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The aim of the present study was to investigate the fiber type-specific abundance of autophagy-related proteins after an overnight fast and following ingestion of a mixed meal in human skeletal muscle. Twelve overweight, healthy young male volunteers underwent a 3-h mixed meal tolerance test following an overnight fast. Blood samples were collected in the overnight-fasted state and throughout the 180-min postmeal period. Skeletal muscle biopsies were collected in the fasted state, and at 30 and 90 min after meal ingestion. Protein content of key autophagy markers and upstream signaling responses were measured in whole muscle and pooled single fibers using immunoblotting. In the fasted state, type I fibers displayed lower LC3B-I but higher LC3B-II abundance and higher LC3B-II/LC3B-I ratio compared with type II fibers (

Objective To investigate the effect of high fat diet-induced insulin resistance on autophagy markers in the liver and skeletal muscle of mice in the fasted state and following an oral glucose bolus. Methods Forty C57BL/6J male mice were fed either a high fat, high sucrose (HFSD, n = 20) or standard chow control (CON, n = 20) diet for 16 weeks. Upon trial completion, mice were gavaged with water or glucose and skeletal muscle and liver were collected 15 min post gavage. Protein abundance and gene expression of autophagy markers and activation of related signalling pathways were assessed. Results Compared to CON, the HFSD intervention increased LC3B-II and p62/SQSTM1 protein abundance in the liver which is indicative of elevated autophagosome content via reduced clearance. These changes coincided with inhibitory autophagy signalling through elevated p-mTOR S2448 and p-ULK1S758. HFSD did not alter autophagy markers in skeletal muscle. Administration of an oral glucose bolus had no effect on autophagy markers or upstream signalling responses in either tissue regardless of diet. Conclusion HFSD induces tissue-specific autophagy impairments, with autophagosome accumulation indicating reduced lysosomal clearance in the liver. In contrast, autophagy markers were unchanged in skeletal muscle, indicating that autophagy is not involved in the development of skeletal muscle insulin resistance.

Endurance training enhances the capacity for fat oxidation during exercise due to increased utilization of intramuscular lipid (IMCL). This study quantitatively investigated the impact of exercise training status on muscle fiber type-specific abundance of regulatory proteins involved in IMCL utilization. Endurance-trained [ n = 7 subjects, peak oxygen consumption (V̇o2peak) 62.6 ± 4.1 (SD) mL·min−1·kg−1] and non-endurance-trained ( n = 8 subjects, V̇o2peak 44.9 ± 5.3 mL·min−1·kg−1) young men completed an incremental exercise test to determine maximal fat oxidation (MFO) and maximal oxygen uptake. Fiber type-specific IMCL content and protein abundance were assessed with immunofluorescence microscopy and immunoblot analysis of pooled single muscle fibers and whole muscle. Endurance-trained individuals displayed a higher MFO rate (0.45 ± 0.15 vs. 0.19 ± 0.07 g/min, P < 0.05), a greater proportion of type I muscle fibers, and higher IMCL content compared with untrained individuals ( P < 0.05). Adipose triglyceride lipase, hormone-sensitive lipase, perilipin 2, perilipin 5, and hydroxyacyl-coenzyme A dehydrogenase abundances were ~2–3-fold higher in type I muscle fibers compared with type IIa fibers ( P < 0.05). Correspondingly, these lipid proteins and oxidative enzymes were higher in endurance-trained individuals when assessed in whole muscle. MFO rate was strongly related to the proportion of type I fibers ( R = 0.81, P < 0.01). The abundance of proteins involved in the regulation of IMCL storage and oxidation is highly muscle fiber type specific. The increased capacity for fat oxidation in endurance-trained individuals corresponded with increased IMCL content and elevated abundance of lipolytic and oxidative enzymes in combination with a greater proportion of type I muscle fibers. NEW & NOTEWORTHY We have utilized contemporary techniques to compare the fiber type-specific characteristics of skeletal muscle from endurance-trained athletes and untrained individuals. We show that type I muscle fibers have a coordinated upregulation of proteins controlling intramuscular lipid storage, mobilization, and oxidation. Furthermore, the enhanced capacity for intramuscular lipid storage and utilization in endurance-trained individuals is related to the increased expression of lipid regulatory proteins combined with a greater proportion of type I muscle fibers.

