Excess Intramyocellular Lipid Does Not Affect Muscle Fiber Biophysical Properties in Mice or People with Metabolically Abnormal Obesity.

Observational studies show correlations between intramyocellular lipid (IMCL) content and muscle strength and contractile function in people with "metabolically abnormal" obesity. However, a clear physiologic mechanism for this association is lacking and causation is debated. We combined immunofluorescent confocal imaging with force measurements on permeabilized muscle fibers from metabolically normal and metabolically abnormal mice and metabolically normal (defined as normal fasting plasma glucose and glucose tolerance) and metabolically abnormal (defined as pre-diabetes and type 2 diabetes) people with overweight/obesity to evaluate relationships among myocellular lipid droplet characteristics (droplet size and density) and biophysical (active contractile and passive viscoelastic) properties. The fiber type specificity of lipid droplet parameters varied between metabolically abnormal and normal mice and among metabolically normal and metabolically abnormal people. However, despite considerable quantities of IMCL in the metabolically abnormal groups, there were no significant differences in peak active tension or passive viscoelasticity between the metabolically abnormal groups and the control group in mice or people. Additionally, there were no significant relationships among IMCL parameters and biophysical variables. Thus, we conclude that IMCL accumulation per se does not impact muscle fiber biophysical properties or physically impede contraction.

Use of computed tomography to identify muscle quality subgroups, spatial mapping, and preliminary relationships to function in those with diabetic peripheral neuropathy.

BACKGROUND: Decreased muscle volume and increased muscle-associated adipose tissue (MAAT, sum of intra and inter-muscular adipose tissue) of the foot intrinsic muscle compartment are associated with deformity, decreased function, and increased risk of ulceration and amputation in those with diabetic peripheral neuropathy (DPN). RESEARCH QUESTION: What is the muscle quality (normal, abnormal muscle, and adipose volumes) of the DPN foot intrinsic compartment, how does it change over time, and is muscle quality related to gait and foot function? METHODS: Computed tomography was performed on the intrinsic foot muscle compartment of 45 subjects with DPN (mean age: 67.2 ± 6.4 years) at baseline and 3.6 years. Images were processed to obtain volumes of MAAT, highly abnormal, mildly abnormal, and normal muscle. For each category, annual rates of change were calculated. Paired t-tests compared baseline and follow-up. Foot function during gait was assessed using 3D motion analysis and the Foot and Ankle Ability Measure. Correlations between muscle compartment and foot function during gait were analyzed using Pearson's correlations. RESULTS: Total muscle volume decreased, driven by a loss of normal muscle and mildly abnormal muscle (p<0.05). MAAT and the adipose-muscle ratio increased. At baseline, 51.5% of the compartment was abnormal muscle or MAAT, increasing to 55.0% at follow-up. Decreased total muscle volume correlated with greater midfoot collapse during gait (r = -0.40, p = 0.02). Greater volumes of highly abnormal muscle correlated with a lower FAAM score (r = -0.33, p = 0.03). SIGNIFICANCE: Muscle volume loss may progress in parallel with MAAT accumulation, impacting contractile performance in individuals with DPN. Only 48.5% of the DPN intrinsic foot muscle compartment consists of normal muscle and greater abnormal muscle is associated with worse foot function. These changes identify an important target for rehabilitative intervention to slow or prevent muscle deterioration and poor foot outcomes.

Insights into post-translational regulation of skeletal muscle contractile function by the acetyltransferases, p300 and CBP.

