Diabetic Skeletal Muscle Research Articles

The effect of leucine supplementation on the skeletal muscles of streptozotocin (STZ)diabetic rats

Skeletal muscle atrophy is one of the serious and less studied diabetic complications.Leucine is an essential amino acid that is transported into most mammalian cells by System L. The current study was conducted to assess the effect of leucine on diabetic skeletal muscles. Forty male Wister rats were allocated into four groups; control, Leucine-treated group was subjected to daily oral supplement of Leucine (1.35 g/kg) for 8 weeks, STZ-diabetic group was treated with single intravenous injection of STZ (45 mg/kg) and STZ-diabetic group supplemented with leucine (1.35 g/kg) for 8 weeks. Body weight and histopathological analysis of soleus muscle were evaluated. There was a significant increase in body weight of leucine group at p> 0.05. Leucine supplementation attenuated loss of soleus muscle mass observed in STZ diabetic rats. These findings may suggest protective effect of leucine against diabetic muscle loss. Leucine supplementation attenuated loss of soleus muscle mass observed in STZ diabetic rats. These findings may suggest protective effect of leucine against diabetic muscle loss.

Protective effects of Liuwei dihuang water extracts on diabetic muscle atrophy

BackgroundLiuwei dihuang (LWDH), a widely used traditional Chinese herbal medicine, has been noticed for its potential on the improvement of diabetic complications including diabetic nephropathy and diabetic encephalopathy. However, whether LWDH can improve the effects of diabetic skeletal muscle atrophy has not yet been reported. PurposeThe present study aimed to investigate the protective effects and mechanisms of the water extract of Liuwei dihuang (LWDH-WE) on skeletal muscle in cellular and animal models of diabetic muscle atrophy. Study designThe muscle protective effects of LWDH-WE on diabetic muscle atrophy and weakness were examined in methylglyoxal (MG)-treated C2C12 myotubes and streptozotocin (STZ)-treated C57BL/6 mice, respectively. MethodsC2C12 myoblasts were differentiated by differentiation medium to form myotube structures. C2C12 myotubes were pre-treated with LWDH-WE 1 h before MG treatment. Diabetic mice were induced by single intraperitoneal injection of STZ (150 mg/kg) in C57BL/6 mice. Cell viability was determined by MTT and LDH assays. Protein expressions were detected by western blots. Morphological changes of cells were observed by an inverted microscope. Mitochondria membrane potential and reactive oxygen species (ROS) production were measured by flow cytometry. Muscle strength was evaluated by measuring grip strength of mice. ResultsIn C2C12 myotubes, LWDH-WE attenuated MG-induced cellular death and oxidative damage accompanied with improving mitochondrial membrane potential, inhibiting NADPH oxidase (Nox) activation, and ROS production. Moreover, LWDH-WE could attenuate MG-induced atrophy of C2C12 myotubes accompanied with regulating protein synthesis (IGF-1R, Akt, mTOR) and protein degradation (FoxO3a, atrogin-1, MuRF-1) signals. In STZ-induced diabetic mice, LWDH-WE improved body weight and skeletal muscle mass of mice. LWDH-WE also enhanced muscle strength of STZ-induced diabetic mice. Furthermore, LWDH-WE enhanced the improvement of insulin on gastrocnemius muscle mass and grip strength in STZ-treated mice. ConclusionLWDH-WE possesses skeletal muscle protection via reducing oxidative damage and regulating protein synthesis and degradation pathways in MG-induced atrophy of C2C12 myotubes. We also reveal the novel protection of LWDH-WE against STZ-induced reduction of muscle mass and muscle strength in mice.

