Jesper Bratz Birk

Exercise enhances skeletal muscle insulin sensitivity, but the signaling mechanisms responsible are poorly understood. Understanding them may open new therapeutic avenues for individuals with limited exercise capacity. Here, we used rapamycin to inhibit mTORC1 in combination with exercise and insulin stimulation in healthy men. A single dose of rapamycin enhanced the insulin-sensitizing effect of exercise by 53% on average compared to placebo. Responses varied widely across individuals (-40% to 218%), and we leveraged this variance through personalized phosphoproteomics to map the mTORC1-dependent signaling network in skeletal muscle. This identified the protein kinase MKNK2 as a candidate downstream effector, which we then targeted for functional validation. Pharmacological inhibition of MKNK2 with eFT508 in insulin-clamped mice reduced both whole-body and skeletal muscle insulin sensitivity, confirming a functional role for MKNK2 activity in muscle glucose uptake. We then used eFT508 in ex vivo incubated human skeletal muscle to map the signaling network downstream of MKNK2, identifying the translational initiator eIF4G1 as a further regulatory node. Together, these findings indicate that exercise-induced insulin sensitization is actively constrained by a negative feedback pathway running from mTORC1 through the translational regulators MKNK2 and eIF4G1, raising the possibility that rapid translation of unidentified target proteins contributes to fine-tuning glucose uptake.

BACKGROUND: Horses are one of the few animals that spontaneously develop atrial fibrillation (AF), making them a powerful model for studying AF mechanisms and treatment effects. Despite the initial effectiveness of treatment in horses and humans, AF-induced atrial remodeling compromises its long-term success. Observational studies have suggested that metformin may reduce the risk of AF, but its effects on progressive AF-induced atrial remodeling have yet to be evaluated in a high-fidelity large animal model. METHODS: Here, we used a longitudinal horse model of tachypacing-induced self-sustained AF to characterize the electrical, molecular, and metabolic atrial changes over 4 months of disease, with and without metformin treatment (30 mg/kg orally, twice daily; initiated before AF induction, N=24 horses). Electrophysiological and multiomic approaches were combined with histology, echocardiography, biochemical, and mitochondrial analyses to evaluate disease progression and treatment response. RESULTS: The horse model replicated critical aspects of AF-induced atrial remodeling observed in Humans, including electrical and structural changes. Despite upregulation of metabolic genes and proteins in AF, no significant ultrastructural mitochondrial changes were detected. Metformin plasma trough levels confirmed stable therapeutic exposure. Metformin-treated horses were protected against early AF stabilization and sustained a less complex AF substrate in the right atrium after 4 months of disease. These protective effects were associated with increased right atrial activity of the metabolic regulator, AMPK (AMP-activated protein kinase), changes in metabolic pathways, and modulation of ion-channel gene expression. CONCLUSIONS: Metformin treatment conferred protection against early AF stabilization and selectively attenuated right atrial substrate complexity in a translationally relevant preclinical model. These findings support metformin as a lead molecule for AF prevention, warranting further mechanistic and clinical studies.

Background: High recurrence rates after atrial fibrillation (AF) treatment may be driven by myocardial changes induced by the arrhythmia itself. Understanding the molecular mechanisms behind these changes is crucial for developing targeted therapies and improving outcomes. Objectives: To characterise the cardiac transcriptome of healthy horses and explore transcriptional changes associated with persistent AF (naturally occurring and tachypacing‐induced). Study Design: Case–control study. Methods: RNA ‐sequencing was performed on atrial and ventricular tissue samples collected from six horses with naturally occurring persistent AF (lasting 2–12 weeks) and six healthy controls. Differential gene expression and pathway enrichment analyses were conducted to identify chamber‐specific differences and molecular pathways associated with AF . Findings were integrated with proteomic data and compared with transcriptional changes in a separate cohort of 10 horses with tachypacing‐induced AF . Atrial metabolic remodelling was further investigated by evaluating the activity of AMP ‐activated protein kinase ( AMPK ), a central metabolic regulator and measuring local glycogen content. Results: The transcriptomes of the four heart chambers had distinct molecular identities. Expression of ion channels and genes encoding calcium handling proteins was largely similar to humans, despite important differences in the ventricular expression of repolarising potassium channels. Persistent AF was associated with minimal ion channel changes but significant upregulation of metabolic, fibrotic and myofibrillar pathways. Metabolic remodeling included upregulation of fatty acid and glycolytic pathways, increased glycogen content in the left atrium and preserved AMPK activity in the right atrium. Transcriptomic profiles of naturally occurring persistent AF correlated well with those of tachypacing‐induced AF . Main Limitations: The study cannot distinguish changes predisposing to AF from those caused by it. Conclusion: Persistent AF was associated with changes in metabolic and fibrotic pathways in the atria, with minimal ion channel remodeling. Targeting these pathways, rather than focusing solely on the electrical disturbance, may improve treatment outcomes in equine AF.

A single bout of exercise improves muscle insulin sensitivity for up to 48 hours via AMPK. Limb ischemia activates AMPK in muscle, and subsequent reperfusion enhances insulin-stimulated vasodilation, potentially eliciting a more pronounced exercise effect with reduced workload. We investigated the combined effect of upper leg intermittent ischemia/reperfusion (IIR) and continuous knee-extension exercise on muscle insulin sensitivity regulation. We found that IIR exercise potentiated AMPK activation and muscle insulin sensitivity. The potentiating effect of IIR exercise on muscle insulin sensitivity was associated with increased insulin-stimulated blood flow in parallel with enhanced phosphorylation of endothelial nitric oxide synthase. Metabolomics analyses demonstrated a suppression of muscle medium-chain acylcarnitines during IIR exercise, which correlated with insulin sensitivity and was consistent with findings in isolated rat muscle treated with decanoyl-l-carnitine. Collectively, combining IIR with low- to moderate-intensity exercise may represent a promising intervention to effectively enhance muscle insulin sensitivity. This approach could offer potential for mitigating muscle insulin resistance in clinical settings and among individuals with lower physical activity levels.