Mice with skeletal muscle-specific and inducible double knockout of the lysine acetyltransferases, p300 (E1A binding protein p300) and CBP (cAMP-response element-binding protein binding protein), referred to as i-mPCKO, demonstrate a dramatic loss of contractile function in skeletal muscle and ultimately die within 7 days. Given that many proteins involved in ATP generation and cross-bridge cycling are acetylated, we investigated whether these processes are dysregulated in skeletal muscle from i-mPCKO mice and, thus, whether they could underlie the rapid loss of muscle contractile function. Just 4-5 days after inducing knockout of p300 and CBP in skeletal muscle from adult i-mPCKO mice, there was ~90% reduction in ex vivo contractile function in the extensor digitorum longus (EDL) and a ~65% reduction in in vivo ankle dorsiflexion torque, as compared to wildtype (WT; i.e. Cre negative) littermates. Despite this profound loss of contractile force in i-mPCKO mice, there were no genotype-driven differences in fatigability during repeated contractions, nor were there genotype differences in mitochondrial-specific pathway enrichment of the proteome, intermyofibrillar mitochondrial volume or mitochondrial respiratory function. As it relates to cross-bridge cycling, remarkably, the overt loss of contractile function in i-mPCKO muscle was reversed in permeabilized fibers supplied with exogenous Ca2+ and ATP, with active tension being similar between i-mPCKO and WT mice, regardless of Ca2+ concentration. Actin-myosin motility was also similar in skeletal muscle from i-mPCKO and WT mice. In conclusion, neither mitochondrial abundance/function, nor actomyosin cross-bridge cycling, are the underlying driver of contractile dysfunction in i-mPCKO mice.

Insights into post-translational regulation of skeletal muscle contractile function by the acetyltransferases, p300 and CBP.

Mice with skeletal muscle-specific inducible double knockout of the lysine acetyltransferases, p300 (E1A binding protein p300) and CBP (cAMP-response element-binding protein binding protein), referred to as i-mPCKO, demonstrate a dramatic loss of contractile function in skeletal muscle and ultimately die within 7 days. Given that many proteins involved in ATP generation and cross-bridge cycling are acetylated, we investigated whether these processes are dysregulated in skeletal muscle from i-mPCKO mice and thus could underlie the rapid loss of muscle contractile function. Just 4-5 days after inducing knockout of p300 and CBP in skeletal muscle from adult i-mPCKO mice, there was ∼90% reduction in ex vivo contractile function in the extensor digitorum longus (EDL) and a ∼65% reduction in in vivo ankle dorsiflexion torque, as compared to wildtype (WT; i.e. Cre negative) littermates. Despite the profound loss of contractile force in i-mPCKO mice, there were no genotype-driven differences in fatigability during repeated contractions, nor were there genotype differences in mitochondrial specific pathway enrichment of the proteome, intermyofibrillar mitochondrial volume or mitochondrial respiratory function. As it relates to cross-bridge cycling, remarkably, the overt loss of contractile function in i-mPCKO muscle was reversed in permeabilized fibers supplied with exogenous Ca 2+ and ATP, with active tension being similar between i-mPCKO and WT mice, regardless of Ca 2+ concentration. Actin-myosin motility was also similar in skeletal muscle from i-mPCKO and WT mice. In conclusion, neither mitochondrial abundance/function, nor actomyosin cross-bridge cycling, are the underlying driver of contractile dysfunction in i-mPCKO mice. New & Noteworthy: The mechanism underlying dramatic loss of muscle contractile function with inducible deletion of both p300 and CBP in skeletal muscle remains unknown. Here we find that impairments in mitochondrial function or cross-bridge cycling are not the underlying mechanism of action. Future work will investigate other aspects of excitation-contraction coupling, such as Ca 2+ handling and membrane excitability, as contractile function could be rescued by permeabilizing skeletal muscle, which provides exogenous Ca 2+ and bypasses membrane depolarization.

Disruption of hepatic mitochondrial pyruvate and amino acid metabolism impairs gluconeogenesis and endurance exercise capacity in mice.

