Acta Physiologica

Brown adipose tissue (BAT) is a unique tissue with mitochondria specialized for thermogenesis via the BAT-specific uncoupling protein 1 (UCP1). Ucp1-/- mice cannot tolerate acute exposure to cold, illustrating the necessity of UCP1 for efficient mitochondrial thermogenesis. However, these mice adapt to low temperatures through a gradual acclimation process, suggesting a high degree of mitochondrial plasticity in brown and beige fat cells. This phenomenon, which remains to be fully elucidated, indicates the potential for these mitochondria to implement effective thermogenic mechanisms in the absence of uncoupling protein 1 (UCP1). Here, we investigated mitochondrial remodeling in beige and brown fat of Ucp1-/- mice to determine how they fulfill their thermogenic role. Upon gradual acclimation to a cold environment, Ucp1-/- mice exhibited body metabolic parameters and temperatures in the interscapular region similar to those of wild-type mice of BAT, highlighting effective thermogenesis. Interestingly, mitochondrial patch-clamp analysis and a mitochondrial Ca2+ swelling assay revealed a dramatic increase in Ca2+ uptake depending on the mitochondrial calcium uniporter (MCU) in BAT mitochondria from Ucp1-/- mice when robust thermogenesis was required. Mitochondrial remodeling was accompanied by markedly increased tethering between mitochondria and the endoplasmic reticulum (ER) in Ucp1-/- mice, confirming a significant restructuring of the contact sites between the ER and mitochondria, likely to adapt to a new Ca2+ homeostasis. Respiratory complexes also underwent significant reorganization, which partly led to a reduction in their assembly. Levels of ATP synthase and its F1 subcomplex increased, suggesting a major source of ATP consumption and energy expenditure. We propose a new role for MCU as a key regulator of mitochondrial plasticity, enabling efficient thermogenesis in beige and brown adipose tissues in the absence of UCP1.

BackgroundSkeletal muscle in wasting conditions often exhibits a common set of phenotypes that include atrophy, mitochondrial respiratory dysfunction, and fragmentation of the acetylcholine receptor (AChR) cluster at the endplate. Mitochondria are frequently implicated in driving muscle pathology in these conditions, although which aspects of mitochondrial function are most relevant is poorly understood. MethodsTo address this gap, we focused on mitochondrial permeability transition (mPT), a well-established pathological mechanism in ischemia-reperfusion injury and neurodegeneration but poorly studied in skeletal muscle. We performed a broad assessment of the consequences of mPT in skeletal muscle, focusing on features that are common in wasting conditions. We then tested whether tumor-host factors could promote mPT and compared differentially expressed genes (DEGs) with mPT and a mouse model of pancreatic cancer cachexia. ResultsInducing mPT in mouse skeletal muscle bundles in a Ca2+ retention capacity assay progressively altered mitochondrial morphology, beginning with cristae swirling and condensation, progressing to mitochondrial cristae displacement, and culminating in breach of the outer mitochondrial membrane; features that are common in wasting conditions. Inducing mPT with Bz423 in single mouse muscle fibers increased mROS and Caspase 3 (Casp3) activity and was prevented by inhibitors of mPT, mROS or Casp3. Incubating single muscle fibers with Bz423 for 24 h reduced fiber diameter by [~]20% which was prevented by inhibiting mPT, mROS, or Casp3. Inducing mPT caused a complex I-specific mitochondrial respiratory impairment and increased co-localization of lysosomes with mitochondria. Inducing mPT also fragmented the AChR cluster at the muscle endplate and was prevented by inhibiting mPT or Casp3. The Ca2+ threshold for mPT and mitochondrial calcein colocalization were reduced by pancreatic tumor-conditioned media in skeletal muscle or C2C12 myoblasts, respectively, and these effects were counteracted by mPT inhibition or cyclophilin D knockout. Finally, there was significant overlap between the transcriptome of mPT and that seen in diaphragm muscle in a mouse model of pancreatic cancer cachexia, particularly during the muscle wasting phase. ConclusionsWe conclude that inducing mPT in skeletal muscle recapitulates muscle phenotypes common with muscle wasting conditions like cachexia. Furthermore, mPT is engaged by tumor-host factors and had significant overlap with DEGs seen during the muscle wasting phase in a mouse model of pancreatic cancer cachexia, warranting further investigation of mPT as a therapeutic target.

