Biology subjects

Wojtaszewski, J. F. P.

Publications and source records attributed to Wojtaszewski, J. F. P..

9 recordsLinked to original sources

bioRxiv2026

Personalized phosphoproteomics establish mTORC1 as a regulator of exercise-induced insulin sensitization in human skeletal muscle

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.

physiology↗

bioRxiv2026

A semantic segmentation model to predict subcellular glycogen localization using transmission electron microscopy images

Transmission electron microscopy (TEM) is the gold standard for assessing subcellular glycogen localization in skeletal muscle fibres, but conventional manual analysis is extremely time-consuming and limits large-scale studies. Here, we developed and validated a deep learning-based semantic segmentation approach to automate quantification of glycogen particles across defined subcellular compartments in human skeletal muscle. Skeletal muscle biopsies were obtained from seven healthy men under conditions of normal, depleted, and supercompensated glycogen content. TEM images were acquired from myofibrillar and subsarcolemmal regions and manually annotated to train two complementary attention U-Net models: a region model identifying subcellular structures (intermyofibrillar space, intramyofibrillar regions including A-band, I-band and Z-disc, and mitochondria) and a glycogen model detecting individual glycogen particles. Combining the two models enabled estimation of compartment-specific glycogen areal densities. Model performance was evaluated against manual point-counting. At the fibre level, estimates based on 10-12 images per region achieved biases below 15% and coefficient of variation below 26% for all compartments. Importantly, model-derived total glycogen volume density showed strong concordance with biochemically determined muscle glycogen content across biopsies. In conclusion, this validated semantic segmentation workflow provides a robust, objective, and highly time-efficient tool for quantifying subcellular glycogen distribution in skeletal muscle. The model substantially reduces analysis time and enables high-throughput investigations of compartmentalized glycogen metabolism, with model weights and code made openly available.

physiology↗

bioRxiv2026

SIX1-dependent myofiber typology and metabolism controls muscle hypertrophy

The different types of muscle fibres respond in a specific way to hypertrophy or atrophy. The mechanisms underlying these heterogeneous adaptations remain poorly understood. Using single-nucleus RNA sequencing, we propose that fast glycolytic fibres show genetic limitations to hypertrophy induced by mechanical overload. We show that a prior fibre transition, achieved by reducing SIX1 protein expression (hypomorphism), enhances and accelerates overload-induced hypertrophy, bypassing the genetic limitations of fast glycolytic fibres. In contrast and unexpectedly, Six1 knockout in myofibers abolished overload-induced hypertrophy and instead caused atrophy of IIb/IIx fibers, despite the induction of a strong slow oxidative phenotype. In particular, Six1 deletion leads to metabolic defects caused by inhibition of glycolysis, AMPK and mitochondrial biogenesis. Our findings highlight the critical role of SIX1/AMPK/glycolysis-dependent aerobic metabolism in muscle growth and suggest that fibre type transitions, coupled with preserved metabolic function, may optimise hypertrophic responses.

physiology↗

bioRxiv2026

Functionally Mature Bioengineered Human Skeletal Muscle Tissues Capture Essential Aspects of Glucose Metabolism

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}2-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.

cell biology↗

bioRxiv2023

The mitochondrial mRNA stabilizing protein, SLIRP, regulates skeletal muscle mitochondrial structure and respiration by exercise-recoverable mechanisms

Summary and graphical abstractDecline 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-1 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/564600v2_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@1cdc6c5org.highwire.dtl.DTLVardef@b4cb1borg.highwire.dtl.DTLVardef@182871corg.highwire.dtl.DTLVardef@7462ea_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

The human AMPKgamma3 R225W mutation does neither enhance basal AMPKgamma3-associated activity nor glycogen in human or mouse skeletal muscle

BackgroundAMP-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 AMPK{gamma}3 subunit is predominantly expressed in fast-twitch muscle fibers. A human AMPK{gamma}3 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 AMPK{gamma}3 being involved in glycogen levels in skeletal muscle. Evidence for such a causative link has never been provided. ResultsWe studied newly obtained muscle biopsies from three human carriers of the AMPK{gamma}3 R225W mutation and matched controls and we developed a novel knock-in mouse model carrying the AMPK{gamma}3 R225W mutation (KI HOM). In all three human pairs, the basal AMPK{gamma}3-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 AMPK{gamma}3-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. ConclusionsThe AMPK{gamma}3 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 AMPK{gamma}3-associated activation by AICAR/muscle contractions, presumably through loss of nucleotide binding.

molecular biology↗

bioRxiv2023

Reducing the mitochondrial oxidative burden alleviates lipid-induced insulin resistance in human skeletal muscle

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 clamp 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 H2O2 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. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/535538v2_ufig1.gif" ALT="Figure 1"> View larger version (86K): org.highwire.dtl.DTLVardef@157d44eorg.highwire.dtl.DTLVardef@13358dborg.highwire.dtl.DTLVardef@e958eorg.highwire.dtl.DTLVardef@17cbc6f_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2022

Microtubule-mediated GLUT4 trafficking is disrupted in insulin resistant skeletal muscle

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.

cell biology↗

bioRxiv2022

Metformin improves glycemia independently of skeletal muscle AMPK via enhanced intestinal glucose clearance

Metformin is an inexpensive oral anti-hyperglycemic agent used worldwide as a first-choice drug for the prevention of type 2 diabetes mellitus (T2DM). Although current view suggests that metformin exerts its anti-hyperglycemic effect by lowering hepatic glucose production, it has been proposed that metformin also reduce hyperglycemia by increasing glucose uptake in skeletal muscle via activation of AMP-activated protein kinase (AMPK). Herein, we demonstrate in lean and diet-induced obese (DIO) male and female mouse models that the anti-hyperglycemic effect of metformin occurs independently of muscle AMPK, and instead relies on elevated intestinal glucose clearance. Furthermore, we report that the AMPK activity is elevated in skeletal muscle from patients with T2DM following chronic metformin treatment, but this is not associated with enhanced peripheral insulin sensitivity. These results argue against existing paradigms and emphasize the non-essential role of muscle AMPK but important role of the intestine for the anti-hyperglycemic effect of metformin.

physiology↗