Exercise robustly increases the glucose demands of skeletal muscle. This demand is met by not only muscle glycogenolysis but also accelerated liver glucose production from hepatic glycogenolysis and gluconeogenesis to fuel mechanical work and prevent hypoglycemia during exercise. Hepatic gluconeogenesis during exercise is dependent on highly coordinated responses within and between muscle and liver. Specifically, exercise increases the rate at which gluconeogenic precursors such as pyruvate/lactate or amino acids are delivered from muscle to the liver, extracted by the liver, and channeled into glucose. Herein, we examined the effects of interrupting hepatic gluconeogenic efficiency and capacity on exercise performance by deleting mitochondrial pyruvate carrier 2 (MPC2) and/or alanine transaminase 2 (ALT2) in the liver of mice. We found that deletion of MPC2 or ALT2 alone did not significantly affect time to exhaustion or postexercise glucose concentrations in treadmill exercise tests, but mice lacking both MPC2 and ALT2 in hepatocytes (double knockout, DKO) reached exhaustion faster and exhibited lower circulating glucose during and after exercise. Use of 2H/1³C metabolic flux analyses demonstrated that DKO mice exhibited lower endogenous glucose production owing to decreased glycogenolysis and gluconeogenesis at rest and during exercise. Decreased gluconeogenesis was accompanied by lower anaplerotic, cataplerotic, and TCA cycle fluxes. Collectively, these findings demonstrate that the transition of the liver to the gluconeogenic mode is critical for preventing hypoglycemia and sustaining performance during exercise. The results also illustrate the need for interorgan cross talk during exercise as described by the Cahill and Cori cycles.NEW & NOTEWORTHY Martino and colleagues examined the effects of inhibiting hepatic gluconeogenesis on exercise performance and systemic metabolism during treadmill exercise in mice. Combined inhibition of gluconeogenesis from lactate/pyruvate and alanine impaired exercise endurance and led to hypoglycemia during and after exercise. In contrast, suppressing either pyruvate-mediated or alanine-mediated gluconeogenesis alone had no effect on these parameters. These findings provide new insight into the molecular nodes that coordinate the metabolic responses of muscle and liver during exercise.

Sarm1 knockout prevents type 1 diabetic bone disease in females independent of neuropathy.

Patients with diabetes have a high risk of developing skeletal diseases accompanied by diabetic peripheral neuropathy (DPN). In this study, we isolated the role of DPN in skeletal disease with global and conditional knockout models of sterile-α and TIR-motif-containing protein-1 (Sarm1). SARM1, an NADase highly expressed in the nervous system, regulates axon degeneration upon a range of insults, including DPN. Global knockout of Sarm1 prevented DPN, but not skeletal disease, in male mice with type 1 diabetes (T1D). Female wild-type mice also developed diabetic bone disease but without DPN. Unexpectedly, global Sarm1 knockout completely protected female mice from T1D-associated bone suppression and skeletal fragility despite comparable muscle atrophy and hyperglycemia. Global Sarm1 knockout rescued bone health through sustained osteoblast function with abrogation of local oxidative stress responses. This was independent of the neural actions of SARM1, as beneficial effects on bone were lost with neural conditional Sarm1 knockout. This study demonstrates that the onset of skeletal disease occurs rapidly in both male and female mice with T1D completely independently of DPN. In addition, this reveals that clinical SARM1 inhibitors, currently being developed for treatment of neuropathy, may also have benefits for diabetic bone through actions outside of the nervous system.

Disruption of Hepatic Mitochondrial Pyruvate and Amino Acid Metabolism Impairs Gluconeogenesis and Endurance Exercise Capacity in Mice.

Exercise robustly increases the glucose demands of skeletal muscle. This demand is met not only by muscle glycogenolysis, but also by accelerated liver glucose production from hepatic glycogenolysis and gluconeogenesis to fuel mechanical work and prevent hypoglycemia during exercise. Hepatic gluconeogenesis during exercise is dependent on highly coordinated responses within and between muscle and liver. Specifically, exercise increases the rate at which gluconeogenic precursors such as pyruvate/lactate or amino acids are delivered from muscle to the liver, extracted by the liver, and channeled into glucose. Herein, we examined the effects of interrupting gluconeogenic efficiency and capacity on exercise performance by deleting hepatic mitochondrial pyruvate carrier 2 (MPC2) and/or alanine transaminase 2 (ALT2) in mice. We found that deletion of MPC2 or ALT2 alone did not significantly affect time to exhaustion or post-exercise glucose concentrations in treadmill exercise tests, but mice lacking both MPC2 and ALT2 in liver (DKO) reached exhaustion faster and exhibited lower circulating glucose during and after exercise. Use of ²H/¹³C metabolic flux analyses demonstrated that DKO mice exhibited lower endogenous glucose production owing to decreased glycogenolysis and gluconeogenesis at rest and during exercise. The decreased gluconeogenesis was accompanied by lower anaplerotic, cataplerotic, and TCA cycle fluxes. Collectively, these findings demonstrate that the transition of the liver to the gluconeogenic mode is critical for preventing hypoglycemia and sustaining performance during exercise. The results also illustrate the need for interorgan crosstalk during exercise as described by the Cahill and Cori cycles.