BackgroundCarnitine plays an obligatory role in energetics owing to its role in the translocation of long-chain fatty acids into the mitochondrion for oxidation. Here, we determined the metabolic and behavioral consequences of systemic carnitine deficiency (SCD) in mice. MethodsFemale C57BL/6J mice were randomized to receive normal drinking water (control, n = 8) or drinking water supplemented with mildronate 4g.L-1 (mildronate, n = 8) for 21 days. Body composition was assessed at baseline and post treatment. Metabolic and behavioral phenotyping was performed continuously over 72 hours following 14 days of control or mildronate treatment. Stable isotope were used to assess whole-body substrate oxidation. Carnitine subfractions were quantified in skeletal muscle and liver, as was mitochondrial respiratory function. Liver and muscle samples also underwent proteomic analysis. ResultsMildronate treatment depleted total carnitine in muscle and liver by [~]97% (P < 0.001) and [~]90% (P < 0.001), respectively. Carnitine depletion was accompanied by lower total energy expenditure (P = 0.01), attributable to lower voluntary wheel running (P = 0.01). Oxidation rates of palmitate (P < 0.01) but not octanoate were lower whereas rates of glucose oxidation were greater in carnitine depleted mice (P < 0.01). Mitochondrial respiratory capacity was unaltered by carnitine deficiency. Carnitine deficiency remodeled muscle and liver proteomes to support lipid oxidation and energy production. SummaryIn mice, carnitine deficiency is characterized by decreased long-chain fatty acid oxidation despite preserved mitochondrial respiratory capacity. Carnitine deficiency resulted in lower voluntary exercise and a concomitant reduction in energy expenditure.

AimSecretin was recently found to play a pivotal role in the renal adaptation to acute base excess. Here, secretin increases pendrin-dependent HCO3- secretion from the beta-intercalated cells in the cortical collecting ducts. Whether secretin and its receptor play a role during prolonged base-loading remains unknown. MethodsUrine and blood acid-base analyses were carried out in secretin receptor (SCTR) KO and WT mice at baseline and after 1 and up to 8 days of base-loading with NaHCO3-enriched drinking water. Changes in pendrin protein abundance and function were assessed by immunoblotting and isolated tubule perfusion experiments. Plasma secretin levels and renal SCTR expression were assessed after 24 hours of acid/base-loading by radioimmunoassay and qPCR, respectively. ResultsSCTR KO mice responded with diminished urine alkalization and a lesser reduction of urinary acid excretion when base-loaded for 48 hours. Concordantly, SCTR KO mice presented with increased blood base retention compared with WTs. Base-loaded SCTR WT and KO mice showed comparable total pendrin protein abundance. Despite this, pendrin function was markedly lower in SCTR KO mice. Base-loaded mice had higher plasma secretin and renal SCTR levels compared with acid-loaded mice. Higher arterial HCO3- associated with higher renal SCTR mRNA expression. ConclusionPlasma secretin and renal SCTR levels are modulated by systemic acid-base status. Loss of the SCTR diminishes renal base excretion capacity and exacerbates systemic base accumulation during prolonged base-loading. These findings further support a central role of secretin and its receptor in the regulation of both acute and prolonged base excess.

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder characterized by skeletal muscle degeneration and cardiomyopathy without a cure. This study examined the therapeutic potential of the sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin (EMPA) on cardiac function in the dystrophin-deficient mdx mouse model of DMD. Male mice were fed control chow or EMPA-containing chow ([~]25 mg/kg/day), and cardiac function was evaluated longitudinally by four-dimensional ultrasound imaging. EMPA did not alter left ventricular mass or chamber volume but preserved ejection fraction (EF) for 12 weeks, maintained significantly higher EF through 24 weeks, and attenuated global impairment of systolic and diastolic myocardial deformation. These functional improvements were accompanied by reduced cardiomyocyte hypertrophy and decreased expression of cardiac stress genes. EMPA reduced mitochondrial DNA damage, increased mitochondrial DNA copy number, and induced transcriptional signatures consistent with enhanced fatty acid and ketone metabolism, contributing to increased myocardial ATP content. Systemically, EMPA improved body mass trajectory, preserved relative lean mass, enhanced skeletal muscle torque, and did not adversely affect renal function. Together, these findings demonstrate that EMPA improves cardiac performance and mitochondrial integrity while enhancing myocardial energy availability in mdx mice, supporting SGLT2 inhibitors as a promising therapeutic strategy for individuals with DMD.

