Skeletal Muscle Of Diabetic Mice Research Articles

The effect of metformin along with high-intensity interval training on gene expression of FoxO1 and Atrogin-1 in type 2 diabetic mice

Background and aims: Muscle atrophy is a complication of type 2 diabetes, in which the expression of atrophy-related genes is increased. The present study aimed to investigate the effect of high-intensity interval training (HIIT) and metformin on gene expression of two atrophy-related genes (i.e., FoxO1 and Atrogin-1) in the skeletal muscle of diabetic mice. Methods: A total of 30 mice (C57BL/6) were assigned to two groups: control (n=6), and high-fat diet (HFD) (n=24). The mice in the HFD group were fed a HDF for 16 weeks. Then, diabetes was induced in mice by HFD. Then, they were divided into 4 groups as follows: (i) diabetic control, (ii) diabetes + metformin (DM), (iii) diabetes + HIIT (DH), and (iv) diabetes + metformin + HIIT (DMH). The DM group took metformin, the HIIT group performed a HIIT program, and the DMH group had both HIIT and metformin. The real-time PCR methods were used to measure the mRNA expression of FoxO1 and Atrogin-1. Results: The findings showed that HFD-induced diabetes caused increases in the expression of FoxO1 (P=0.0006) and Atrogin-1 (P=0.0008), and HIIT could restrain these increments (P=0.086, P=0.041). However, the decreasing effect of metformin on the expression of these genes was not significant (P=0.15) and the combined effect of HIIT and metformin on the expression of these genes was not greater than the individual effect of HIIT (P=0.64). Conclusion: These results indicated that HIIT (but not metformin) may prevent type 2 diabetes-induced downregulation of FoxO1 and Atrogin-1 in skeletal muscle, and metformin could not affect the impact of the training on these atrophy-related genes.

Low-Carbohydrate Diet Modulates Glucose–Lipid Utilization in Skeletal Muscle of Diabetic Mice

Type 2 diabetes is associated with many complications, including skeletal muscle atrophy. Ketogenic diets and low-carbohydrate diets (LCD) have recently been introduced as dietary interventions in patients with diabetes, but their effects on glucose and lipid metabolism in skeletal muscle have not been studied. In the current study, we compared the effects of LCD and ketogenic diet on glucose and lipid metabolism in skeletal muscle of diabetic mice. C57BL/6J mice with type 2 diabetes, constructed by a high-fat diet combined with streptozotocin, were fed a standard diet, a high-fat diet, an LCD, or a ketogenic diet for 14 weeks, respectively. Here, we found that the LCD, rather than the ketogenic diet, retained skeletal muscle weight and suppressed the expression of atrophy-related genes in diabetic mice. In addition, the LCD had more glycolytic/type IIb myofiber content and inhibited forkhead box O1 and pyruvate dehydrogenase kinase 4 expression, leading to improved glucose utilization. However, the ketogenic diet maintained more oxidative/type I myofibers. Moreover, compared with the ketogenic diet, the LCD decreased intramuscular triglycerides content and muscle lipolysis, suggesting improvement in lipid metabolism. Taken together, these data suggested that the LCD improved glucose utilization, and inhibited lipolysis and atrophy in skeletal muscle of diabetic mice, while the ketogenic diet showed metabolic disorders in skeletal muscle.

MiR-539-5p regulates Srebf1 transcription in the skeletal muscle of diabetic mice by targeting DNA methyltransferase 3b

Aberrant DNA methylation is associated with diabetes, but the precise regulatory events that control the levels and activity of DNA methyltransferases (DNMTs) is not well understood. Here we show that miR-539-5p targets Dnmt3b and regulates its cellular levels. miR-539-5p and Dnmt3b show inverse patterns of expression in skeletal muscle of diabetic mice. By binding to the 3′ UTR of Dnmt3b, miR-539-5p downregulates its levels in C2C12 cells and in human primary skeletal muscle cells. miR-539-5p-Dnmt3b interaction regulates Srebf1 transcription by altering methylation at CpG islands within Srebf1 in C2C12 cells. Dnmt3b inhibition alone was sufficient to upregulate Srebf1 transcription. In vivo antagonism of miR-539-5p in normal mice induced hyperglycemia and hyperinsulinemia and impaired oral glucose tolerance. These mice had elevated Dnmt3b and decreased Srebf1 levels in skeletal muscle. db/db mice injected with miR-539-5p mimics showed improved circulatory glucose and cholesterol levels. Oral glucose tolerance improved together with normalization of Dnmt3b and Srebf1 levels in skeletal muscle. Our results support a critical role of miR-539-5p and Dnmt3b in aberrant skeletal muscle metabolism during diabetes by regulating Srebf1 transcription; modulating the miR-539-5p-Dnmt3b axis might have therapeutic potential for addressing altered skeletal muscle physiology during insulin resistance and type 2 diabetes.