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration.

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

Skeletal muscle delimited myopathy and verapamil toxicity in SUR2 mutant mouse models of AIMS.

ABCC9-related intellectual disability and myopathy syndrome (AIMS) arises from loss-of-function (LoF) mutations in the ABCC9 gene, which encodes the SUR2 subunit of ATP-sensitive potassium (KATP ) channels. KATP channels are found throughout the cardiovascular system and skeletal muscle and couple cellular metabolism to excitability. AIMS individuals show fatigability, muscle spasms, and cardiac dysfunction. We found reduced exercise performance in mouse models of AIMS harboring premature stop codons in ABCC9. Given the roles of KATP channels in all muscles, we sought to determine how myopathy arises using tissue-selective suppression of KATP and found that LoF in skeletal muscle, specifically, underlies myopathy. In isolated muscle, SUR2 LoF results in abnormal generation of unstimulated forces, potentially explaining painful spasms in AIMS. We sought to determine whether excessive Ca2+ influx through CaV 1.1 channels was responsible for myopathology but found that the Ca2+ channel blocker verapamil unexpectedly resulted in premature death of AIMS mice and that rendering CaV 1.1 channels nonpermeable by mutation failed to reverse pathology; results which caution against the use of calcium channel blockers in AIMS.

Development and expansion of intramuscular adipose tissue is not dependent on UCP-1-lineage cells in mice.

Accumulation of adipose tissue within and outside of skeletal muscle is associated with orthopedic injury and metabolic disease, where it is thought to impede muscle function. The close juxtaposition between this adipose and myofibers has led to hypotheses that paracrine interactions between the two regulate local physiology. Recent work suggests that intramuscular adipose tissue (IMAT) may have features of beige or brown fat, indicated by the expression of uncoupling protein-1 (UCP-1). However, this is contested by other studies. Clarification of this point is needed to inform our understanding of the relationship between IMAT and muscle health. To achieve this, we examined the effects of constitutive UCP-1+ cell ablation (UCP1-DTA) on IMAT development and homeostasis. IMAT developed normally in UCP1-DTA mice, with no significant differences in quantity compared with wild-type littermates. Likewise, IMAT accumulation in response to glycerol-induced injury was similar between genotypes, with no significant differences in adipocyte size, quantity, or dispersion. This suggests that neither physiological nor pathological IMAT express UCP-1 and that the development of IMAT does not depend on UCP-1 lineage cells. In response to ß3-adrenergic stimulation, we find minor, localized UCP-1 positivity in wildtype IMAT, but the bulk of the adipocytes are unresponsive. In contrast, two depots of muscle-adjacent (epi-muscular) adipose tissue have reduced mass in UCP1-DTA mice and UCP-1 positivity in wildtype littermates, comparable to traditional beige and brown adipose depots. Taken together this evidence strongly supports a white adipose phenotype for mouse IMAT and a brown/beige phenotype for some adipose outside the muscle boundary.

Site-1 protease inhibits mitochondrial respiration by controlling the TGF-ß target gene Mss51.

The mitochondrial response to changes in cellular energy demand is necessary for cellular adaptation and organ function. Many genes are essential in orchestrating this response, including the transforming growth factor (TGF)-ß1 target gene Mss51, an inhibitor of skeletal muscle mitochondrial respiration. Although Mss51 is implicated in the pathophysiology of obesity and musculoskeletal disease, how Mss51 is regulated is not entirely understood. Site-1 protease (S1P) is a key activator of several transcription factors required for cellular adaptation. However, the role of S1P in muscle is unknown. Here, we identify S1P as a negative regulator of muscle mass and mitochondrial respiration. S1P disruption in mouse skeletal muscle reduces Mss51 expression and increases muscle mass and mitochondrial respiration. The effects of S1P deficiency on mitochondrial activity are counteracted by overexpressing Mss51, suggesting that one way S1P inhibits respiration is by regulating Mss51. These discoveries expand our understanding of TGF-ß signaling and S1P function.