Humans have, throughout history, faced periods of starvation necessitating increased physical effort to gather food. To explore adaptations in muscle function, 13 participants (7 males and 6 females) fasted for seven days. They lost 4.6 ± 0.3 kg lean and 1.4 ± 0.1 kg fat mass. Maximal isometric and isokinetic strength remained unchanged, while peak oxygen uptake decreased by 13%. Muscle glycogen was halved, while expression of electron transport chain proteins was unchanged. Pyruvate dehydrogenase kinase 4 (PDK4) expression increased 13-fold, accompanied by inhibitory pyruvate dehydrogenase phosphorylation, reduced carbohydrate oxidation and decreased exercise endurance capacity. Fasting had no impact on 5’ AMP-activated protein kinase (AMPK) activity, challenging its proposed role in muscle protein degradation. The participants maintained muscle strength and oxidative enzymes in skeletal muscle during fasting but carbohydrate oxidation and high-intensity endurance capacity were reduced.

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.

Background: AMP-activated protein kinase (AMPK) is activated during cellular energy perturbation. AMPK is composed of three subunits and several variants of AMPK complexes are expressed in skeletal muscle. The regulatory AMPKγ3 subunit is predominantly expressed in fast-twitch muscle fibers. A human AMPKγ3 R225W mutation has been described in two families. In cultured cells derived from R225W carrier muscle, the mutation was reported to increase total AMPK activity. In addition, elevated glycogen levels were observed in skeletal muscle. This led to the idea of AMPKγ3 being involved in glycogen levels in skeletal muscle. Evidence for such a causative link has never been provided. Results: We studied newly obtained muscle biopsies from three human carriers of the AMPKγ3 R225W mutation and matched controls and we developed a novel knock-in mouse model carrying the AMPKγ3 R225W mutation (KI HOM). In all three human pairs, the basal AMPKγ3-associated activity was reduced when assayed in the absence of exogenous AMP. No difference was observed when assayed under AMP saturation, which was supported by findings in muscle of KI HOM mice. Furthermore, the well-established stimulatory effects of AICAR/muscle contraction on AMPKγ3-associated activity were absent in muscle from KI HOM mice. Muscle glycogen levels were not affected by the mutation in human carriers or in KI HOM mice. Conclusions: The AMPKγ3 R225W mutation does not impact AMPK-associated activity in mature human skeletal muscle and the mutation is not linked to glycogen accumulation. The R225W mutation ablates AMPKγ3-associated activation by AICAR/muscle contractions, presumably through loss of nucleotide binding.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-22015-4

Skeletal muscle conveys several of the health-promoting effects of exercise; yet the underlying mechanisms are not fully elucidated. Studying skeletal muscle is challenging due to its different fiber types and the presence of non-muscle cells. This can be circumvented by isolation of single muscle fibers. Here, we develop a workflow enabling proteomics analysis of pools of isolated muscle fibers from freeze-dried human muscle biopsies. We identify more than 4000 proteins in slow- and fast-twitch muscle fibers. Exercise training alters expression of 237 and 172 proteins in slow- and fast-twitch muscle fibers, respectively. Interestingly, expression levels of secreted proteins and proteins involved in transcription, mitochondrial metabolism, Ca²⁺ signaling, and fat and glucose metabolism adapts to training in a fiber type-specific manner. Our data provide a resource to elucidate molecular mechanisms underlying muscle function and health, and our workflow allows fiber type-specific proteomic analyses of snap-frozen non-embedded human muscle biopsies.

Key points: Rodent studies suggest muscle fibre type‐specific insulin response in the recovery from exercise. The current study investigates muscle fibre type‐specific insulin action in the recovery from exercise in healthy subjects. In type I and type II muscle fibres, key proteins in glucose metabolism are similarly regulated by insulin during recovery from exercise. Our findings imply that both type I and type II muscle fibres contribute to the phenomenon of increased insulin sensitivity in the recovery from a single bout of exercise in humans. Human skeletal muscle consists of slow‐twitch (type I) and fast‐twitch (type II) muscle fibres. Muscle insulin action, regulating glucose uptake and metabolism, is improved following a single exercise bout. Rodent studies suggest that this phenomenon is confined to specific muscle fibre types. Whether this phenomenon is also confined to specific fibre types in humans has not been described. To investigate this, nine healthy men underwent a euglycaemic hyperinsulinaemic clamp (EHC) in the recovery from a single bout of one‐legged knee‐extensor exercise. Pools of type I and type II fibres were prepared from muscle biopsies taken in the rested and prior exercised leg before and after the EHC. AMPK γ3 and TBC1D4 – two key proteins regulating muscle insulin action following exercise – were higher expressed in type II than type I fibres. However, phosphor‐regulation of TBC1D4 was similar between fibre types when related to the total amount of TBC1D4 protein. The activating dephosphorylation of glycogen synthase was also similar in the two fibre types. Thus, insulin‐induced regulation of key proteins important for transport and intracellular flux of glucose towards glycogen storage in the recovery from exercise, does not differ between fibre types. In conclusion, the insulin‐sensitizing effect of a single bout of exercise includes both type I and type II fibres in human skeletal muscle. This may be an important observation for future pharmacological strategies targeting muscle insulin sensitivity in humans.