BackgroundDiabetes-associated neurodegeneration is amplified by methylglyoxal (MGO)-driven dicarbonyl stress linking hyperglycemia to neuronal insulin resistance and maladaptive neuroinflammation. We tested the neuroprotective activity of MNP-021, a non-electrophilic TRPA1 modulator, in neurons and glial cells in vitro. MethodsSH-SY5Y neurons were pretreated with MNP-021 and challenged with MGO, then profiled by high-content imaging, RNA-seq, Seahorse OCR/ECAR, glycolytic stress assays and AKT/ERK/CREB immunoblotting {+/-} insulin. In parallel, HMC3 glial cells were treated with MNP-021, exposed to LPS/TNF- or A{beta}(25-35) and tested for viability and inflammatory markers by ELISA and qRT-PCR. ResultsMGO increased nucleus-to-cytoplasm area ratio by 49% and dysregulated glucose handling, increasing 2-NBDG uptake by [~]25%, with GLUT1/GLUT4 membrane redistribution; MNP-021 normalized morphology, uptake, and transporter localization without cytotoxicity up to 10 {micro}M. RNA-seq identified 754 MGO-deregulated genes, including ISR/metabolic nodes (GCK, SESN2, PHGDH/PSAT1, PCK2); MNP-021 buffered stress-induced transcription with limited baseline effects, remodeled mitochondrial redox readouts consistent with controlled ROS signaling, while improving mitochondrial content/architecture and blunting stress-evoked compensatory glycolysis. MNP-021 restored pro-survival signaling (pAKT/pERK and nuclear pCREB), including insulin responsiveness during MGO exposure. MNP-021 reduced IL-6/TNF- release while increasing IL-10 and ARG1 ([~]1.9-fold vs LPS/TNF-) in HMC3 glial cells, shifting them toward a pro-resolving IL-10/ARG1 program with reduced A{beta}(25-35)-evoked cytokine release with GLP-1 remaining very low ([&le;]10 pg/mL) and not significantly increased in this system. ConclusionsMNP-021 coordinates transcriptomic restraint, transporter-level glucose handling, mitochondrial resilience, and pro-survival/pro-resolving signaling across neuron-microglia compartments, supporting TRPA1-tuned small-molecule modulation as a candidate strategy against dicarbonyl-linked neuro-metabolic stress.

Pancreatic islets contain -, {beta}-, {gamma}- and {delta}-cells as sensors and actuators regulating glucose homeostasis. Despite the known importance of -cells, they are seemingly required for glucose tolerance only under metabolic stress. In an inducible model of -cell ablation in mice (GluDTR), glucose tolerance was considerably decreased by physiological addition of amino-acids mimicking meals. Analysis of islet {beta}-cell secretion and electrical activities using microelectrode arrays (MEA) detected only minor differences in GluDTR mice for glucose but revealed a major reduction upon addition of amino acids. Analysis of functional islet {beta}-cell networks by high density MEA revealed leading regions in different locations, a high degree of synchrony and the activation of large cell clusters. The characteristics of leading regions were preserved in GluDTR islets, but synchrony, cluster size and signal propagation speed were largely reduced. Thus, even without metabolic stress, -cells are required for nutrient homeostasis by regulating the dynamics of {beta}-cell networks. TeaserIslet -cells are required for meal tolerance by adjusting synchrony, cluster size and signal propagation of {beta}-cell networks.

Maintenance of skeletal muscle function is essential for functional independence, quality of life and healthspan. Muscle RING-finger protein-1 (MuRF1) negatively regulates muscle function and mass through ubiquitination and degradation of muscle proteins. Accordingly, genetic and pharmacological inhibition of MuRF1 attenuates muscle wasting and weakness under catabolic stress. To explore the potential of MuRF1 inhibitors (e.g., MyoMed-205) to improve muscle health, we investigated here the long-term effects of MyoMed-205 on functional capacity and muscle physiology in rats under basal conditions. Wistar rats were randomized to control or MyoMed-205 groups and were followed for 4 or 8 weeks. Body weight, food and water intake, and exercise capacity were monitored weekly. At each endpoint, the soleus muscle was collected for histological analyses. MyoMed-205-treated rats showed normal basic survival-related behaviors and body growth. After 8 weeks, MyoMed-205-treated animals exhibited enhanced exercise capacity (speed (m/min): +45%, p = 0.01; endurance (min): +47%, p = 0.03; and distance covered (m): +87%, p = 0.04) compared with baseline performance. Conversely, no differences were found in soleus fiber type distribution, cross-sectional area, or lipid and collagen content. Our findings indicate that MyoMed-205 enhances functional exercise capacity independently of changes in soleus muscle structure in rats under basal conditions.