Tenotomy-induced muscle atrophy is sex-specific and independent of NFκB.

The nuclear factor-κB (NFκB) pathway is a major thoroughfare for skeletal muscle atrophy and is driven by diverse stimuli. Targeted inhibition of NFκB through its canonical mediator IKKß effectively mitigates loss of muscle mass across many conditions, from denervation to unloading to cancer. In this study, we used gain- and loss-of-function mouse models to examine the role of NFκB in muscle atrophy following rotator cuff tenotomy - a model of chronic rotator cuff tear. IKKß was knocked down or constitutively activated in muscle-specific inducible transgenic mice to elicit a twofold gain or loss of NFκB signaling. Surprisingly, neither knockdown of IKKß nor overexpression of caIKKß significantly altered the loss of muscle mass following tenotomy. This finding was consistent across measures of morphological adaptation (fiber cross-sectional area, fiber length, fiber number), tissue pathology (fibrosis and fatty infiltration), and intracellular signaling (ubiquitin-proteasome, autophagy). Intriguingly, late-stage tenotomy-induced atrophy was exacerbated in male mice compared with female mice. This sex specificity was driven by ongoing decreases in fiber cross-sectional area, which paralleled the accumulation of large autophagic vesicles in male, but not female muscle. These findings suggest that tenotomy-induced atrophy is not dependent on NFκB and instead may be regulated by autophagy in a sex-specific manner.

Muscle atrophy ­ the gradual loss of muscle mass ­ follows injuries to our muscles, tendons, or joints. During atrophy, muscles shrink and become weaker, which can interfere with everyday activities and, ultimately, decrease quality of life. Rotator cuff tears are a common example of such injuries. A rotator cuff is group of four muscles that come together as tendons to form a cuff that normally stabilises our shoulders and allows us to lift and move our arms over our heads. Rotator cuff tears can result from an injury or may be caused by ageing-related wear and tear of the tendon. A signalling protein, called NFκB, is thought to be involved in muscle atrophy. When the NFκB signal is switched on, it interacts with genes that are thought to speed up the loss of muscle mass. However, NFκB's precise role in atrophy and recovery after muscle injury is still poorly understood, particularly following injuries where a tendon is cut or torn. Meyer et al. therefore set out to determine whether or not NFκB played a role in the muscle atrophy following rotator cuff tears. Meyer et al. used genetically engineered mice in which NFκB's signal could be turned off at the time of rotator cuff injury, and specifically in muscle cells (but not other parts of the body). The experiments revealed that stopping NFκß signalling in these mice did not reduce muscle atrophy after a rotator cuff injury: the levels of atrophy, muscle performance, and muscle composition were the same regardless of whether the NFκß signal was active. The sex of the mice did, however, affect muscle atrophy, specifically the way in which they lost muscle mass. In male mice, the size of muscle cells decreased, while in female mice, the number of muscle cells decreased. Muscle cells in male mice (but not in females) also accumulated abnormally high amounts of protein, which is an indication of a mechanism of muscle breakdown called autophagy. These results shed new light on the way that we lose muscle mass after injury, and how that could vary depending on the individual. Meyer et al. hope that this study will help guide the development of new, more effective treatments for muscle atrophy, and ultimately contribute to therapies tailored to the characteristics of the patient and the type of injury.

Effect of Veau Class on Levator Veli Palatini Muscle Composition.