The Effect of Gembili Starch (Dioscorea esculenta) and Eubacterium rectal Supplementation on Skeletal Muscle Peroxisome Proliferator-Activated Receptor γ Coactivator 1α (Pgc-1α) Expression in Diabetic Mice Models

BACKGROUND: Gembili or Dioscorea esculenta is a local food that is produced by several areas in Indonesia. Few studies have reported its health benefits for diabetes mellitus but a little is understood about its mechanism of action. PGC-1α is a transcriptional coactivator for genes that involved in energy metabolism and increased expression of this gene has previously been associated with improved insulin sensitivity. AIM: The objective of this study was to investigate the effect of Gembili starch and Gembili starch with butirogenic bacteria Eubacterium rectal on PGC-1α expression in skeletal muscle of diabetic mice. MATERIALS AND METHODS: Three months old male diabetic Wistar mice were divided into groups based on dietary supplement: Gembili starch only; Gembili starch with E. rectal; and E. rectal only. Positive (diabetic mice) and negative (non-diabetic) control groups were used in this study. After 4 weeks of supplementation, mice were sacrificed and muscle tissue was taken from musculus vastus latissimus. Plasma blood glucose was measured before and after intervention. PGC-1α expression was measured with immunohistochemistry and quantified by dividing cells that produce PGC-1α with total cells. RESULTS: Plasma blood glucose was reduced after invention in group that received Gembili starch only (p < 0.001); Gembili starch with E. rectal (p < 0.001); and E. rectal only (p < 0.001). The protein expression of PGC-1α in diabetic mice receiving Gembili starch only was significantly higher compared to control (p < 0.05). CONCLUSION: This study shown that Gembili starch supplementation was able to improve glucose control in diabetic mice and this effect was obtained perhaps through PGC-1α activation. Further study is needed to investigate the effect of Gembili starch supplementation on fat metabolism.

RNA sequencing reveals potential interacting networks between the altered transcriptome and ncRNome in the skeletal muscle of diabetic mice.

For a global epidemic like Type 2 diabetes mellitus (T2DM), while impaired gene regulation is identified as a primary cause of aberrant cellular physiology; in the past few years, non-coding RNAs (ncRNAs) have emerged as important regulators of cellular metabolism. However, there are no reports of comprehensive in-depth cross-talk between these regulatory elements and the potential consequences in the skeletal muscle during diabetes. Here, using RNA sequencing, we identified 465 mRNAs and 12 long non-coding RNAs (lncRNAs), to be differentially regulated in the skeletal muscle of diabetic mice and pathway enrichment analysis of these altered transcripts revealed pathways of insulin, FOXO and AMP-activated protein kinase (AMPK) signaling to be majorly over-represented. Construction of networks showed that these pathways significantly interact with each other that might underlie aberrant skeletal muscle metabolism during diabetes. Gene–gene interaction network depicted strong interactions among several differentially expressed genes (DEGs) namely, Prkab2, Irs1, Pfkfb3, Socs2 etc. Seven altered lncRNAs depicted multiple interactions with the altered transcripts, suggesting possible regulatory roles of these lncRNAs. Inverse patterns of expression were observed between several of the deregulated microRNAs (miRNAs) and the differentially expressed transcripts in the tissues. Towards validation, overexpression of miR-381-3p and miR-539-5p in skeletal muscle C2C12 cells significantly decreased the transcript levels of their targets, Nfkbia, Pik3r1 and Pi3kr1, Cdkn2d, respectively. Collectively, the findings provide a comprehensive understanding of the interactions and cross-talk between the ncRNome and transcriptome in the skeletal muscle during diabetes and put forth potential therapeutic options for improving insulin sensitivity.

Pathway attenuation of fatty acid beta-oxidation in the skeletal muscle of a type 2 diabetic mouse model.