Mitochondrial Adaptation in Insulin Resistant Human Skeletal Muscle

Introduction: The integrity, morphology, structure and functionality of skeletal muscle mitochondria in patients with type 2 diabetes (T2D) is heavily disputed. Thus, the purpose of this investigation was to characterize mitochondrial phenotypes across the insulin resistance spectrum. Methods: 30 sedentary adults aged 18-55 were grouped as lean (BMI 21±0.6, N=13), obese (BMI 32±0.9, N=12), or T2D (BMI 33±2.5, N=5) based on oral glucose tolerance tests and body mass index (BMI). All subjects completed a 3-day, inpatient stay consisting of cardio-metabolic testing, body composition analysis and a hyperinsulinemic-euglycemic clamp study. On the day of the clamp, muscle specimens were obtained basally and under insulin-stimulated conditions. Mitochondrial membrane potential (δψm) was measured by confocal microscopy using the cationic fluorophore TMRM. Oxidative phosphorylation (OXPHOS) was assessed in permeabilized fibers via high resolution respirometry (Oroboros Oxygraph-2k). Muscle mitochondrial ultrastructure and content were determined by transmission electron microscopy (TEM) of fixed muscle sections. Results: There was a significant, step-wise decrease in insulin-stimulated glucose uptake across the metabolic spectrum (P<0.001). No differences in resting δψm were observed, however, insulin stimulation depolarized δψm in all groups (P<0.05). Those with T2D exhibited increased basal complex III (P<0.05) and IV (P<0.05) activity vs. lean and obese controls. Furthermore, obese controls and T2D displayed augmented complex IV responsiveness to insulin stimulation (P<0.01). Threshold analysis of muscle sections revealed a progressive loss of mitochondrial content, as well as accumulation of lipid droplets across the metabolic spectrum (P<0.05). Conclusion: Collectively, these findings provide direct evidence of mitochondrial adaptation in insulin resistant and diabetic skeletal muscle. Disclosure C.L. Axelrod: None. C.E. Fealy: None. A. Mulya: None. E. Huang: None. H. Fujioka: None. B. Burguera: None. C.L. Hoppel: None. J.P. Kirwan: None.

Insulin Receptor-WNK1 Signaling Targeting Glut4 Trafficking Are Blunted In Diabetic Skeletal Muscle

Insulin Receptor-WNK1 Signaling Targeting Glut4 Trafficking Are Blunted In Diabetic Skeletal Muscle

Patterns of Suppressed Mitochondrial Respiration in Isolated Muscle Fibers from Type 2 Diabetics

The quality of skeletal muscle is markedly influenced by mitochondrial function. Aberrant mitochondrial function has been associated with reduced contractile and metabolic dysfunction in skeletal muscle, potentially leading to insulin resistance and type 2 diabetes. However, the direct relationship between mitochondrial dysfunction and type 2 diabetes remains to be fully understood. In the present study, vastus lateralis muscle biopsies were obtained from individuals with type 2 diabetes (N = 7) and non‐diabetic, age, gender and weight‐matched controls (N = 7). Maximal mitochondrial oxygen consumption was assessed in isolated muscle fibers and normalized to wet tissue weight. Preliminary findings illustrate that reductions in carbohydratedependent ADP‐stimulated respiration (~30%) were sustained in muscle fibers from type 2 diabetics, even with the addition of the Krebs Cycle anaplerotic substrates, glutamate (~30%) and succinate (~32%). Interestingly, trends of similar magnitudes persisted for lipid substrate metabolism, as fatty acid‐dependent ADP‐stimulated respiration also tended to be lower (~35%) compared to controls. Furthermore, rotenone‐treated respiration in diabetic skeletal muscle tended to be reduced (~37%). To conclude, these initial findings provide support for a systematic impairment in the oxidative metabolism of both carbohydrates and lipids in skeletal muscle of type 2 diabetic individuals. In addition, findings from rotenone‐treated conditions indicate that investigating functional deficits of Complex I of the Electron Transport Chain (ETC) may provide a more insightful understanding of the relationship between mitochondrial respiratory control and type 2 diabetes.Support or Funding Information*This study is supported by funds from the American Diabetic Association, ADA‐1‐16‐ICIS‐015, (D.P.C.) and start‐up funds from the Department of Health and Human Physiology at the University of Iowa (V.A.L.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tocotrienol-rich fraction supplementation reduces hyperglycemia-induced skeletal muscle damage through regulation of insulin signaling and oxidative stress in type 2 diabetic mice

