MicroRNA-194 modulates glucose metabolism and its skeletal muscle expression is reduced in diabetes

BACKGROUND: The regulation of microRNAs (miRNAs) at different stages of the progression of type 2 diabetes mellitus (T2DM) and their role in glucose homeostasis was investigated. METHODS: Microarrays were used to assess miRNA expression in skeletal muscle biopsies taken from healthy individuals and patients with pre-diabetes or T2DM, and insulin resistant offspring of rat dams fed a high fat diet during pregnancy. RESULTS: Twenty-three miRNAs were differentially expressed in patients with T2DM, and 7 in the insulin resistant rat offspring compared to their controls. Among these, only one miRNA was similarly regulated: miR-194 expression was significantly reduced by 25 to 50% in both the rat model and in human with pre-diabetes and established diabetes. Knockdown of miR-194 in L6 skeletal muscle cells induced an increase in basal and insulin-stimulated glucose uptake and glycogen synthesis. This occurred in conjunction with an increased glycolysis, indicated by elevated lactate production. Moreover, oxidative capacity was also increased as we found an enhanced glucose oxidation in presence of the mitochondrial uncoupler FCCP. When miR-194 was down-regulated in vitro, western blot analysis showed an increased phosphorylation of AKT and GSK3β in response to insulin, and an increase in expression of proteins controlling mitochondrial oxidative phosphorylation. CONCLUSIONS: Type 2 diabetes mellitus is associated with regulation of several miRNAs in skeletal muscle. Interestingly, miR-194 was a unique miRNA that appeared regulated across different stages of the disease progression, from the early stages of insulin resistance to the development of T2DM. We have shown miR-194 is involved in multiple aspects of skeletal muscle glucose metabolism from uptake, through to glycolysis, glycogenesis and glucose oxidation, potentially via mechanisms involving AKT, GSK3 and oxidative phosphorylation. MiR-194 could be down-regulated in patients with early features of diabetes as an adaptive response to facilitate tissue glucose uptake and metabolism in the face of insulin resistance

Frequent interruptions of sedentary time modulates contraction- and insulin-stimulated glucose uptake pathways in muscle:Ancillary analysis from randomized clinical trials

Epidemiological studies have observed associations between frequent interruptions of sitting time with physical activity bouts and beneficial metabolic outcomes, even in individuals who regularly exercise. Frequent interruptions to prolonged sitting reduce postprandial plasma glucose. Here we studied potential skeletal muscle mechanisms accounting for this improved control of glycemia in overweight adults under conditions of one day uninterrupted sitting and sitting interrupted with light-intensity or moderate-intensity walking every 20-min (n = 8); and, after three days of either uninterrupted sitting or light-intensity walking interruptions (n = 5). Contraction- and insulin-mediated glucose uptake signaling pathways as well as changes in oxidative phosphorylation proteins were examined. We showed that 1) both interventions reduce postprandial glucose concentration, 2) acute interruptions to sitting over one day stimulate the contraction-mediated glucose uptake pathway, 3) both acute interruptions to sitting with moderate-intensity activity over one day and light-intensity activity over three days induce a transition to modulation of the insulin-signaling pathway, in association with increased capacity for glucose transport. Only the moderate-intensity interruptions resulted in greater capacity for glycogen synthesis and likely for ATP production. These observations contribute to a mechanistic explanation of improved postprandial glucose metabolism with regular interruptions to sitting time, a promising preventive strategy for metabolic diseases