Whether catabolic abnormalities of fatty acids exist in the skeletal muscle of type 2 diabetes mellitus (T2DM) has not been determined. In this study, we postulated that a systematic evaluation of the protein abundance and metabolic activity related to fatty acids in the skeletal muscle tissues of a T2DM mouse model was feasible to address this question. Mitochondria were extracted from wild-type (WT) and db/db mice followed by quantitative analysis of the proteins involved in mitochondrial fatty acid oxidation (mFAO). The pathway activity of mFAO in skeletal muscle tissues was monitored in vitro using mass spectrometry, and tissue lipidomic analysis was conducted in profiling and target mode to distinguish the levels of long-chain acylcarnitines between WT and db/db mice. Two proteins related to the mFAO pathway were significantly downregulated in the skeletal muscle mitochondria of db/db mice. The measurement of mFAO pathway activity in vitro revealed that the abundance of long-chain acylcarnitines (C14 to C18) in db/db mice was lower than that in WT mice, and the determination of acylcarnitines in skeletal muscle tissues in vivo revealed that most long-chain acylcarnitines were decreased in db/db mice. The findings of lower abundance of ACAD9 and CPT1B, reduced activity of the mFAO pathway in vitro and decreased acylcarnitines in vivo firmly support that the mFAO pathway in the skeletal muscle of diabetic mice is attenuated, possibly resulting in cell/tissue dysfunction in diabetes.

The Flavonoid Kaempferol Ameliorates Streptozotocin-Induced Diabetes by Suppressing Hepatic Glucose Production.

In diabetes mellitus, the excessive rate of glucose production from the liver is considered a primary contributor for the development of hyperglycemia, in particular, fasting hyperglycemia. In this study, we investigated whether kaempferol, a flavonol present in several medicinal herbs and foods, can be used to ameliorate diabetes in an animal model of insulin deficiency and further explored the mechanism underlying the anti-diabetic effect of this flavonol. We demonstrate that oral administration of kaempferol (50 mg/kg/day) to streptozotocin-induced diabetic mice significantly improved hyperglycemia and reduced the incidence of overt diabetes from 100% to 77.8%. This outcome was accompanied by a reduction in hepatic glucose production and an increase in glucose oxidation in the muscle of the diabetic mice, whereas body weight, calorie intake, body composition, and plasma insulin and glucagon levels were not altered. Consistently, treatment with kaempferol restored hexokinase activity in the liver and skeletal muscle of diabetic mice while suppressed hepatic pyruvate carboxylase activity and gluconeogenesis. These results suggest that kaempferol may exert antidiabetic action via promoting glucose metabolism in skeletal muscle and inhibiting gluconeogenesis in the liver.

Resveratrol Improves Muscle Atrophy by Modulating Mitochondrial Quality Control in STZ-Induced Diabetic Mice.

ScopeIn this study, we aim to determine the effects of resveratrol (RSV) on muscle atrophy in streptozocin‐induced diabetic mice and to explore mitochondrial quality control (MQC) as a possible mechanism.Methods and resultsThe experimental mice were fed either a control diet or an identical diet containing 0.04% RSV for 8 weeks. Examinations were subsequently carried out, including the effects of RSV on muscle atrophy and muscle function, as well as on the signaling pathways related to protein degradation and MQC processes. The results show that RSV supplementation improves muscle atrophy and muscle function, attenuates the increase in ubiquitin and muscle RING‐finger protein‐1 (MuRF‐1), and simultaneously attenuates LC3‐II and cleaved caspase‐3 in the skeletal muscle of diabetic mice. Moreover, RSV treatment of diabetic mice results in an increase in mitochondrial biogenesis and inhibition of the activation of mitophagy in skeletal muscle. RSV also protects skeletal muscle against excess mitochondrial fusion and fission in the diabetic mice.ConclusionThe results suggest that RSV ameliorates diabetes‐induced skeletal muscle atrophy by modulating MQC.

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.

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.