Chronic hyperglycemia induces impairment of muscle growth and development of diabetes mellitus (DM). Since skeletal muscle is the major site for disposal of ingested glucose, impaired glucose metabolism causes imbalance between protein synthesis and degradation which adversely affects physical mobility.In this study, we investigated the effect of tocotrienol-rich fraction (TRF) supplementation on skeletal muscle damage in diabetic mice. Diabetes was induced by a high-fat diet with streptozotocin (STZ) injection (100 mg/kg) in male C57BL/6J mice. After diabetes was induced (fasting blood glucose levels≥250 mg/dl), normal control (CON) and diabetic control (DMC) groups were administrated with olive oil, while TRF treatment groups were administrated with TRF (dissolved in olive oil) at low dose (100 mg/kg BW, LT) or high dose (300 mg/kg BW, HT) by oral gavage for 12 weeks.TRF supplementation ameliorated muscle atrophy, plasma insulin concentration and homeostatic model assessment estimated insulin resistance in diabetic mice. Moreover, TRF treatment up-regulated IRS-1 and Akt levels accompanied by increased translocation of GLUT4. Furthermore, TRF increased mitochondrial biogenesis by activating SIRT1, SIRT3 and AMPK in diabetic skeletal muscle. These changes were in part mechanistically explained by reduced levels of skeletal muscle proteins related to oxidative stress (4-hydroxynonenal, protein carbonyls, Nrf2 and HO-1), inflammation (NFkB, MCP-1, IL-6 and TNF-α), and apoptosis (Bax, Bcl₂ and caspase-3) in diabetic mice. Taken together, these results suggest that TRF might be useful as a beneficial nutraceutical to prevent skeletal muscle atrophy associated with diabetes by regulating insulin signaling via AMPK/SIRT1/PGC1α pathways in type 2 diabetic mice.

Exercise training impacts exercise tolerance and bioenergetics in gastrocnemius muscle of non-obese type-2 diabetic Goto-Kakizaki rat in vivo

The functional and bioenergetics impact of regular physical activity upon type-2 diabetic skeletal muscle independently of confounding factors of overweight remains undocumented. Here, gastrocnemius muscle energy fluxes, mitochondrial capacity and mechanical performance were assessed noninvasively and longitudinally in non-obese diabetic Goto-Kakizaki rats using magnetic resonance (MR) imaging and dynamic 31-phosphorus MR spectroscopy (31P-MRS) throughout a 6-min fatiguing bout of exercise performed before, in the middle (4-week) and at the end of an 8-week training protocol consisting in 60-min daily run on a treadmill. The training protocol reduced plasmatic insulin level (−61%) whereas blood glucose and non-esterified fatty acids levels remained unaffected, thereby indicating an improvement of insulin sensitivity. It also increased muscle mitochondrial citrate synthase activity (+45%) but this increase did not enhance oxidative ATP synthesis capacity in working muscle in vivo while glycolytic ATP production was increased (+33%). On the other hand, the training protocol impaired maximal force-generating capacity (−9%), total amount of force produced (−12%) and increased ATP cost of contraction (+32%) during the fatiguing exercise. Importantly, these deleterious effects were transiently worsened in the middle of the 8-week period, in association with reduced oxidative capacity and increased basal [Pi]/[PCr] ratio (an in vivo biomarker of muscle damage). These data demonstrate that the beneficial effect of regular training on insulin sensitivity in non-obese diabetic rat occurs separately from any improvement in muscle mitochondrial function and might be linked to an increased capacity for metabolizing glucose through anaerobic process in exercising muscle.

Coriolus versicolor aqueous extract ameliorates insulin resistance with PI3K/Akt and p38 MAPK signaling pathways involved in diabetic skeletal muscle.