OBJECTIVE: To examine levator veli palatini muscle composition in patients with nonsyndromic cleft palate and investigate the impact of Veau class. DESIGN: Prospective cohort study. SETTING: Tertiary care academic hospital. PATIENTS/PARTICIPANTS: Thirteen patients with nonsyndromic cleft palate were recruited. INTERVENTIONS: During primary palatoplasty, a sample of levator veli palatini muscle was excised and prepared for histological analysis. MAIN OUTCOME MEASURES: Fat and collagen content were determined utilizing Oil Red and Sirius red stains, respectively, while muscle fiber cross-sectional areas were calculated from H&E-stained samples, with analysis using histomorphometric methods. Immunofluorescent staining of myosin heavy chain isoforms was performed. RESULTS: Patients underwent repair at 10.8 months of age (interquartile range [IQR] 10.2-12.9). Fat content of the levator veli palatini muscle was low in both groups, ranging from 0% to 5.2%. Collagen content ranged from 8.5% to 39.8%; neither fat nor collagen content showed an association with Veau classes. Mean muscle fiber cross-sectional area decreased with increasing Veau class, from 808 µm2 (range 692-995 µm2) in Veau II to 651 µm2 (range 232-750 µm2) in Veau III (P = .02). There was also a nonsignificant decrease in proportion of type I muscle fibers with increasing Veau class (44.3% [range 31.4%-84.4%] in Veau II vs 35.3% [range 17.4%-61.3%] in Veau III). CONCLUSIONS: Muscle fiber area in levator veli palatini muscles decreases in Veau III clefts in comparison to Veau II. The impact of these differences in velopharyngeal dysfunction requires further analysis of a larger cohort.

Leptin mediates the regulation of muscle mass and strength by adipose tissue.

Adipose tissue secretes numerous cytokines (termed 'adipokines') that have known or hypothesized actions on skeletal muscle. The majority of adipokines have been implicated in the pathological link between excess adipose and muscle insulin resistance, but approximately half also have documented in vitro effects on myogenesis and/or hypertrophy. This complexity suggests a potential dual role for adipokines in the regulation of muscle mass in homeostasis and the development of pathology. In this study, we used lipodystrophic 'fat-free' mice to demonstrate that adipose tissue is indeed necessary for the development of normal muscle mass and strength. Fat-free mice had significantly reduced mass (∼15%) and peak contractile tension (∼20%) of fast-twitch muscles, a slowing of contractile dynamics and decreased cross-sectional area of fast twitch fibres compared to wild-type littermates. These deficits in mass and contractile tension were fully rescued by reconstitution of ∼10% of normal adipose mass, indicating that this phenotype is the direct consequence of absent adipose. We then showed that the rescue is solely mediated by the adipokine leptin, as similar reconstitution of adipose from leptin-knockout mice fails to rescue mass or strength. Together, these data indicate that the development of muscle mass and strength in wild-type mice is dependent on adipose-secreted leptin. This finding extends our current understanding of the multiple roles of adipokines in physiology as well as disease pathophysiology to include a critical role for the adipokine leptin in muscle homeostasis. KEY POINTS: Adipose-derived cytokines (adipokines) have long been implicated in the pathogenesis of insulin resistance in obesity but likely have other under-appreciated roles in muscle physiology. Here we use a fat-free mouse to show that adipose tissue is necessary for the normal development of muscle mass and strength. Through add-back of genetically modified adipose tissue we show that leptin is the key adipokine mediating this regulation. This expands our understanding of leptin's role in adipose-muscle signalling to include development and homeostasis and adds the surprising finding that leptin is the sole mediator of the maintenance of muscle mass and strength by adipose tissue.

Geometric modeling predicts architectural adaptations are not responsible for the force deficit following tenotomy in the rotator cuff.

Tenotomy, or the severing of then tendinous connection between muscle and bone, is an experimental model frequently used to assess muscular changes in response to unloading with retraction. It is most translationally relevant to rotator cuff (RC) tendon tears as these frequently progress to chronic muscle retraction and thus most recent tenotomy animal models have used RC muscles. Tenotomy induces chronic changes to muscle architecture including reduced muscle mass, muscle length, fiber length and pennation angle. However, most RC studies evaluating the physiological consequences of tenotomy do not account for changes in architecture, in part because these are difficult to measure in RC muscles. This is a critical omission as architectural changes can dramatically impact muscle force generating capacity. In this work, we develop a geometric model to predict changes in fiber length and pennation angle of the mouse supraspinatus and infraspinatus muscles given a measured muscle length. We then validate this model with detailed architectural measurements in tenotomized muscles and show a close match between predicted and experimental values. Finally, using this model, we find that predicted changes in architecture cannot explain the force deficit in tenotomized muscle. The contributions of this work are 1) a simple geometric model that predicts changes in architecture with retraction, validated in the mouse RC but with potential application across muscles and species and 2) data indicating that architectural adaptation is not solely responsible for the force deficit with tenotomy which suggests future research should focus on intrinsic changes to the myofiber.