BackgroundIn the context of increased salt intake in the world population, the understanding of the mechanisms that contribute to its correct renal excretion and therefore, avoid variation of blood volume and blood pressure is of major importance. MethodsMolecular, ex vivo microperfusion on isolated tubules, and integrative analysis, was used to identify, characterize and investigate a Na+ secretion pathway in the collecting duct. ResultsIn collecting duct of mice, salt load induced an increase of the type A intercalated cells (AIC) number, an overexpression of the H(Na),K-ATPase type 2 (HKA2) catalytic subunit Atp12a and a stimulation of the bumetanide-sensitive Na+ secretion in isolated and microperfused tubules. Surprisingly, HKA2KO mice fed a high-salt diet exhibit a strong dysregulation of their Na+ and water balance with a pronounced loss of Na+ and fluid, alkalosis, hypokalemia and low blood pressure. This Bartter-like phenotype is due to an over-inhibition of the thick ascending limb (TAL) related to an elevated PGE2 production. ConclusionOur findings establish that activation of Na+ secretion in AIC act as the fine-tuning knob in the regulation of renal Na+ excretion in response to high salt intake. Its absence is overcompensated by an inhibition of the Na+ transport system of the TAL.

Metabolic dysfunction-associated steatotic liver disease (MASLD) afflicts more than one-third of adults globally, contributing significantly to an increased cardiovascular disease risk. Further, patients with severe liver disease experience muscle weakness (sarcopenic obesity) and fatigue. Hypoxia-inducible factor 2 (HIF2) accumulates in the livers of MASLD patients and has been implicated in disease progression. Here we sought to understand the role of hepatic HIF2 in mediating hepatic and extra-hepatic features of MASLD. Using a well-validated obese mouse model of MASLD, we investigated the impact of hepatocyte-specific HIF2 deletion (hHIF2-/-) on hepatic, cardiac and skeletal muscle metabolism, and cardiac function. Over 28 weeks, mice were exposed to a high-fat, high-fructose, high-cholesterol (GAN) diet, which induced obesity alongside hepatic steatosis, fibrosis and inflammation. In contrast to observations in lean mouse models of liver disease, hHIF2-/- did not protect against MASLD, despite greater hepatic NADH-supported mitochondrial respiration and higher intracellular sphingomyelin levels. Instead, in the hearts of GAN-fed mice, hHIF2-/- caused diacylglycerol accumulation independent of diet, accumulation of long-chain acyl-carnitines and exacerbation of ceramide accumulation. Langendorff-perfused hearts from hHIF2-/- mice showed systolic and diastolic dysfunction, including 24% lower left ventricular developed pressure and 34% lower maximal rate of relaxation (dP/dtmin). However, isolated hearts from hHIF2-/- mice were protected against MASLD-associated sympathetic dominance, determined using autonomic receptor agonist stimulation. Both GAN-feeding and hHIF2-/- were associated with lower lean mass (14% and 5.4% lower than respective controls), whilst hHIF2-/- enhanced OXPHOS-associated protein levels in gastrocnemius muscle. Overall, hHIF2-/- resulted in detrimental extra-hepatic effects, including myocardial lipid accumulation, impaired cardiac function, and loss of whole-body lean mass, with no apparent protection against MASLD disease progression.

Metabolic syndrome (MetS), characterized by abdominal obesity, insulin resistance, dyslipidemia, and hypertension, affects a substantial proportion of the global population and increases the risk for cardiovascular disease, diabetes, and metabolic dysfunction-associated steatotic liver disease (MASLD). Despite its prevalence, there are currently no effective pharmacological therapies targeting MetS, highlighting the need to identify novel etiological mechanisms, particularly within the gastrointestinal (GI) tract. Using a mouse model of MetS and healthy lean controls, we assessed the colonic microenvironment through metabolomic, transcriptomic, and microbiome analyses. Colonic organoids were cultured to further explore epithelial alterations. Additionally, human MetS fecal metabolomics data were cross-compared with the mouse model to validate translational relevance. MetS mice exhibited upregulation of colonic anabolic pathways, including glycolysis, the pentose phosphate pathway, and the tryptophan/kynurenine pathway, without evidence of intestinal inflammation. Microbiome analysis revealed an increased abundance of the genus Lactobacillus in MS NASH mice. Colonic organoids from MetS mice showed altered goblet cell differentiation. Comparative analysis with human MetS fecal metabolomics demonstrated similar dysregulated pathways, underscoring the translational relevance of these findings. Our study reveals significant metabolic and microbial alterations in the colon of MS NASH mice, implicating a dysfunctional GI tract as a potential etiological factor in MetS. These findings highlight specific metabolic pathways and microbial signatures that could serve as future therapeutic targets for MetS. NEW & NOTEWORTHYThis study identifies the colon as a metabolically active tissue affected in metabolic syndrome. Despite the absence of intestinal inflammation, MS NASH mice displayed altered colonic metabolism and microbiota composition, with conserved metabolite changes matching those seen in humans with metabolic syndrome. These findings highlight colonic metabolic dysfunction as a potential driver of gut dysbiosis and disease progression in metabolic syndrome and MASLD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/716131v1_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@1b7c685org.highwire.dtl.DTLVardef@4a832aorg.highwire.dtl.DTLVardef@1e95c66org.highwire.dtl.DTLVardef@1b14209_HPS_FORMAT_FIGEXP M_FIG C_FIG