Patients with type 2 diabetes mellitus (T2DM) are usually with poor immunity and easier to suffer from cancer and microbial infections. Herein, we report an efficient anti-diabetic medicinal mushroom, Coriolus versicolor (CV). This study aimed to investigate the anti-diabetic and anti-insulin-resistance effects of CV aqueous extract in myoblasts (L6 cells) and skeletal muscle of T2DM rat. Our results showed that CV extract treatment significantly reduced blood glucose levels of T2DM rats, whereas CV extract increased glucose consumption in insulin resistant L6 cells. Besides, the translocation and expression of glucose transporter 4 were enhanced by CV extract, which indicated that CV extract was effective in diabetic skeletal muscle. Moreover, CV extract treatments resulted in remarkable anti-insulin-resistance effects, which was reflected by the change of gene and protein expression levels in PI3K/Akt and p38 MAPK pathways. PI3K inhibitor, LY29004, and p38 MAPK inhibitor, SB203580 confirmed it further. In conclusion, our results demonstrated that the CV extract exhibited anti-diabetic and anti-insulin-resistance effects in diabetic skeletal muscle, and the effects were mediated by PI3K/Akt and p38 MAPK pathways. These findings are remarkable when considering the use of commercially available CV by diabetic patients who also suffer from cancer or microbial infections.

Resistance training increases fibroblast growth factor-21 and irisin levels in the skeletal muscle of Zucker diabetic fatty rats.

[Purpose]Although the fibroblast growth factor-21 (FGF-21) and irisin roles are well demonstrated in metabolic disease, there have been no reports investigating the effect of resistance exercise on FGF-21 and irisin levels in diabetic skeletal muscles. Therefore, this study aimed to investigate the change of FGF-21 and irisin levels in various skeletal muscles, and their association with muscle strength, following 8 weeks of resistance training using Zucker diabetic fatty rats (type 2 diabetic animal models).[Methods]Twenty-four male lean (Zucker lean control, ZLC) and diabetic (Zucker diabetic fatty, ZDF) rats (age, 8 weeks old) were separated into 3 groups, lean control (ZLC-Con, n=8), diabetic control (ZDF-Con, n=8), and diabetic exercise-trained groups (ZDF-Ex, n=8). The rats in ZDF-Ex were trained to climb a 1-m vertical (85 degrees inclined) ladder with weights. Resistance training was performed with 10 repetitions/day for 12 weeks (3 days/week). The skeletal muscle levels of FGF-21 and irisin were measured using enzyme-linked immunosorbent assays.[Results]The levels of FGF-21 in the soleus (SOL) and extensor digitorum longus muscles of ZDF-Ex were higher (p<0.05) compared to levels in ZDF-Con. Additionally, we found a significantly higher irisin level in the SOL muscles of ZDF-Ex compared to that in ZDF-Con. Moreover, we found that the levels of FGF-21 (R=0.532, p=0.02) and irisin (R=0.498, p=0.03) had significant correlations with grip strength.[Conclusion]Based on these results, resistance training may be an efficient intervention for increasing FGF-21 and irisin levels in type 2 diabetic (T2DM) skeletal muscles.

Developmentally regulated alternative splicing is perturbed in type 1 diabetic skeletal muscle.

Type 1 diabetic patients can develop skeletal muscle weakness and atrophy by molecular mechanisms that are not well understood. Alternative splicing (AS) is critical for gene expression in the skeletal muscle, and its dysregulation is implicated in muscle weakness and atrophy. Therefore, we investigated whether AS patterns are affected in type 1 diabetic skeletal muscle contributing to skeletal muscle defects. AS patterns were determined by reverse transcription-polymerase chain reaction and levels of RNA binding proteins were assessed by Western blot in type 1 diabetic mouse skeletal muscle and during normal mouse skeletal muscle development. Five genes with critical functions in the skeletal muscle are misspliced in type 1 diabetic skeletal muscle, resembling their AS patterns at embryonic stages. AS of these genes undergoes dramatic transitions during skeletal muscle development, correlating with changes in specific RNA binding proteins. Embryonic spliced variants are inappropriately expressed in type 1 diabetic skeletal muscle. Muscle Nerve 56: 744-749, 2017.

Erythropoietin Reduces Insulin Resistance via Regulation of Its Receptor-Mediated Signaling Pathways in db/db Mice Skeletal Muscle.

