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

The effect of nitric oxide donor sodium nitroprusside on glucose uptake in human primary skeletal muscle cells

Nitric oxide (NO) has been implicated as an important signaling molecule in the insulin-independent, contraction-mediated glucose uptake pathway and may represent a novel strategy for blood glucose control in patients with type 2 diabetes (T2DM). The current study sought to determine whether the NO donor, sodium nitroprusside (SNP) increases glucose uptake in primary human skeletal muscle cells (HSkMC) derived from both healthy individuals and patients with T2DM. Vastus lateralis muscle cell cultures were derived from seven males with T2DM (aged 54 &plusmn; 2 years, BMI 31.7 &plusmn; 1.2 kg/m2, fasting plasma glucose 9.52 &plusmn; 0.80 mmol/L) and eight healthy individuals (aged 46 &plusmn; 2 years, BMI 27.1 &plusmn; 1.5 kg/m2, fasting plasma glucose 4.69 &plusmn; 0.12 mmol/L). Cultures were treated with both therapeutic (0.2 and 2 &mu;M) and supratherapeutic (3, 10 and 30 mM) concentrations of SNP. An additional NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) was also examined at a concentration of 50 &mu;M. Glucose uptake was significantly increased following both 30 and 60 min incubations with the supratherapeutic SNP treatments (P = 0.03) but not the therapeutic SNP doses (P = 0.60) or SNAP (P = 0.54). There was no difference in the response between the healthy and T2DM cell lines with any treatment or dose. The current study demonstrates that glucose uptake is elevated by supratherapeutic, but not therapeutic doses of SNP in human primary skeletal muscle cells derived from both healthy volunteers and patients with T2D. These data confirm that nitric oxide donors have potential therapeutic utility to increase glucose uptake in humans, but that SNP only achieves this in supratherapeutic doses. Further study to delineate mechanisms and the therapeutic window is warranted.<br /

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

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

Differentially expressed microRNAs identified by microarray.

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