Carbenoxolone enhances peripheral insulin sensitivity and GLUT4 expression in skeletal muscle of obese rats: Potential participation of UBC9 protein.

AIM: This study investigates the insulin sensitizer effect of carbenoxolone (CBX) and potentially involved peripheral mechanisms. MAIN METHODS: Taking glucose transporter 4 (GLUT4) as a marker of glucose disposal, we investigated the CBX effects on whole-body insulin sensitivity and solute carrier 2a4 (Slc2a4)/GLUT4 expression in visceral (VAT) and subcutaneous (SAT) adipose tissues and soleus muscle of monosodium glutamate (MSG)-induced obese rats. Sterol regulatory element binding protein (SREBP1), an enhancer of Slc2a4 expression was analyzed through mRNA content and SREBP1-binding to Slc2a4 promoter. Finally, the small ubiquitin-modifier conjugating enzyme 9 (UBC9), whose low content indicates accelerated GLUT4 degradation was analyzed in soleus. KEY FINDINGS: Hypercorticosteronemia, hyperinsulinemia and low glucose decay rate in the insulin tolerance test of obese rats were restored by CBX (P < 0.05). Slc2a4/GLUT4 increased in SAT (P < 0.05) and decreased in VAT (P < 0.01) of obese rats. In soleus, obesity increased Slc2a4 but decreased GLUT4 (P < 0.01), possibly by accelerating GLUT4 degradation, as suggested by decreased UBC9 (P < 0.01). CBX restored both UBC9 and GLUT4 contents. SREBP1 did not participate in the Slc2a4 transcriptional regulation. SIGNIFICANCE: The insulin sensitizer effect of CBX involves the increase of GLUT4 expression in soleus, indicating an increased glucose disposal in skeletal muscle. This observation reinforces the skeletal muscle as the main site of insulin-induced glucose uptake and sheds new light on the metabolic effects of 11ßHSD1 inhibitors, since most of the studies so far have focused on its effects on liver and adipose tissues.

Estrogen Receptor 1 (ESR1) Enhances Slc2a4/GLUT4 Expression by a SP1 Cooperative Mechanism.

Background: Estrogens are involved in glycemic regulation, playing an important role in the development and/or progression of insulin resistance. For that, estrogens regulate the expression of the glucose transporter protein GLUT4 (codified by the solute carrier family 2 member 4 gene, Slc2a4), thus modulating adipose and muscle glucose disposal. This regulation is a balance between ESR1-mediated enhancer effect and ESR2-mediated repressor effect on Slc2a4 gene. However, molecular mechanisms involved in these effects are poorly understood. Since the specificity protein 1 (SP1) participates in several ESR-mediated genomic regulations, the aim of the present study is to investigate the participation of SP1 in the ESR1/2-mediated regulation of Slc2a4 gene. Methods: Differentiated 3T3-L1 adipocytes were 24-hour challenged with 10 nM estradiol (E2) and 10 nM ESR1 agonist (PPT) or 100 nM ESR2 agonist (DPN), added or not with E2. Slc2a4 and Sp1 mRNAs (RT-qPCR), total GLUT4 and nuclear ESR1, ESR2 and SP1 proteins (Western blotting), SP1 binding activity into Slc2a4 promoter (EMSA), and nuclear complexation of SP1/ESR1 (immunoprecipitation) were analyzed. Results: E2 and PPT increased (25-50%) whereas DPN reduced (20-45%) Slc2a4 and GLUT4 expression. Nuclear content of ESR1 and ESR2 remained unchanged. Nuclear content of SP1 increased (50 to 90%) by PPT and DPN added or not with E2; the highest effect observed with PPT alone. PPT also increased the nuclear content of SP1/ESR1 complex and the SP1 binding into the Slc2a4 promoter. Conclusions: ESR1 activation in adipocytes increased the nuclear content of SP1 protein, the SP1/ESR1 interaction and SP1 binding into the Slc2a4 gene promoter, culminating with increased Slc2a4/GLUT4 expression. No involvement of SP1 seems to occur in ESR2-mediated repressor effect on Slc2a4. We expect that this ESR1/SP1 cooperative effect can contribute to the development of new approaches for prevention or treatment of insulin resistance and diabetes mellitus.

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.

Carbohydrate- and lipid-enriched meals acutely disrupt glycemic homeostasis by inducing transient insulin resistance in rats.

Chronic intake of high-carbohydrate or high-lipid diets is a well-known insulin resistance inducer. This study investigates the immediate effect (1-6 h) of a carbohydrate- or lipid-enriched meal on insulin sensitivity. Fasted rats were refed with standard, carbohydrate-enriched (C), or lipid-enriched (L) meal. Plasma insulin, glucose, and non-esterified fatty acids (NEFA) were measured at 1, 2, 4, and 6 h of refeeding. The glucose-insulin index showed that either carbohydrates or lipids decreased insulin sensitivity at 2 h of refeeding. At this time point, insulin tolerance tests (ITTs) and glucose tolerance tests (GTTs) detected insulin resistance in C rats, while GTT confirmed it in L rats. Reduced glycogen and phosphorylated AKT and GSK3 content revealed hepatic insulin resistance in C rats. Reduced glucose uptake in skeletal muscle subjected to the fatty acid concentration that mimics the high NEFA level of L rats suggests insulin resistance in these animals is mainly in muscle. In conclusion, carbohydrate- or lipid-enriched meals acutely disrupt glycemic homeostasis, inducing a transient insulin resistance, which seems to involve liver and skeletal muscle, respectively. Thus, the insulin resistance observed when those types of diets are chronically consumed may be an evolution of repeated episodes of this transient insulin resistance.