p300 or CBP is required for insulin-stimulated glucose uptake in skeletal muscle and adipocytes.

While current thinking posits that insulin signaling to glucose transporter 4 (GLUT4) exocytic translocation and glucose uptake in skeletal muscle and adipocytes is controlled by phosphorylation-based signaling, many proteins in this pathway are acetylated on lysine residues. However, the importance of acetylation and lysine acetyltransferases to insulin-stimulated glucose uptake is incompletely defined. Here, we demonstrate that combined loss of the acetyltransferases E1A binding protein p300 (p300) and cAMP response element binding protein binding protein (CBP) in mouse skeletal muscle caused a complete loss of insulin-stimulated glucose uptake. Similarly, brief (i.e., 1 hour) pharmacological inhibition of p300/CBP acetyltransferase activity recapitulated this phenotype in human and rodent myotubes, 3T3-L1 adipocytes, and mouse muscle. Mechanistically, these effects were due to p300/CBP-mediated regulation of GLUT4 exocytic translocation and occurred downstream of Akt signaling. Taken together, we highlight a fundamental role for acetylation and p300/CBP in the direct regulation of insulin-stimulated glucose transport in skeletal muscle and adipocytes.

A unique sarcopenic progression in the mouse rotator cuff.

BACKGROUND: In response to chronic injury, the muscles of the rotator cuff (RC) experience a unique degeneration characterized by extensive fatty infiltration and loss of contractile function. Human studies suggest this degeneration is also a feature of RC sarcopenia and may precede RC injury. In this study, we investigated whether RC muscles exhibit a similar unique sarcopenia in the mouse. METHODS: Male and female mice were subdivided into four age groups: 3, 9, 18, and 24 months. The supraspinatus (SS) and infraspinatus muscles of the RC and the tibialis anterior (TA) muscle of the hindlimb were assessed. Muscle mass, contractile function, fibre cross-sectional areas and numbers, fatty infiltration, and fibrosis were assessed at each time point. Targeted transcriptional analyses were performed to assess the role of metabolic and inflammatory derangement in the pathology. RESULTS: The 24-month-aged female mice exhibited decreased mass (25% lower than at 9 and 18 months, P < 0.01) in all muscles tested. However, only RC muscles also exhibited decreased contractile tension at this time point (20% lower than at 18 months, P < 0.005). Similarly, only female RC muscles exhibited increased fatty infiltration at 24 months (20% higher than 9 months, P < 0.05) and had elevated transcriptional markers of adipogenesis (2.4-fold higher Pparg and 3.8-fold higher Adipoq expression compared with 9 months, P < 0.001). Unbiased metabolic transcriptional profiling identified up-regulation of the antigen presentation (Z scores of 2.3 and 1.9 for SS and TA, respectively) and cytokine and chemokine signalling (Z scores of 3.1 and 2.4 for SS and TA, respectively) pathways in 24 month female muscle compared with 9. Further transcriptional analysis supported increased expression of pro-adipogenic inflammatory signals (6.3-fold increase in Il6 and 5.0-fold increase in Anxa2, P < 0.01) and increased presence of fibro-adipogenic progenitors (2.5-fold) in the 24-month-aged female RC compared with 9 months that together exacerbate fatty infiltration. CONCLUSIONS: These data indicate that female mice replicate the unique sarcopenic pathology in the ageing human RC. Furthermore, they suggest that the exacerbated fatty infiltration is due to an interaction between higher resident fibro-adipogenic progenitor numbers and an elevated systemic inflammation in aged female mice. We conclude that female mouse RC muscle is a novel system to study both human RC degeneration and the signals that regulate sarcopenic fatty infiltration in general, which is prevalent in humans but largely absent from the rodent hindlimb.

