Dysregulation between TRIM63/FBXO32 expression and soleus muscle wasting in diabetic rats: potential role of miR-1-3p, -29a/b-3p, and -133a/b-3p.

Diabetes mellitus (DM) induces a variable degree of muscle sarcopenia, which may be related to protein degradation and to the expression of both E3 ubiquitin ligases and some specific microRNAs (miRNAs). The present study investigated the effect of diabetes and acute muscle contraction upon the TRIM63 and FBXO32 expression as well as the potential involvement of some miRNAs. Diabetes was induced by streptozotocin and studied after 30 days. Soleus muscles were harvested, stimulated to contract in vitro for twitch tension analysis (0.5 Hz), 30 min later for tetanic analysis (100 Hz), and 30 min later were frozen. TRIM63 and FBXO32 proteins were quantified by western blotting; Trim63 mRNA, Fbxo32 mRNA, miR-1-3p, miR-29a-3p, miR-29b-3p, miR-133a-3p, and miR-133b-3p were quantified by qPCR. Diabetes induced sarcopenia by decreasing (P < 0.05) muscle weight/tibia length index, maximum tetanic contraction and relaxation rates, and absolute twitch and tetanic forces (P < 0.05). Diabetes decreased (P < 0.05) the Trim63 and Fbxo32 mRNAs (30%) and respective proteins (60%), and increased (P < 0.01) the miR-29b-3p (2.5-fold). In muscle from diabetic rats, acute contractile stimulus increased TRIM63 protein, miR-1-3p, miR-29a-3p, and miR-133a/b-3p, but decreased miR-29b-3p (P < 0.05). Independent of the metabolic condition, after muscle contraction, both TRIM63 and FBXO32 proteins correlated significantly with miR-1-3p, miR-29a/b-3p, and miR-133a/b-3p. All diabetes-induced regulations were reversed by insulin treatment. Concluding, the results depict that muscle wasting in long-term insulinopenic condition may not be accompanied by increased proteolysis, pointing out the protein synthesis as an important modulator of muscle sarcopenia in DM.

Insulin acutely triggers transcription of Slc2a4 gene: participation of the AT-rich, E-box and NFKB-binding sites.

AIMS: The insulin-sensitive glucose transporter protein GLUT4 (solute carrier family 2 member 4 (Slc2a4) gene) plays a key role in glycemic homeostasis. Decreased GLUT4 expression is a current feature in insulin resistant conditions such as diabetes, and the restoration of GLUT4 content improves glycemic control. This study investigated the effect of insulin upon Slc2a4/GLUT4 expression, focusing on the AT-rich element, E-box and nuclear factor NF-kappa-B (NFKB) site. MAIN METHODS: Rat soleus muscles were incubated during 180 min with insulin, added or not with wortmannin (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (PI3K)-inhibitor), ML9 (serine/threonine protein kinase (AKT) inhibitor) and tumor necrosis factor (TNF, GLUT4 repressor), and processed for analysis of GLUT4 protein (Western blotting); Slc2a4, myocyte enhancer factor 2a/d (Mef2a/d), hypoxia inducible factor 1a (Hif1a), myogenic differentiation 1 (Myod1) and nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (Nfkb1) messenger ribonucleic acids (mRNAs) (polymerase chain reaction (PCR)); and AT-rich- (myocyte-specific enhancer factor 2 (MEF2)-binding site), E-box- (hypoxia inducible factor 1 alpha (HIF1A)- and myoblast determination protein 1 (MYOD1)-binding site), and NFKB-binding activity (electrophoretic mobility assay). KEY FINDINGS: Insulin increased Slc2a4 mRNA expression (140%) and nuclear proteins binding to AT-rich and E-box elements (~90%), all effects were prevented by wortmannin and ML9. Insulin also increased Mef2a/d and Myod1 mRNA expression, suggesting the participation of these transcriptional factors in the Slc2a4 enhancing effect. Conversely, insulin decreased Nfkb1 mRNA expression and protein binding to the NFKB-site (~50%). Furthermore, TNF-induced inhibition of GLUT4 expression (~40%) was prevented by insulin in an NFKB-binding repressing mechanism. GLUT4 protein paralleled the Slc2a4 mRNA regulations. SIGNIFICANCE: Insulin enhances the Slc2a4/GLUT4 expression in the skeletal muscle by activating AT-rich and E-box elements, in a PI3K/AKT-dependent mechanism, and repressing NFKB-site activity as well. These results unravel how post-prandial increase of insulin may guarantee GLUT4 expression, and how the insulin signaling impairment can participate in insulin resistance-induced repression of GLUT4.