Erythropoietin (EPO) can reduce insulin resistance (IR) in adipocytes; however, it is unknown whether EPO can decrease IR in skeletal muscle. Here we investigated whether EPO could reduce IR in type 2 diabetic mouse skeletal muscle and its possible signaling mechanisms of action. Twelve-week-old db/db diabetic mice were employed in this study. Systemic use of EPO improved glucose profiles in type 2 diabetic mice after 4 and 8 weeks treatment. EPO up-regulated EPOR protein expression in skeletal muscle, and subsequently activated downstream signaling molecules such as JAK2, IRS-1, PI3K, AKT, and eNOS. We next constructed lentivirally-delivered shRNAs against EPOR and transfected skeletal muscle cells to knockdown EPOR. EPOR knockdown inhibited EPO induced JAK2, IRS-1, PI3K, AKT, eNOS signaling transduction, autophagy and Glut 4 translocation, as well as promoted apoptosis in skeletal muscle. Thus, EPO reduces skeletal muscle IR in type 2 diabetic mice via its specific receptor, EPOR. Possible mechanisms involved in its action may include increased autophagy and reduced apoptosis in type 2 diabetic skeletal muscles, which provides a new strategy for the treatment of IR.

Transcriptional profiles of type 2 diabetes in human skeletal muscle reveal insulin resistance, metabolic defects, apoptosis, and molecular signatures of immune activation in response to infections

Skeletal muscle insulin resistance is considered to be the primary defect involved in type 2 diabetes mellitus (T2DM). Despite transcriptome studies in limited T2DM human subjects suggesting an association of T2DM with impaired oxidative phosphorylation in muscle, its molecular pathogenesis remains largely unknown. To identify dysregulated genes and gene networks that are associated with T2DM in human skeletal muscle, we examined expression patterns of 56,318 transcribed genes on 92 T2DM cases and 184 gender-, age- and race-matched non-diabetic controls from the Genotype-Tissue Expression (GTEx) database. RNA-Sequencing data suggest that diabetic skeletal muscle is characterized by decreased expression of genes that are related to insulin resistance (IRS2, MTOR, SLC2A4, and PPARA), carbohydrate, energy, and amino acid metabolism pathways (NDUFS1, NDUFA10, NDUFB4, NDUFB5, NDUFA5, NDUFB10, SDHB, SDHC, ATP5H, ATP5A, and ATP5J). Up-regulated genes in T2DM are mainly enriched in apoptosis pathways (TP53, GADD45A, TNFRSF10B, TP53AIP1, and PMAIP1), and notably include immune-related pathways suggestive of a response to various infectious diseases (C2, CFB, C4A, C4B, C1S, C1R, C3, HLA-DRA, HLA-DMA, HLA-DOA, and HLA-DPB1). These results confirm the essential regulation of impaired insulin signaling and oxidative phosphorylation in the muscle of T2DM patients, and provide novel molecular insights into the pathophysiological mechanisms of T2DM.

Doxorubicin Induces Inflammatory Modulation and Metabolic Dysregulation in Diabetic Skeletal Muscle

Anti-cancer agent doxorubicin (DOX) has been demonstrated to worsen insulin signaling, engender muscle atrophy, trigger pro-inflammation, and induce a shift to anaerobic glycolytic metabolism in skeletal muscle. The myotoxicity of DOX in diabetic skeletal muscle remains largely unclear. This study examined the effects of DOX on insulin signaling, muscle atrophy, pro-/anti-inflammatory microenvironment, and glycolysis metabolic regulation in skeletal muscle of db/db diabetic and db/+ non-diabetic mice. Non-diabetic db/+ mice and diabetic db/db mice were randomly assigned to the following groups: db/+CON, db/+DOX, db/dbCON, and db/dbDOX. Mice in db/+DOX and db/dbDOX groups were intraperitoneally injected with DOX at a dose of 15 mg per kg body weight whereas mice in db/+CON and db/dbCON groups were injected with the same volume of saline instead of DOX. Gastrocnemius was immediately harvested, weighed, washed with cold phosphate buffered saline, frozen in liquid nitrogen, and stored at −80°C for later analysis. The effects of DOX on diabetic muscle were neither seen in insulin signaling markers (Glut4, pIRS1Ser636∕639, and pAktSer473) nor muscle atrophy markers (muscle mass, MuRF1 and MAFbx). However, DOX exposure resulted in enhancement of pro-inflammatory favoring microenvironment (as indicated by TNF-α, HIFα and pNFκBp65) accompanied by diminution of anti-inflammatory favoring microenvironment (as indicated by IL15, PGC1α and pAMPKβ1Ser108). Metabolism of diabetic muscle was shifted to anaerobic glycolysis after DOX exposure as demonstrated by our analyses of PDK4, LDH and pACCSer79. Our results demonstrated that there might be a link between inflammatory modulation and the dysregulation of aerobic glycolytic metabolism in DOX-injured diabetic skeletal muscle. These findings help to understand the pathogenesis of DOX-induced myotoxicity in diabetic muscle.