Short-term heat acclimation (HA) induces cardiovascular and fluid-regulatory adaptations, but its impact on markers of renal tubular injury and acute kidney injury risk (AKI) during exercise-heat stress remains unclear. Fourteen healthy endurance athletes were randomised to five days of isothermic HA (HOT; n = 7; 32 {degrees}C, 70% relative humidity; target core temperature [&ge;]38.5 {degrees}C), or matched exercise in thermoneutral conditions (TEMP, n = 7). Heat stress tests (HST; 45 min cycling at 32 {degrees}C, 70% RH) were performed pre- and post-intervention. Blood biomarkers of kidney tubular stress (NGAL, KIM-1), fluid-regulation (copeptin, serum osmolality) and sympathetic activity (plasma normetanephrine) were measured at rest and immediately post-HST. HA reduced resting heart rate (-8 {+/-} 5 bpm, p = 0.007, d = 1.0), increased plasma volume (+7.3 {+/-} 5.1%, p = 0.022) and sweat loss (+500 {+/-} 539 mL, p = 0.018, d = 1.1). Copeptin rose during the pre-intervention HST in both groups (HOT: +11 {+/-} 6; TEMP: +12 {+/-} 13 pmol{middle dot}L-1, p < 0.05), but not post-intervention. NGAL increased only in TEMP during HST1 (+45 {+/-} 29 g{middle dot}L-1, p = 0.030), while KIM-1 remained unchanged. No group x time interactions were observed for any biomarkers (p > 0.05). Five days of HA improved cardiovascular and thermoregulatory responses but did not alter renal stress markers or fluid-regulatory responses during exercise in the heat. These findings suggest short-term HA enhances heat tolerance without reducing acute renal biomarker responses under hot, humid conditions. New & NoteworthyFive days of isothermic heat acclimation improved cardiovascular and thermoregulatory responses, related to a lower resting heart rate, plasma volume expansion, and greater sweat loss. However, these benefits did not reduce renal tubular stress markers (NGAL, KIM-1), fluid-regulatory strain (copeptin), or sympathetic activity (normetanephrine) during exercise in the heat. Short-term heat acclimation lowers cardiovascular strain but does not mitigate renal biomarker responses, suggesting kidney stress risk remains unchanged in hot, humid conditions.

The acceleration of hepatic lipid disposal during acute exercise has been proposed as a contributor to the anti-steatotic effects of exercise training. Ketogenesis, which produces acetoacetate (AcAc) and {beta}-hydroxybutyrate ({beta}OHB) from fatty acids, is among the lipid disposal pathways stimulated by exercise. This study tested the hypothesis that hepatic ketogenesis is necessary for exercise training to lower liver lipids. Liver-specific 3-hydroxymethylglutaryl-CoA synthase 2 knockout (HMGCS2 KO) mice and wild type (WT) littermates underwent sedentary, acute exercise, and exercise training protocols. Liver ketone bodies and lipids were determined via mass spectrometry platforms. Stable isotope infusions in conscious, unrestrained mice defined mitochondrial oxidative fluxes at rest and during exercise. Loss of hepatic HMGCS2 decreased liver AcAc and {beta}OHB concentrations and impaired their increase during exercise. Liver triacylglycerides (TAGs) were comparable between genotypes at rest (i.e., ad libitum fed and short fasted conditions). In contrast, liver TAGs were elevated in HMGCS2 KO mice following acute, non-exhaustive exercise. Liver TCA cycle flux was higher in KO mice at rest. During exercise, TCA cycle flux increased in both WT and KO mice but was not different between genotypes with greater exercise duration. This suggests that enhanced disposal of lipids via the TCA cycle may prevent liver lipid accumulation in HMGCS2 KO mice under sedentary conditions, but not during exercise. Unexpectedly, exercise training decreased liver TAGs similarly in both HMGCS2 KO and WT mice. In conclusion, hepatic ketogenesis supports liver lipid homeostasis during acute exercise, but is not required for exercise training to lower liver lipids. NEW & NOTEWORTHYExercise training has been proposed to mitigate liver steatosis partly through enhanced hepatic lipid disposal. During acute exercise, the disposal of fatty acids to ketone bodies is stimulated. This study tested the hypothesis that hepatic ketogenesis was required for exercise training to reduce liver fat in mice. The results show that hepatic ketogenesis is needed to prevent lipid accumulation during acute exercise, but is not necessary for exercise training to lower liver lipids.