Effects of breaking up prolonged sitting on skeletal muscle gene expression

Breaking up prolonged sitting has been beneficially associated with cardiometabolic risk markers in both observational and intervention studies. We aimed to define the acute transcriptional events induced in skeletal muscle by breaks in sedentary time. Overweight/obese adults participated in a randomized three-period, three-treatment crossover trial in an acute setting. The three 5-h interventions were performed in the postprandial state after a standardized test drink and included seated position with no activity and seated with 2-min bouts of light- or moderate-intensity treadmill walking every 20 min. Vastus lateralis biopsies were obtained in eight participants after each treatment, and gene expression was examined using microarrays validated with real-time quantitative PCR. There were 75 differentially expressed genes between the three conditions. Pathway analysis indicated the main biological functions affected were related to small-molecule biochemistry, cellular development, growth and proliferation, and carbohydrate metabolism. Interestingly, differentially expressed genes were also linked to cardiovascular disease. For example, relative to prolonged sitting, activity bouts increased expression of nicotamide N-methyltransferase, which modulates anti-inflammatory and anti-oxidative pathways and triglyceride metabolism. Activity bouts also altered expression of 10 genes involved in carbohydrate metabolism, including increased expression of dynein light chain, which may regulate translocation of the GLUT-4 glucose transporter. In addition, breaking up sedentary time reversed the effects of chronic inactivity on expression of some specific genes. This study provides insight into the muscle regulatory systems and molecular processes underlying the physiological benefits induced by interrupting prolonged sitting

MiR-194 expression in the skeletal muscle of mice fed a high fat diet.

<p>MiR-194 expression was measured by qPCR in the skeletal muscle from mice fed a chow or a high fat diet for 8 weeks (n = 9–10 per group). Values are expressed as mean ± SEM. <i>P</i>-values were determined using Student’s t-test (*p<0.05 vs chow fed mice).</p

miR-194 expression in the skeletal muscle of human participants and rat offspring and correlations with HOMA-IR.

<p>miR-194 expression was validated by qPCR in human (A) and rat samples (B) (n = 4–6 per group). Values are expressed as mean ± SEM. <i>P</i>-values were determined using 1-way ANOVA followed by Student-Newman-Keuls <i>post-hoc</i> test for human data (*p<0.05 vs healthy), or Student’s t-test for rat data (*p<0.05 vs control). Correlation between miR-194 expression and HOMA-IR in human (C) and rat (D) was assessed using Pearson’s or Spearman’s correlation test as appropriate. R and p-values are indicated on the graphs.</p

Phenotypic characteristics.

<p>Phenotypic characteristics.</p

Effect of miR-194 inhibition on AKT and GSK3β expression in L6 cells.

<p>Expression of the phosphorylated and total forms of AKT (A) and GSK3β (B) were measured by Western blot in L6 cells 48 hours after transfection with a miR-194 inhibitor. Ratios phosphorylated:total are expressed as mean ± SEM (n = 3 per group). Representative blots are shown below the graphs. <i>P</i>-values were determined using 2-way ANOVA (normally distributed data) or Kruskal-Wallis 1-way ANOVA on ranks (non-normally distributed data) followed by Student-Newman-Keuls <i>post-hoc</i> tests (* p<0.05 vs. control, # p<0.05 vs. basal).</p

Differentially expressed microRNAs identified by microarray.

<p>Differentially expressed microRNAs identified by microarray.</p

Effect of miR-194 inhibition on glucose homeostasis in L6 cells.

<p>Basal or insulin-stimulated glucose uptake (A), glycogen synthesis (B) and lactate production (C) were assayed in L6 cells 48 hours after transfection with a miR-194 inhibitor. Glucose oxidation (D) was also measured in these transfected cells under basal conditions or after treatment with the mitochondrial uncoupler FCCP. Values are expressed as mean ± SEM of 4–6 individual experiments, with 4 technical replicates. <i>P</i>-values were determined using 2-way ANOVA (normally distributed data) or Kruskal-Wallis 1-way ANOVA on ranks (non-normally distributed data) followed by Student-Newman-Keuls <i>post-hoc</i> tests (* p<0.05 vs. control, # p<0.05 vs. basal).</p

Effect of miR-194 inhibition on protein expression of the oxidative phosphorylation complexes expression in L6 cells.

<p>Expression of the oxidative phosphorylation (OXPHOS) complexes I to V of the electron transport chain was measured by western blot in L6 cells 48 hours after transfection with a miR-194 inhibitor. Data are expressed as mean ± SEM (n = 3 per group). Representative blots are shown. <i>P</i>-values were determined using t- tests (*p<0.05 and **p<0.01 vs control).</p