Statin Therapy Alters Lipid Storage in Diabetic Skeletal Muscle.

While statins significantly reduce cholesterol levels and thereby reduce the risk of cardiovascular disease, the development of myopathy with statin use is a significant clinical side effect. Recent guidelines recommend increasing inclusion criteria for statin treatment in diabetic individuals; however, the impact of statins on skeletal muscle health in those with diabetes (who already suffer from impairments in muscle health) is ill defined. Here, we investigate the effects of fluvastatin treatment on muscle health in wild type (WT) and streptozotocin (STZ)-induced diabetic mice. WT and STZ-diabetic mice received diet enriched with 600 mg/kg fluvastatin or control chow for 24 days. Muscle morphology, intra and extracellular lipid levels, and lipid transporter content were investigated. Our findings indicate that short-term fluvastatin administration induced a myopathy that was not exacerbated by the presence of STZ-induced diabetes. Fluvastatin significantly increased ectopic lipid deposition within the muscle of STZ-diabetic animals, findings that were not seen with diabetes or statin treatment alone. Consistent with this observation, only fluvastatin-treated diabetic mice downregulated protein expression of lipid transporters FAT/CD36 and FABPpm in their skeletal muscle. No differences in FAT/CD36 or FABPpm mRNA content were observed. Altered lipid compartmentalization resultant of a downregulation in lipid transporter content in STZ-induced diabetic skeletal muscle was apparent in the current investigation. Given the association between ectopic lipid deposition in skeletal muscle and the development of insulin-resistance, our findings highlight the necessity for more thorough investigations into the impact of statins in humans with diabetes.

Histone deacetylase inhibition regulates miR-449a levels in skeletal muscle cells

ABSTRACTmicroRNAs (miRNAs) are small non-coding RNAs that regulate cellular processes by fine-tuning the levels of their target mRNAs. However, the regulatory elements determining cellular miRNA levels are not well studied. Previously, we had described an altered miRNA signature in the skeletal muscle of db/db mice. Here, we sought to explore the role of epigenetic mechanisms in altering these miRNAs. We show that histone deacetylase (HDAC) protein levels and activity are upregulated in the skeletal muscle of diabetic mice. In C2C12 cells, HDAC inhibition using suberoylanilide hydroxamic acid (SAHA) altered the levels of 24 miRNAs: 15 were downregulated and 9 were upregulated. miR-449a, an intronic miRNA localized within the Cdc20b gene, while being downregulated in the skeletal muscle of diabetic mice, was the most highly upregulated during HDAC inhibition. The host gene, Cdc20b, was also significantly upregulated during HDAC inhibition. Bioinformatics analyses identified a common promoter for both Cdc20b and miR-449a that harbors significant histone acetylation marks, suggesting the possibility of regulation by histone acetylation-deacetylation. These observations suggest an inverse correlation between miR-449a levels and HDAC activity, in both SAHA-treated skeletal muscle cells and db/db mice skeletal muscle. Further, in SAHA-treated C2C12 cells, we observed augmented occupancy of acetylated histones on the Cdc20b/miR-449a promoter, which possibly promotes their upregulation. In vivo injection of SAHA to db/db mice significantly restored skeletal muscle miR-449a levels. Our results provide insights into the potential regulatory role of epigenetic histone acetylation of the miR-449a promoter that may regulate its expression in the diabetic skeletal muscle.

Irisin improves fatty acid oxidation and glucose utilization in type 2 diabetes by regulating the AMPK signaling pathway.