Lysosomal function is essential for cardiac proteostasis and cellular health, yet its regulation during ageing remains poorly defined. We hypothesised that ageing alters both the abundance of acidic organelles and the machinery supporting their acidification. Using fluorescence-based In Vivo Imaging Systems (IVIS) with Lysotracker Red in young (2-4 months) and aged (18 months) mouse hearts, we quantified whole-heart acidic-vesicle signals and assessed expression of lysosomal and autophagy-related genes (Lamp2, Atp6v1a, Sqstm1, Cd63, Atg12, Nfe2l2, M6pr) by RT-qPCR. Whole-heart labelled Lysotracker fluorescence did not differ significantly between age groups, indicating preservation of the total acidic-vesicle pool. No changes in Atp6v1a and Lamp2 expression suggest acidification capacity and structural stability are maintained, whereas the minor, upregulation of Sqstm1 might indicate increased autophagic demand and altered vesicle trafficking, which warrants further investigation. No statistical significant changes in M6pr, Atg12, or Nfe2l2 were detected, suggesting transcriptional stability in enzyme trafficking, core autophagy, and oxidative stress pathways. Regionally, atria showed higher Lysotracker signal than ventricles, consistent with known enrichment of acidic vesicular stores in atrial physiology. These findings highlight the utility of IVIS imaging of Lysotracker-labelled hearts, providing rapid whole-organ assessment of acidic vesicle distribution, albeit with limited depth resolution. Complementary techniques such as RT-qPCR analysis is essential to interpret IVIS findings, enabling insight into underlying molecular changes in lysosomal and autophagy pathways during cardiac ageing.

Skeletal muscle is the most abundant tissue in the human body and is essential for locomotion and the regulation of whole-body metabolism. The maintenance of skeletal muscle mass is essential for health, yet the molecular and signaling mechanisms that control skeletal muscle mass remain poorly understood. Transforming growth factor-{beta}-activated kinase 1 (TAK1) is a key signaling protein that regulates multiple intracellular pathways. Recent studies have demonstrated that TAK1 is a critical regulator of skeletal muscle mass. However, the mechanisms by which TAK1 regulates muscle mass and whether its role is sex dependent remain incompletely understood. In this study, we show that targeted inactivation of TAK1 induces muscle atrophy more rapidly in male than in female mice. Loss of TAK1 activity also abolished mechanical overload-induced phosphorylation of p70S6K and rpS6, and the induction of myofiber hypertrophy in both sexes. RNA-Seq analysis further revealed that TAK1 inactivation in skeletal muscle disrupts the gene expression of various molecules involved in catabolic processes, calcium signaling, muscle structure development, and aerobic respiration. Moreover, TAK1 inactivation impairs fatty acid oxidation and promotes lipid accumulation in skeletal muscle of adult mice in a sex-independent manner. Collectively, our findings demonstrate that TAK1 regulates skeletal muscle mass and growth by coordinating distinct intracellular pathways in both male and female mice.

{beta}2-Adrenergic receptor (Adr{beta}2) is the most abundant form of adrenergic receptors in skeletal muscle. Our previous studies have shown that the ventromedial hypothalamic nucleus (VMH) regulates metabolic benefits of exercise, potentially by skeletal muscle Adr{beta}2. Although a large body of literature has shown the importance of Adr{beta}2 on skeletal muscle physiology, it remains unexplored whether skeletal muscle Adr{beta}2 contributes to metabolic benefits of exercise, such as prevention of diet-induced obesity (DIO). Here, we generated mice lacking Adr{beta}2 in skeletal muscle cells (SKMAdr{beta}2) and tested whether SKMAdr{beta}2 is required for metabolic benefits of exercise on DIO. Deletion of SKMAdr{beta}2 completely abolished the induction of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1) in skeletal muscle by {beta}2-agonist, which is a potent activator of Pgc-1. Exercise upregulates Pgc-1, which regulates a broad range of skeletal muscle physiology, including hypertrophy and mitochondrial function. Deletion of SKMAdr{beta}2 hampers augmented Pgc-1 in skeletal muscle by a single bout of exercise. Intriguingly, we found that deletion of SKMAdr{beta}2 increased endurance capacity. Further, our data showed that body weight in DIO mice lacking SKMAdr{beta}2 is comparable to that of control DIO mice during exercise training, suggesting that deletion of SKMAdr{beta}2 did not affect the metabolic benefits of exercise in DIO. Collectively, our data indicate that SKMAdr{beta}2 contributes to exercise-induced transcriptional changes and endurance capacity, however, it is not required for exercise benefits on bodyweight in DIO mice.