Aims/Hypothesis Current drugs used to treat metabolic diseases such as obesity and type 2 diabetes have limited efficacy in directly normalising muscle insulin action, while exercise is effective in enhancing this aspect of metabolic disease. We recently identified Scriptaid as a compound that can replicate aspects of the exercise adaptive response, through disruption of the class IIa HDAC corepressor complex. The Aim of this study was to determine the effect of Scriptaid on muscle insulin action in obesity. Materials and Methods Diet-induced obese mice were administered Scriptaid (1 mg/kg) via daily i.p. injection for four weeks. Whole body and skeletal muscle metabolic phenotyping of mice was performed in addition to echocardiography to assess cardiac morphology and function. Results Scriptaid treatment had no effect on body weight or composition, but did increase energy expenditure, supported by increased lipid oxidation, while food intake was also increased. Scriptaid enhanced the expression of oxidative genes and proteins, increased fatty acid oxidation and reduced triglycerides and diacylglycerides in skeletal muscle. Furthermore, ex vivo insulin-stimulated glucose uptake by skeletal muscle was enhanced. Surprisingly, heart weight was reduced in Scriptaid-treated mice and was associated with enhanced expression of genes involved in oxidative metabolism in the heart. Scriptaid also improved indices of both diastolic and systolic cardiac function. Conclusion These data show that pharmacological targeting of the class IIa HDACs corepressor complex with Scriptaid could be used to enhance muscle insulin action and cardiac function in obesity.

Objective A greater capacity for fat oxidation in endurance trained athletes is linked to greater utilisation of intramuscular lipid (IMCL). IMCL breakdown occurs only in type I muscle fibres yet little is known about the fibre type specific abundance of lipid regulatory proteins. We explored the impact of endurance training on the maximal rate fat oxidation, muscle fibre type and muscle fibre type specific abundance of proteins regulating IMCL metabolism. Methods Endurance trained (n=7, 28 ± 3 years, VO2max62.6 ± 1.6 ml·min-1·kg-1) and untrained (n=8, 25 ± 1 years, VO2max44.9 ± 1.9 ml·min-1·kg-1) males performed an incremental exercise test to determine maximal fat oxidation rate. Muscle fibre type composition and fibre type-specific IMCL content was assessed with immunofluorescence microscopy and protein abundance was analysed with immunoblotting on pooled single muscle fibres and whole muscle. Results Endurance trained individuals displayed a higher peak fat oxidation rate (0.49 ± 0.05 vs. 0.20 ± 0.03 g·min-1, P

To determine the effect of Scriptaid, a compound that can replicate aspects of the exercise adaptive response through disruption of the class IIa histone deacetylase (HDAC) corepressor complex, on muscle insulin action in obesity.Diet-induced obese mice were administered Scriptaid (1 mg/kg) via daily intraperitoneal injection for 4 weeks. Whole-body and skeletal muscle metabolic phenotyping of mice was performed, in addition to echocardiography, to assess cardiac morphology and function.Scriptaid treatment had no effect on body weight or composition, but did increase energy expenditure, supported by increased lipid oxidation, while food intake was also increased. Scriptaid enhanced the expression of oxidative genes and proteins, increased fatty acid oxidation and reduced triglycerides and diacylglycerides in skeletal muscle. Furthermore, ex vivo insulin-stimulated glucose uptake by skeletal muscle was enhanced. Surprisingly, heart weight was reduced in Scriptaid-treated mice and was associated with enhanced expression of genes involved in oxidative metabolism in the heart. Scriptaid also improved indices of both diastolic and systolic cardiac function.These data show that pharmacological targeting of the class IIa HDAC corepressor complex with Scriptaid could be used to enhance muscle insulin action and cardiac function in obesity.

We previously used Gene Expression Signature technology to identify methazolamide (MTZ) and related compounds with insulin sensitizing activity in vitro. The effects of these compounds were investigated in diabetic db/db mice, insulin-resistant diet-induced obese (DIO) mice, and rats with streptozotocin (STZ)-induced diabetes. MTZ reduced fasting blood glucose and HbA1c levels in db/db mice, improved glucose tolerance in DIO mice, and enhanced the glucose-lowering effects of exogenous insulin administration in rats with STZ-induced diabetes. Hyperinsulinemic-euglycemic clamps in DIO mice revealed that MTZ increased glucose infusion rate and suppressed endogenous glucose production. Whole-body or cellular oxygen consumption rate was not altered, suggesting MTZ may inhibit glucose production by different mechanism(s) to metformin. In support of this, MTZ enhanced the glucose-lowering effects of metformin in db/db mice. MTZ is known to be a carbonic anhydrase inhibitor (CAI); however, CAIs acetazolamide, ethoxyzolamide, dichlorphenamide, chlorthalidone, and furosemide were not effective in vivo. Our results demonstrate that MTZ acts as an insulin sensitizer that suppresses hepatic glucose production in vivo. The antidiabetic effect of MTZ does not appear to be a function of its known activity as a CAI. The additive glucose-lowering effect of MTZ together with metformin highlights the potential utility for the management of type 2 diabetes.

