Adipocyte-derived factors impair insulin signaling in differentiated human vascular smooth muscle cells via the upregulation of miR-143

AbstractCardiovascular complications are common in patients with type 2 diabetes. Adipokines have been implicated in the induction of proliferative and pro-atherogenic alterations in human vascular smooth muscle cells (hVSMC). Other reports demonstrated the importance of the miRNA cluster miR-143/145 in the regulation of VSMC homeostasis and insulin sensitivity. Here we investigated whether the detrimental effects of adipokines on hVSMC function could be ascribed to alterations in miR-143/145 expression. The exposure of hVSMC to conditioned media (CM) from primary human subcutaneous adipocytes increased the expression of smooth muscle α-actin (SMA), and the miR-143/145 cluster, but markedly impaired the insulin-mediated phosphorylation of Akt and its substrate endothelial nitric oxide synthase (eNOS). Furthermore, CM promoted the phosphorylation of SMAD2 and p38, which have both been linked to miR-143/145 induction. Accordingly, the induction of miR-143/145 as well as the inhibition of insulin-mediated Akt- and eNOS-phosphorylation was prevented when hVSMC were treated with pharmacological inhibitors for Alk-4/5/7 and p38 before the addition of CM. The transfection of hVSMC with precursor miR-143, but not with precursor miR-145, resulted in impaired insulin-mediated phosphorylation of Akt and eNOS. This inhibition of insulin signaling by CM and miR-143 is associated with a reduction in the expression of the oxysterol-binding protein-related protein 8 (ORP8). Finally, the knock-down of ORP8 resulted in impaired insulin-mediated phosphorylation of Akt in hVSMC. Thus, the detrimental effects of adipocyte-derived conditioned media on insulin action in primary hVSMC can be ascribed to the Alk- and p38-dependent induction of miR-143 and subsequent downregulation of ORP8

Protein levels of Sfrp5 and Wnt5a in primary human adipocytes and skeletal muscle cells.

<p>Lysates from differentiated primary human adipocytes and skeletal muscle cells were analyzed for protein abundance of Sfrp5 (A) and Wnt5a (B) by Western blotting. Phosphorylation signals were normalized for GAPDH protein abundance and expressed as mean ± standard error of the mean of independent experiments (adipocytes; n = 6; skeletal muscle: n = 4) using cells from different donors. The values obtained for adipocytes were set at 100%.</p

Effect of Sfrp5 on insulin signaling in primary human skeletal muscle cells.

<p>Primary human skeletal muscle cells were kept untreated, exposed to Sfrp5 for 24/ml MCP-1 for 18 h with or without a 4 h preincubation with 100 ng/ml Sfrp5. Then, when indicated (+) cells were stimulated with insulin (10 min; 100 nM). Cell lysates were analyzed for phosphorylation of Akt-Thr308 (A), Akt-Ser473 (B), GSK3α-Ser21 (C) and PRAS40-Thr246 (D) by Western blotting. Phosphorylation signals were normalized for GAPDH protein abundance and expressed as mean ± standard error of the mean of five independent experiments using cells from different donors. The values obtained for Sfrp5-untreated, insulin-treated cells were considered as control and set at 100%. Differences among groups were calculated by two-way ANOVA followed by Bonferroni multiple comparison analysis. #, indicates <i>p</i><0.05 for the effect of insulin stimulation; ***, <i>p</i><0.001; **, <i>p</i><0.01; *, <i>p</i><0.05 versus untreated cells.</p

Effect of Sfrp5 on glucose uptake in primary human adipocytes.

<p>Mature adipocytes were kept untreated, exposed to 100/ml Sfrp5 for 24 h, when indicated (+) stimulated with insulin (30 min; 100 nM), whereafter glucose uptake was determined. The incorporated amount of 2-deoxy-D-[1-³H]glucose was normalized for protein content and expressed as mean ± standard error of the mean of four independent experiments using cells from different donors. The values obtained for Sfrp5-untreated, insulin-treated cells were considered as control and set at 100%. Differences among groups were calculated by two-way ANOVA followed by Bonferroni multiple comparison analysis. #, indicates <i>p</i><0.05 for the effect of insulin stimulation; *, <i>p</i><0.05 for the effect of Sfrp5.</p