Rosana Cristina Tieko Mori

Intensive insulin treatment induces insulin resistance in diabetic rats by impairing glucose metabolism-related mechanisms in muscle and liver

Insulin replacement is the only effective therapy to manage hyperglycemia in type 1 diabetes mellitus (T1DM). Nevertheless, intensive insulin therapy has inadvertently led to insulin resistance. This study investigates mechanisms involved in the insulin resistance induced by hyperinsulinization. Wistar rats were rendered diabetic by alloxan injection, and 2 weeks later received saline or different doses of neutral protamine Hagedorn insulin (1.5, 3, 6, and 9 U/day) over 7 days. Insulinopenic-untreated rats and 6U- and 9U-treated rats developed insulin resistance, whereas 3U-treated rats revealed the highest grade of insulin sensitivity, but did not achieve good glycemic control as 6U- and 9U-treated rats did. This insulin sensitivity profile was in agreement with glucose transporter 4 expression and translocation in skeletal muscle, and insulin signaling, phosphoenolpyruvate carboxykinase/glucose-6-phosphatase expression and glycogen storage in the liver. Under the expectation that insulin resistance develops in hyperinsulinized diabetic patients, we believe insulin sensitizer approaches should be considered in treating T1DM.

Glimepiride as insulin sensitizer: increased liver and muscle responses to insulin

Aim:  Glimepiride, a low‐potency insulin secretagogue, is as efficient on glycaemic control as other sulphonylureas, suggesting an additional insulin‐sensitizer role. The aim of the present study was to confirm the insulin‐sensitizer role of glimepiride and to show extra‐pancreatic effects of the drug.

SLC2A2 gene expression in kidney of diabetic rats is regulated by HNF-1α and HNF-3β

We hypothesize that, in kidney of diabetic rats, hepatocyte nuclear factors (HNF-1alpha and HNF-3beta) play a critical role in the overexpression of solute carrier 2A2 (SLC2A2) gene. Diabetic rats submitted or not to rapid (up to 12h) and short-term (1, 4 and 6 days) insulin treatment were investigated. Twofold increase in GLUT2 mRNA was observed in diabetic, accompanied by significant increases in HNF-1alpha and HNF-3beta expression and binding activity. Additional 2-fold increase in GLUT2 mRNA and HNF-3beta expression/activity was observed in 12-h insulin-treated rats. Six-day insulin treatment decreased GLUT2 mRNA and HNF-1alpha expression and activity to levels of non-diabetic rats, whereas HNF-3beta decreased to levels of non-insulin-treated diabetic rats. Our results provide evidence for a link between the overexpression of SLC2A2 gene and the transcriptional activity of HNF-1alpha and HNF-3beta in kidney of diabetic rats. Furthermore, recovery of SLC2A2 gene after 6-day insulin treatment also involves HNF-1alpha and HNF-3beta activity.

The Na+/glucose cotransporters: from genes to therapy

Glucose enters eukaryotic cells via two types of membrane-associated carrier proteins, the Na(+)/glucose cotransporters (SGLT) and the facilitative glucose transporters (GLUT). The SGLT family consists of six members. Among them, the SGLT1 and SGLT2 proteins, encoded by the solute carrier genes SLC5A1 and SLC5A2, respectively, are believed to be the most important ones and have been extensively explored in studies focusing on glucose fluxes under both physiological and pathological conditions. This review considers the regulation of the expression of the SGLT promoted by protein kinases and transcription factors, as well as the alterations determined by diets of different compositions and by pathologies such as diabetes. It also considers congenital defects of sugar metabolism caused by aberrant expression of the SGLT1 in glucose-galactose malabsorption and the SGLT2 in familial renal glycosuria. Finally, it covers some pharmacological compounds that are being currently studied focusing on the interest of controlling glycemia by antagonizing SGLT in renal and intestinal tissues.

β‐Adrenergic activity preserves GLUT4 protein in glycolytic fibers in fasting

Glucose transporter 4 (GLUT4) expression in adipose tissue decreases during fasting. In skeletal muscle, we hypothesized that GLUT4 expression might be maintained in a β‐adrenergic–dependent way to ensure energy disposal for contractile function. Herein we investigate β‐blockade or β‐stimulation effects on GLUT4 expression in oxidative (soleus) and glycolytic [extensor digitorum longus (EDL)] muscles of fasted rats. Fasting increased GLUT4 mRNA in soleus (24%) and EDL (40%), but the protein content increased only in soleus (30%). β1–β2‐, and β1–β2–β3‐blockade decreased (20–30%) GLUT4 mRNA content in both muscles, although GLUT4 protein decreased only in EDL. When mRNA and GLUT4 protein regulations were discrepant, changes in the mRNA poly(A) tail length were detected, indicating a posttranscriptional modulation of gene expression. These results show that β‐adrenergic activity regulates GLUT4 gene expression in skeletal muscle during fasting, highlighting its participation in preservation of GLUT4 protein in glycolytic muscle. Muscle Nerve, 2009

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.