It has been reported that irisin regulated exercise-mediated adipocyte browning; however, the systematical effects of irisin on the metabolism of glucose and lipid in diabetes are largely unknown. In the present study, we investigated the role and underlying mechanism of irisin in glucose utilization and lipid metabolism in diabetic mice. A mouse model of diabetes was established by feeding C57BL/6 mice with high-fat diet. The diabetic mice were then treated with irisin. To mimic type 2 diabetes in vitro, myocytes and hepatocytes were cultured in a medium of high glucose and high fat. Glucose uptake, fatty acid oxidation and the expression of related protein were evaluated. Irisin improved glucose tolerance and glucose uptake as evidenced by increased (18)F-FDG accumulation and GLUT4 translocation in diabetic skeletal muscle. Irisin also increased glucose uptake in myocytes cultured in high glucose/high fatty acid medium. In contrast, irisin reduced the expression of PEPCK and G6Pase, which are involved in gluconeogenesis, in diabetic liver. Consistently, irisin reduced fat weight and serum total cholesterol and triglyceride levels in diabetic mice, but increased acetyl coenzyme A carboxylase-β phosphorylation in muscle tissue and uncoupling protein 1 expression in fat tissue. In addition, irisin increased the oxidation of fatty acid in myocytes. Knockdown of the adenosine monophosphate (AMP)-activated protein kinase (AMPK) attenuated the effects of irisin on glucose uptake and fatty acid β-oxidation in myocytes. Similarly, inhibition of AMPK by a specific inhibitor reduced the effects of irisin on PEPCK and G6Pase expression in hepatocytes. Our results suggest that irisin has an essential role in glucose utilization and lipid metabolism, and irisin is a promising pharmacological target for the treatment of diabetes and its complications.

Unacylated ghrelin restores insulin and autophagic signaling in skeletal muscle of diabetic mice.

Impairment of insulin signaling in skeletal muscle detrimentally affects insulin-stimulated disposal of glucose. Restoration of insulin signaling in skeletal muscle is important as muscle is one of the major sites for disposal of blood glucose. Recently, unacylated ghrelin (UnAG) has received attention in diabetic research due to its favorable actions on improving glucose tolerance, glycemic control, and insulin sensitivity. The investigation of UnAG has entered phase Ib clinical trial in type 2 diabetes and phase II clinical trial in hyperphagia in Prader-Willi syndrome. Nonetheless, the precise mechanisms responsible for the anti-diabetic actions of UnAG remain incompletely understood. In this study, we examined the effects of UnAG on restoring the impaired insulin signaling in skeletal muscle of db/db diabetic mice. Our results demonstrated that UnAG effectively restored the impaired insulin signaling in diabetic muscle. UnAG decreased insulin receptor substrate (IRS) phosphorylation, increased protein kinase B (Akt) phosphorylation, and, hence, suppressed mTOR signaling. Consequently, UnAG enhanced Glut4 localization and increased PDH activity in the diabetic skeletal muscle. Intriguingly, our data indicated that UnAG normalized the suppressed autophagic signaling in diabetic muscle. In conclusion, our findings illustrated that UnAG restored the impaired insulin and autophagic signaling in skeletal muscle of diabetic mice, which are valuable to understand the underlying mechanisms of the anti-diabetic action of UnAG at peripheral skeletal muscle level.

Hyperglycemia induced expression, phosphorylation, and translocation of αB-crystallin in rat skeletal muscle.