Subcutaneous Adipose Tissue Metabolic Function and Insulin Sensitivity in People With Obesity.

We used stable isotope-labeled glucose and palmitate tracer infusions, a hyperinsulinemic-euglycemic clamp, positron emission tomography of muscles and adipose tissue after [18F]fluorodeoxyglucose and [15O]water injections, and subcutaneous adipose tissue (SAT) biopsy to test the hypotheses that 1) increased glucose uptake in SAT is responsible for high insulin-stimulated whole-body glucose uptake in people with obesity who are insulin sensitive and 2) putative SAT factors thought to cause insulin resistance are present in people with obesity who are insulin resistant but not in those who are insulin sensitive. We found that high insulin-stimulated whole-body glucose uptake in insulin-sensitive participants with obesity was not due to channeling of glucose into SAT but, rather, was due to high insulin-stimulated muscle glucose uptake. Furthermore, insulin-stimulated muscle glucose uptake was not different between insulin-sensitive obese and lean participants even though adipocytes were larger, SAT perfusion and oxygenation were lower, and markers of SAT inflammation, fatty acid appearance in plasma in relation to fat-free mass, and plasma fatty acid concentration were higher in the insulin-sensitive obese than in lean participants. In addition, we observed only marginal or no differences in adipocyte size, SAT perfusion and oxygenation, and markers of SAT inflammation between insulin-resistant and insulin-sensitive obese participants. Plasma fatty acid concentration was also not different between insulin-sensitive and insulin-resistant obese participants, even though SAT was resistant to the inhibitory effect of insulin on lipolysis in the insulin-resistant obese group. These data suggest that several putative SAT factors commonly implicated in causing insulin resistance are normal consequences of SAT expansion unrelated to insulin resistance.

Harnessing adipose stem cell diversity in regenerative medicine.

Since the first isolation of mesenchymal stem cells from lipoaspirate in the early 2000s, adipose tissue has been a darling of regenerative medicine. It is abundant, easy to access, and contains high concentrations of stem cells (ADSCs) exhibiting multipotency, proregenerative paracrine signaling, and immunomodulation-a winning combination for stem cell-based therapeutics. While basic science, preclinical and clinical findings back up the translational potential of ADSCs, the vast majority of these used cells from a single location-subcutaneous abdominal fat. New data highlight incredible diversity in the adipose morphology and function in different anatomical locations or depots. Even in isolation, ADSCs retain a memory of this diversity, suggesting that the optimal adipose source material for ADSC isolation may be application specific. This review discusses our current understanding of the heterogeneity in the adipose organ, how that heterogeneity translates into depot-specific ADSC characteristics, and how atypical ADSC populations might be harnessed for regenerative medicine applications. While our understanding of the breadth of ADSC heterogeneity is still in its infancy, clear trends are emerging for application-specific sourcing to improve regenerative outcomes.

Adipose tissue is a critical regulator of osteoarthritis.

Osteoarthritis (OA), the leading cause of pain and disability worldwide, disproportionally affects individuals with obesity. The mechanisms by which obesity leads to the onset and progression of OA are unclear due to the complex interactions among the metabolic, biomechanical, and inflammatory factors that accompany increased adiposity. We used a murine preclinical model of lipodystrophy (LD) to examine the direct contribution of adipose tissue to OA. Knee joints of LD mice were protected from spontaneous or posttraumatic OA, on either a chow or high-fat diet, despite similar body weight and the presence of systemic inflammation. These findings indicate that adipose tissue itself plays a critical role in the pathophysiology of OA. Susceptibility to posttraumatic OA was reintroduced into LD mice using implantation of a small adipose tissue depot derived from wild-type animals or mouse embryonic fibroblasts that undergo spontaneous adipogenesis, implicating paracrine signaling from fat, rather than body weight, as a mediator of joint degeneration.