Beta-adrenergic blockade increases GLUT4 and improves glycemic control in insulin-treated diabetic Wistar rats

OBJECTIVE Unequivocal modulation of glycemic homeostasis by chronic beta-adrenergic blockade in diabetes has never been demonstrated. This study investigates the participation of beta-adrenergic system in glycemic control and muscle glucose transporter GLUT4 expression in insulin-treated diabetic rats. METHODS Insulin-treated diabetic Wistar (W) or spontaneously hypertensive rats (SHR) were additionally treated with propranolol, and glycemic homeostasis and expression of some target mRNAs and proteins in soleus and extensor digitorum longus (EDL) muscles were analyzed. RESULTS Insulin improved glycemic control in both strains. Importantly, in W, propranolol promoted a further improvement in glycemic control, which was accompanied by decreased PKA and Tnf expression, and increased Slc2a4 and GLUT4 in EDL. Those effects were not observed in diabetic-SHR. DISCUSSION Propranolol-induced decrease in beta-adrenergic activity in skeletal muscles of insulin-treated diabetic Wistar rats increases GLUT4 expression in EDL, improving glycemic control. These outcomes represent a positive effect of nonselective beta-blockade, which might be extended to autonomic neuropathy.

Recovery of insulin sensitivity and Slc2a4 mRNA expression depend on T3 hormone during refeeding

OBJECTIVE GLUT4 protein, encoded by the Slc2a4 gene, plays a key role in muscle glucose uptake, and its expression decreases in muscles under insulin resistance. Slc2a4/GLUT4 decreases with fasting and rapidly increases with refeeding and the same occurs to plasma glucose, amino acids, insulin and T3. Thus, they might be potential regulators of the Slc2a4 gene, which makes them promising targets for strategies to improve GLUT4 expression. Herein, we investigate the role of metabolic-hormonal parameters triggered by refeeding upon the Slc2a4 expression. MATERIALS/METHODS Plasma glucose/insulin/T3, and gastrocnemius Slc2a4 mRNA contents were measured in rats studied at the end of 48-h fasting, and subsequently at: i) 2-4h after spontaneous refeeding; ii) 2-4h after T3 injection, without refeeding; and iii) 0.5-2h after intravenous infusion of insulin, insulin+glucose and insulin+amino acids, without refeeding. RESULTS Refeeding increased plasma glucose/insulin/T3 and muscle Slc2a4 mRNA, reverting insulin resistance. Post-fasting infusions surprisingly induced a further Slc2a4 mRNA decrease (~20%, P<0.05 vs. fasting), but T3 injection induced a ~2-fold increase in Slc2a4 mRNA, 2-4h later (P<0.001). Moreover, T3 increased glycemia and insulinemia to the 2h-refed rats levels, suggesting that T3 elevation is a key factor to the mechanisms of metabolic balance during refeeding. CONCLUSIONS Refeeding induces a rapid increase in muscle Slc2a4 expression, not associated with increased plasma glucose, insulin or amino acids, but highly correlated to increased plasma T3 concentration. This result points out T3 hormone as a powerful Slc2a4 enhancer, an effect that may be acutely explored in situations of insulin resistance.

Estradiol-induced regulation of GLUT4 in 3T3-L1 cells: involvement of ESR1 and AKT activation

Impaired insulin-stimulated glucose uptake involves reduced expression of the GLUT4 (solute carrier family 2 facilitated glucose transporter member 4, SLC2A4 gene). 17β-estradiol (E2) modulates SLC2A4/GLUT4 expression, but the involved mechanisms are unclear. Although E2 exerts biological effects by binding to estrogen receptors 1/2 (ESR1/2), which are nuclear transcriptional factors; extranuclear effects have also been proposed. We hypothesize that E2 regulates GLUT4 through an extranuclear ESR1 mechanism. Thus, we investigated the effects of E2 upon (1) subcellular distribution of ESRs and the proto-oncogene tyrosine-protein kinases (SRC) involvement; (2) serine/threonine-protein kinase (AKT) activation; (3) Slc2a4/GLUT4 expression and (4) GLUT4 subcellular distribution and glucose uptake in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were cultivated or not with E2 for 24 h, and additionally treated or not with ESR1-selective agonist (PPT), ESR1-selective antagonist (MPP) or selective SRC inhibitor (PP2). Subcellular distribution of ESR1, ESR2 and GLUT4 was analyzed by immunocytochemistry; Slc2a4 mRNA and GLUT4 were quantified by qPCR and Western blotting, respectively; plasma membrane GLUT4 translocation and glucose uptake were analyzed under insulin stimulus for 20 min or not. E2 induced (1) translocation of ESR1, but not of ESR2, from nucleus to plasma membrane and AKT phosphorylation, effects mimicked by PPT and blocked by MPP and PP2; (2) increased Slc2a4/GLUT4 expression and (3) increased insulin-stimulated GLUT4 translocation and glucose uptake. In conclusion, E2 treatment promoted a SRC-mediated nucleus-plasma membrane shuttle of ESR1, and increased AKT phosphorylation, Slc2a4/GLUT4 expression and plasma membrane GLUT4 translocation; consequently, improving insulin-stimulated glucose uptake. These results unravel mechanisms through which estrogen improves insulin sensitivity.