an AMPK-dependent manner. Lastly, chronic AICAR treatment elevated hexokinase II protein content and normalized phosphorylation of p70S6K(thr389) (mTORC1 substrate) and ACC(ser212) (AMPK substrate) and rescued cancer-induced insulin intolerance. ConclusionsProtein contents of AMPK subunits were upregulated in skeletal muscle of patients with NSCLC. AMPK activation seemed protectively inferred by AMPK-deficient mice developing metabolic dysfunction in response to cancer, including AMPK-dependent regulation of multiple proteins crucial for glucose metabolism. These observations highlight the potential for targeting AMPK to counter cancer-associated metabolic dysfunction and possibly cachexia.