Exercise is a cornerstone therapy for diabetes because working skeletal muscles take up glucose at dramatically greater rates than postprandial insulin-stimulated glucose uptake and, notably, do so without a requirement for insulin. This remarkable ability of working muscles is preserved in diabetes, when muscles become resistant to insulin. However, the mechanism of insulin-independent glucose uptake by working muscles is not fully understood. Here we describe a previously unrecognized glucose uptake pathway in muscle, which we refer to as "mSGLT" based on shared properties with the Sodium Glucose Linked Transporter family. In contrast to the abundant GLUT4 transporter, mSGLT is not regulated by insulin, requires Na,K-ATPase-2 activity, and transports the hexose -methyl-D-glucoside (MDG), a glucose derivative that is handled by SGLTs but not GLUT4. The mSGLT pathway and GLUT transport pathways are independent and additive. In addition to exercise, mSGLT imports glucose under other conditions of adrenergic stimulation, which inhibits pancreatic insulin release and reduces the insulin sensitivity of muscle. SGLT2-specific antibodies recognize a protein in muscle of similar size to the kidney SGLT2; this protein localizes to the muscle t-tubules, together with Na,K-ATPase-2 and MAP17, the regulatory subunit of SGLT2. However, skeletal muscles do not express a full-length transcript of Slc5a2 (SGLT2), and SGLT2-specific inhibitors do not inhibit mSGLT with high affinity. The novel transporter may be a muscle variant of Slc5a2 that results from post-transcriptional or post-translational mechanisms. mSGLT and its regulation offer potential muscle-specific therapeutic targets for treating hyperglycemia and other conditions when insulin-stimulated glucose disposal into muscle is impaired.

AimThe mammalian tracheal epithelium is composed by different cell types unevenly distributed along the proximal-distal axis. Nevertheless, variations in expression and function of ion channels and transporters participating in fluid absorption and secretion had never been studied separately in proximal and distal sections of the mouse trachea. In this work, we aim to characterize basal and stimulated absorption and secretion of fluid obtained from proximal and distal trachea from the same animal. MethodsUssing chamber experiments were performed using a custom-made tissue slider that allowed the mounting small tracheal sections, where response to agonists and blockers was recorded. The role of the NKCC1 co-transporter was studied using the Slc12a2-/- mouse. A genetically tomato-induced mouse model was used to assess co-expression of NKCC1 and ASCL3 by immunofluorescence. Animals were instilled with different interleukins (ILs) to determine changes in absorption, secretion and mucus properties. ResultsProximal trachea didnt participate in sodium absorption but exhibited higher cAMP- and succinate-induced anion secretion than the distal section. NBCe1-dependent bicarbonate and TMEM16A-driven chloride secretion was significantly higher in the distal section. NKCC1+ cells were found in the submucosal glands (SMGs) and abundant patches of NKCC1+ cells in the distal region. Isolated NKCC1+ cells co-expressing ASCL3 were also detected. ILs treatment changed the electrophysiological properties of the distal but not the proximal trachea. ConclusionsOur experiments determined that the mouse trachea organizes its functions differentially in the proximal and distal sections, based in the functional distribution of channels, transporters and receptors. While the distal trachea drastically changed its responses to agonists inducing anion secretion the proximal trachea was unperturbed by the action of ILs.