αB-Crystallin (αBC) is a member of the small heat shock protein family that responds to a variety of stress and prevents the aggregation of partially unfolded proteins. Chronic hyperglycemia created during diabetes results in skeletal muscle atrophy and leads to diabetic myopathy. The aim of this study was to investigate the role of αBC under chronic hyperglycemia in rat skeletal muscle. Diabetes was induced in Wistar rats by a single i.p injection of streptozotocin and maintained for a period of 12 weeks at the end of which the animals were sacrificed and the muscle was collected. The protein levels of αBC and its phosphorylation status in gastrocnemius muscle were analyzed by immunoblotting. The translocation of phosphorylated αBC was analyzed by detergent solubility assay, co-immunoprecipitation (Co-IP), and immunohistochemistry. The cell death was analyzed by TUNEL assay and by apoptotic markers. The interaction of αBC with Bax was analyzed by Co-IP. Chronic hyperglycemia significantly increased the protein levels of αBC and its phosphorylation at S59 by activation of p38 mitogen-activated protein kinase (p38MAPK) and at S45 by activation of the extracellular regulated protein kinase 1/2 (ERK1/2). Further, phosphorylated αBC translocated and interacted with desmin indicating that phosphorylated αBC forms might be involved in protection of sarcomere structures from disruption in chronic hyperglycemia. Further, Co-IP studies showed an impaired interaction of αBC with Bax which could be one of the possible factors for increased cell death as evidenced by TUNEL assay in diabetic muscle. These results suggest that an increased expression, phosphorylation, translocation of αBC, and its involvement in apoptosis might play a significant role in maintenance of cytoskeletal architecture and protection of cells from apoptosis in diabetic skeletal muscle.

Functional Expression of WNK Kinases and Insulin Signaling Effectors in Diabetic Skeletal Muscle

WNK (with‐no‐lysine [K]) kinases (WNK1‐4) are serine/threonine protein kinases with an atypical placement of the catalytic lysine and tissue‐specific expression. WNK1, ubiquitously expressed including skeletal muscle, is known as a downstream effector of insulin signaling and WNKs have been implicated in protein trafficking highly correlated with insulin resistance or diabetes. However, expression pattern and functional role of WNKs in skeletal muscle and linkage between WNKs and insulin signaling effectors such as PKB/Akt and glucose transports in skeletal muscle with type II diabetes are ill‐defined. In the present study, WNK1 and WNK2, not WNK4 are expressed in C2C12. The expression levels of WNK1 and WNK2 significantly decreased in db/db mice, a type II diabetic model, compared to that of wild type mice. Phosphorylated Akt in skeletal muscle decreased in db/db mice supporting insulin resistance in skeletal muscle. Insulin stimulates Akt followed by WNK1 phosphorylation. Insulin stimulation of WNK1 is blunted by phosphoinositide‐3‐kinase inhibitors. Together, an expression of GLUT4, a downstream effector of Akt and/or WNK1, markedly reduced in db/db mice, whereas GLUT1 expression level is not altered suggesting that GLUT1 and GLUT4 are differently regulated in type II diabetic skeletal muscle. Therefore, these results may provide new insights for increased susceptibility to insulin resistance and diabetes in skeletal muscle.[This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (NRF‐2013R1A1A2060764)]

Increased pyruvate dehydrogenase kinase expression in cultured myotubes from obese and diabetic individuals.

To investigate the mechanisms of impairments in oxidative metabolism in obese and diabetic (T2DM) skeletal muscle, this study analysed the adaptive expression of genes involved in fatty acid (FA) oxidation and mitochondrial biogenesis in primary myotubes treated with elevated FAs. Muscle samples from obese or obese T2DM donors were stored or processed into human primary skeletal muscle myotubes, which were treated for 6h with a saturated (palmitic acid) or a monounsaturated (oleic acid) FA with or without a polyunsaturated FA (eicosapentaenoic acid: EPA). Real-time PCR analysis was used to determine mRNA expression. Basal pyruvate dehydrogenase kinase 4 (PDK4) mRNA expression in whole muscle samples from obese and T2DM subjects was increased compared to lean (P<0.05; n=13-20/group). In obese- and T2DM-derived myotubes, oleic acid treatment alone and in combination with EPA increased PDK4 mRNA expression compared to control (P<0.05; n=7/group), whereas palmitic acid alone and in combination with EPA only increased PDK4 mRNA in T2DM-derived myotubes compared to control (P<0.05; n=7/group). EPA alone did not alter mRNA expression of PDK4. These findings show that FAs induce the expression of PDK4 mRNA, which was increased in myotubes cultured from obese and T2DM donors. This persistent difference in PDK4 expression, present after culturing, suggests a fundamental alteration in the FA-mediated gene expression. This may in turn translate to differences in the regulation of oxidative substrate flux to impact on insulin sensitivity.