Inhibition of xanthine oxidase reduces hyperglycemia-induced oxidative stress and improves mitochondrial alterations in skeletal muscle of diabetic mice

Reactive oxygen species (ROS) have been widely implicated in the pathogenesis of diabetes and more recently in mitochondrial alterations in skeletal muscle of diabetic mice. However, so far the exact sources of ROS in skeletal muscle have remained elusive. Aiming at better understanding the causes of mitochondrial alterations in diabetic muscle, we designed this study to characterize the sites of ROS production in skeletal muscle of streptozotocin (STZ)-induced diabetic mice. Hyperglycemic STZ mice showed increased markers of systemic and muscular oxidative stress, as evidenced by increased circulating H(2)O(2) and muscle carbonylated protein levels. Interestingly, insulin treatment reduced hyperglycemia and improved systemic and muscular oxidative stress in STZ mice. We demonstrated that increased oxidative stress in muscle of STZ mice is associated with an increase of xanthine oxidase (XO) expression and activity and is mediated by an induction of H(2)O(2) production by both mitochondria and XO. Finally, treatment of STZ mice, as well as high-fat and high-sucrose diet-fed mice, with oxypurinol reduced markers of systemic and muscular oxidative stress and prevented structural and functional mitochondrial alterations, confirming the in vivo relevance of XO in ROS production in diabetic mice. These data indicate that mitochondria and XO are the major sources of hyperglycemia-induced ROS production in skeletal muscle and that the inhibition of XO reduces oxidative stress and improves mitochondrial alterations in diabetic muscle.

Comment on: Biscetti et al. (2010) High-Mobility Group Box-1 Protein Promotes Angiogenesis After Peripheral Ischemia in Diabetic Mice Through a VEGF-Dependent Mechanism. Diabetes;59:1496–1505

In a recent Diabetes article, Biscetti et al. (1) report that high-mobility group box-1 (HMGB1) protein levels are reduced in the skeletal muscle of diabetic mice and that exogenous HMGB1 administration, following the induction of hindlimb ischemia, promotes angiogenesis through a VEGF-dependent mechanism. With regard to the angiogenic effect of HMGB1, we have previously demonstrated that both HMGB1 administration and …

Major Urinary Protein-1 Increases Energy Expenditure and Improves Glucose Intolerance through Enhancing Mitochondrial Function in Skeletal Muscle of Diabetic Mice

Major urinary protein-1 (MUP-1) is a low molecular weight secreted protein produced predominantly from the liver. Structurally it belongs to the lipocalin family, which carries small hydrophobic ligands such as pheromones. However, the physiological functions of MUP-1 remain poorly understood. Here we provide evidence demonstrating that MUP-1 is an important player in regulating energy expenditure and metabolism in mice. Both microarray and real-time PCR analysis demonstrated that the MUP-1 mRNA abundance in the liver of db/db obese mice was reduced by approximately 30-fold compared with their lean littermates, whereas this change was partially reversed by treatment with the insulin-sensitizing drug rosiglitazone. In both dietary and genetic obese mice, the circulating concentrations of MUP-1 were markedly decreased compared with the lean controls. Chronic elevation of circulating MUP-1 in db/db mice, using an osmotic pump-based protein delivery system, increased energy expenditure and locomotor activity, raised core body temperature, and decreased glucose intolerance as well as insulin resistance. At the molecular level, MUP-1-mediated improvement in metabolic profiles was accompanied by increased expression of genes involved in mitochondrial biogenesis, elevated mitochondrial oxidative capacity, decreased triglyceride accumulation, and enhanced insulin-evoked Akt signaling in skeletal muscle but not in liver. Altogether, these findings raise the possibility that MUP-1 deficiency might contribute to the metabolic dysregulation in obese/diabetic mice, and suggest that the beneficial metabolic effects of MUP-1 are attributed in part to its ability in increasing mitochondrial function in skeletal muscle.

Amino Acid Supplements Improve Native Antioxidant Enzyme Expression in the Skeletal Muscle of Diabetic Mice

Oxidative stress plays an important role in the pathogenesis of diabetic complications. We investigated the effects of a specific oral mixture of amino acid (AA) supplements on the antioxidant defense system, superoxide dismutase (SOD), and heat shock proteins (HSPs: HspB1, similar to Hsp 20 kDa, and HspB7) in the soleus muscle of streptozotocin (STZ)-diabetic mice by bidimensional electrophoresis and mass spectrometry. Four groups of 5 mice were considered: nondiabetic control mice, nondiabetic mice given AA supplements (0.1 g/kg per day for 15 days), diabetic mice (induced with STZ 65 mg/kg), and diabetic mice given AAs. AA supplements in the nondiabetic animals were associated with a statistical increase of SOD and no changes in expression of HSPs. Diabetes mellitus decreased antioxidant SOD and increased cellular stress as demonstrated by the overall upregulated HSPs. Administration of AAs counteracted the effects of diabetes, producing upregulation of SOD and downregulation of HSPs. These data suggest a role for AA supplements in controlling the antioxidant defense system and reducing the oxidative stress in diabetic skeletal muscle.