BackgroundDiabetes mellitus leads to skeletal muscle dysfunction associated with loss of strength, impaired blood perfusion, lipid accumulation, and inflammation. The opening of large-pore channels has been linked to increased membrane permeability and inflammatory signaling in several pathologies. Boldine, an alkaloid from Peumus boldus, blocks large-pore channel activity and exhibits antioxidant and anti-inflammatory properties. This study evaluated whether boldine prevents skeletal muscle alterations induced by diabetes and explored potential underlying mechanisms. MethodsDiabetes was induced in male C57BL/6J mice using streptozotocin (STZ, 40 mg/kg/day for 5 days). Diabetic mice were treated with boldine (50 mg/kg/day) for four weeks. Muscle strength and resting membrane potential were analyzed in vivo. Also, right gastrocnemius muscle blood perfusion at basal and after acetylcholine (10 M) stimulation were analyzed in vivo. Lipid accumulation was assessed using Oil Red O staining, and CD31 immunodetection was used to evaluate capillary density. mRNA levels of NLRP3 were evaluated in muscle by qPCR. In human myoblasts (AB1167) cultured under low (8 mM) or high glucose (25 mM) conditions, with or without boldine, membrane permeability (ethidium uptake), intracellular Ca{superscript 2} (Fura-2), nitric oxide (DAF-FM), and levels of NLRP3 and Casp1 (qPCR) and reactivity PPAR{gamma} (Immunofluorescence) were determined. ResultsSTZ mice showed reduced muscle strength and depolarized resting membrane potential, both prevented by boldine. Basal muscle perfusion was [~]20% lower in diabetic mice (160.1 {+/-} 17.2 vs. 199.1 {+/-} 13.8 units), whereas boldine preserved perfusion (184.6 {+/-} 14.3 units). Oil Red O-positive fibers increased to 52.4 {+/-} 3.6% in diabetic mice and decreased to 15.2 {+/-} 4.1% with boldine (control: 3.1 {+/-} 1.3%; p<0.05). NLRP3 mRNA increased 17.7 {+/-} 2.8-fold in diabetic muscle and was reduced by [~]50% with boldine. In myoblasts, high glucose increased ethidium uptake, nitric oxide production, NLRP3 and caspase-1 expression, and nuclear PPAR{gamma} ([~]45% positive nuclei); all effects were prevented by boldine. ConclusionsBoldine preserves skeletal muscle function and vascular reactivity in diabetes and prevents lipid accumulation and inflammasome activation both in vivo and in vitro. These effects are associated with inhibition of large-pore channel activity and attenuation of downstream calcium-dependent, inflammatory, and adipogenic pathways, supporting boldine as a promising therapeutic candidate for diabetes-associated skeletal muscle dysfunction. Graphical abstractIn myoblasts, high glucose activates large-pore channels, elevating cytoplasmic Ca{superscript 2} concentration and nitric oxide generation, which increases the activity of Cx-formed hemichannels, raises the levels of inflammasome components, and promotes lipid accumulation. In STZ-diabetic mice, de novo expression of large-pore channels in skeletal muscles contributes to reduced blood perfusion, accumulation of intramuscular fat, muscle weakness, and reduced resting membrane potential of myofibers. Boldine inhibits large-pore channel activity, preventing these alterations and preserving muscle physiology in vivo. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/707704v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@19179b4org.highwire.dtl.DTLVardef@1cd3d21org.highwire.dtl.DTLVardef@16851d6org.highwire.dtl.DTLVardef@1d4e77c_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundDyspnea and exercise intolerance are the primary clinical symptoms of heart failure. Heart failure patients experience frequent hypoxemic episodes, yet underlying mechanisms and relevance remain poorly understood. In a cohort of heart failure patients and multiple animal models, we identify pulmonary capillary rarefaction driven by excessive autophagy in endothelial cells as a novel mechanism of hypoxemia and cardiac disease progression. MethodsA cohort of heart failure with preserved ejection fraction (HFpEF) patients was analyzed for parameters of left ventricular (LV) dysfunction and pulmonary gas exchange. Morphological and cellular mechanisms of impaired pulmonary oxygenation were assessed in three animal models of heart failure, namely two HFpEF models, SU5416-treated ZSF1 obese rats and high fat diet/L-NAME treated mice, and in rats subjected to aortic banding. Lung microvascular rarefaction was quantified by micro-computed tomography, stereology, flow cytometry and dye efflux. Cellular mechanisms of capillary loss were analyzed by single-cell transcriptomics, electron microscopy and immunofluorescence, and in mice with endothelial-specific deletion of the autophagy gene Atg7 (Atg7EN-KO). ResultsIn 234 HFpEF patients, advancing NYHA class was associated with progressive worsening of arterial oxygen saturation at rest and during exercise and a reduced lung diffusing capacity. Impaired gas diffusion correlated with indices of LV diastolic dysfunction. Impaired oxygenation and reduced exercise capacity were similarly evident in animal models of left heart disease, which showed a distinct loss of pulmonary microvessels and capillaries. Lung microvascular endothelial cells in HFpEF showed characteristics of increased autophagic flux and apoptosis. Relative to their wild type HFpEF controls, Atg7EN-KO mice had less capillary loss, restored normoxemia, improved exercise tolerance, and mitigated LV diastolic dysfunction. Additional studies in HFpEF mice corroborated the functional relevance of impaired gas exchange for the progression of left heart disease by demonstrating that additional hypoxia aggravated, whereas moderate hyperoxia improved LV function. ConclusionOur findings identify pulmonary microvascular rarefaction as a novel pathomechanism in heart failure that i) contributes to dyspnea and exercise intolerance, ii) impairs pulmonary gas exchange and iii) accelerates LV disease progression. Strategies targeting this axis such as moderate oxygen therapy may mitigate cardiopulmonary morbidity in heart failure. Clinical Trial RegistrationRegistered in the DRKS (Deutsches Register fur klinische Studien) as trial# DRKS00032974 at https://drks.de/search/en/trial/DRKS00032974.