Zinc accumulation may impair cellular bioenergetics, which is associated with neuronal apoptosis. We simultaneously assessed anaerobic and aerobic metabolism in live cells to characterise this effect and hypothesised that the omega 3 fatty acid docosahexaenoic acid (DHA) would protect against any zinc mediated alterations in bioenergetics. In this study we observed a decrease in cellular oxygen consumption, but not glycolytic rate, following chronic zinc exposure, which was specific for neuronal cells. This was due to impaired ATP turnover, without any other effects on mitochondrial function, and was restored by DHA. DHA had no further effects on bioenergetics. These data suggest that zinc disrupts bioenergetics at a point distal to the respiratory chain, which is restored by DHA.

Skeletal muscle tissue undergoes adaptive changes in response to stress and the genes that control these processes are incompletely characterised. NDRG2 (N-myc downstream-regulated gene 2), a stress- and growth-related gene, was investigated in skeletal muscle growth and adaption. While NDRG2 expression levels were found to be up-regulated in both differentiated human and mouse myotubes compared with undifferentiated myoblasts, the suppression of NDRG2 in C2C12 myoblasts resulted in slowed myoblast proliferation. The increased expression levels of the cell cycle inhibitors, p21 Waf1/Cip1 and p27 Kip1, and of various muscle differentiation markers in NDRG2-deficient myoblasts indicate that a lack of NDRG2 promoted cell cycle exiting and the onset of myogenesis. Furthermore, the analysis of NDRG2 regulation in C2C12 myotubes treated with catabolic and anabolic agents and in skeletal muscle from human subjects following resistance exercise training revealed NDRG2 gene expression to be down-regulated during hypertrophic conditions, and conversely, up-regulated during muscle atrophy. Together, these data demonstrate that NDRG2 expression is highly responsive to different stress conditions in skeletal muscle and suggest that the level of NDRG2 expression may be critical to myoblast growth and differentiation.

Some gene deletions or mutations have little effect on metabolism and metabolic adaptation because of redundancy and/or compensation in metabolic pathways. The mechanisms for redundancy and/or compensation in metabolic adaptation in mammalian cells are unidentified. Here, we show that in mouse muscle and myogenic cells, compensatory regulation of the histone deacetylase (HDAC5) transcriptional repressor maintains metabolic integrity. HDAC5 phosphorylation regulated the expression of diverse metabolic genes and glucose metabolism in mouse C2C12 myogenic cells. However, loss of AMP-activated protein kinase (AMPK), a HDAC5 kinase, in muscle did not affect HDAC5 phosphorylation in mouse skeletal muscle during exercise, but resulted in a compensatory increase (32.6%) in the activation of protein kinase D (PKD), an alternate HDAC5 kinase. Constitutive PKD activation in mouse C2C12 myogenic cells regulated metabolic genes and glucose metabolism. Although aspects of this response were HDAC5 phosphorylation dependent, blocking HDAC5 phosphorylation when PKD was active engaged an alternative compensatory adaptive mechanism, which involved post-transcriptional reductions in HDAC5 mRNA (−93.1%) and protein. This enhanced the expression of a specific subset of metabolic genes and mitochondrial metabolism. These data show that compensatory regulation of HDAC5 maintains metabolic integrity in mammalian cells and reinforces the importance of preserving the cellular metabolic adaptive response.—McGee, S. L., Swinton, C., Morrison, S., Gaur, V., Campbell, D. E., Jorgensen, S. B., Kemp, B. E., Baar, K., Steinberg, G. R., Hargreaves, M. Compensatory regulation of HDAC5 in muscle maintains metabolic adaptive responses and metabolism in response to energetic stress.

Insulin resistance is a heterogeneous disorder caused by a range of genetic and environmental factors, and we hypothesize that its etiology varies considerably between individuals. This heterogeneity provides significant challenges to the development of effective therapeutic regimes for long-term management of type 2 diabetes. We describe a novel strategy, using large-scale gene expression profiling, to develop a gene expression signature (GES) that reflects the overall state of insulin resistance in cells and patients. The GES was developed from 3T3-L1 adipocytes that were made “insulin resistant” by treatment with tumor necrosis factor-α (TNF-α) and then reversed with aspirin and troglitazone (“resensitized”). The GES consisted of five genes whose expression levels best discriminated between the insulin-resistant and insulin-resensitized states. We then used this GES to screen a compound library for agents that affected the GES genes in 3T3-L1 adipocytes in a way that most closely resembled the changes seen when insulin resistance was successfully reversed with aspirin and troglitazone. This screen identified both known and new insulin-sensitizing compounds including nonsteroidal anti-inflammatory agents, β-adrenergic antagonists, β-lactams, and sodium channel blockers. We tested the biological relevance of this GES in participants in the San Antonio Family Heart Study ( n = 1,240) and showed that patients with the lowest GES scores were more insulin resistant (according to HOMA_IR and fasting plasma insulin levels; P < 0.001). These findings show that GES technology can be used for both the discovery of insulin-sensitizing compounds and the characterization of patients into subtypes of insulin resistance according to GES scores, opening the possibility of developing a personalized medicine approach to type 2 diabetes.