901. Recovery from Type 1 Diabetes by Insulin and Glucokinase Expression in Skeletal Muscle

Type 1 diabetes results from autoimmune destruction of insulin-producing |[beta]|-cells in pancreatic islets. Chronic hyperglycemia, due to failure to maintain proper glycemic control by insulin treatment, leads to the development of diabetes-specific microvascular pathology in the retina and renal glomerulus, and neurological and macrovascular complications. Skeletal muscle is the most important site of glucose removal from blood: it accounts for about 70% of glucose disposal after a meal. Skeletal muscle can be engineered to produce basal levels of insulin and express the liver enzyme glucokinase (GK), both of which increase glucose removal from blood. To obtain long-term expression of insulin and GK genes we examined the use of adeno-associated (AAV-1 serotype) viral vectors to transduce skeletal muscle. Co-expression of insulin and GK in skeletal muscle of diabetic mice treated with AAV1 vectors, restored normoglycemia and reverted all metabolic alterations. Moreover, the normalization of glycemia was parallel to normalization of fluid and food intake. Furthermore, these Ins+GK-treated diabetic mice did not develop secondary complications, such as peripheral neuropathy and nephropathy. Thus, we report evidence that a novel viral gene-therapy approach provides blood glucose concentration-dependent regulation of glucose homeostasis in diabetic animals. This is achieved by a combination of basal insulin production and the |[ldquo]|glucose sensor|[rdquo]| activity of GK in skeletal muscle. The significance of this work is that since normalization of glycemia is achieved, secondary complications of type 1 diabetes may not develop. This would greatly improve the quality of life of diabetic patients.

Coordinated patterns of gene expression for substrate and energy metabolism in skeletal muscle of diabetic mice.

Metabolic abnormalities underlying diabetes are primarily the result of the lack of adequate insulin action and the associated changes in protein phosphorylation and gene expression. To define the full set of alterations in gene expression in skeletal muscle caused by diabetes and the loss of insulin action, we have used Affymetrix oligonucleotide microarrays and streptozotocin-diabetic mice. Of the genes studied, 235 were identified as changed in diabetes, with 129 genes up-regulated and 106 down-regulated. Analysis revealed a coordinated regulation at key steps in glucose and lipid metabolism, mitochondrial electron transport, transcriptional regulation, and protein trafficking. mRNAs for all of the enzymes of the fatty acid beta-oxidation pathway were increased, whereas those for GLUT4, hexokinase II, the E1 component of the pyruvate dehydrogenase complex, and subunits of all four complexes of the mitochondrial electron transport chain were all coordinately down-regulated. Only about half of the alterations in gene expression in diabetic mice could be corrected toward normal after 3 days of insulin treatment and euglycemia. These data point to as of yet undefined mechanisms for highly coordinated regulation of gene expression by insulin and potential new targets for therapy of diabetes mellitus.

Myocyte Enhancer Factor 2 (MEF2)-Binding Site Is Required forGLUT4 Gene Expression in Transgenic Mice: REGULATION OF MEF2 DNA BINDING ACTIVITY IN INSULIN-DEFICIENT DIABETES

We have previously demonstrated that important regulatory elements responsible for regulated expression of the human GLUT4 promoter are located between -1154 and -412 relative to transcription initiation (Olson, A. L., and Pessin, J. E. (1995) J. Biol. Chem. 270, 23491-23495). Through further analysis of this promoter regulatory region, we have identified a perfectly conserved myocyte enhancer factor 2 (MEF2)-binding domain (-CTAAAAATAG-) that is necessary, but not sufficient, to support tissue-specific expression of a chloramphenicol acetyltransferase reporter gene in transgenic mice. Biochemical analysis of this DNA element demonstrated the formation of a specific DNA-protein complex using nuclear extracts isolated from heart, hindquarter skeletal muscle, and adipose tissue but not from liver. DNA binding studies indicated that this element functionally interacted with the MEF2A and/or MEF2C MADS family of DNA binding transcription factors. MEF2 DNA binding activity was substantially reduced in nuclear extracts isolated from both heart and skeletal muscle of diabetic mice, which correlated with decreased transcription rate of the GLUT4 gene. MEF2 binding activity completely recovered to control levels following insulin treatment. Together these data demonstrated that MEF2 binding activity is necessary for regulation of the GLUT4 gene promoter in muscle and adipose tissue.