Dual SGLT1/SGLT2 Inhibitor Phlorizin Ameliorates Non-Alcoholic Fatty Liver Disease and Hepatic Glucose Production in Type 2 Diabetic Mice.

PURPOSE: NAFLD is a hepatic component of type 2 diabetes mellitus (T2D), in which impaired hepatic glucose production plays an important role. Inhibitors of sodium glucose transporter 2 (SGLT2) reduce glycemia and exert beneficial effects on diabetic complications. Recently, dual SGLT1/2 inhibition has been proposed to be more effective in reducing glycemia. We hypothesized that improving hepatic glucose metabolism induced by SGLT1/2 inhibition could be accompanied by beneficial effects on NAFLD progression. METHODS: Glycemic homeostasis, hepatic glucose production and NAFLD features were investigated in obese T2D mice, treated with SGLT1/2 inhibitor phlorizin for 1 week. RESULTS: T2D increased glycemia; insulinemia; hepatic expression of phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase) and glucose transporter 2 (Slc2a2 gene); hepatocyte nuclear factors 1A/4A/3B-binding activity in Slc2a2; endogenous glucose production; liver weight, plasma transaminase concentration as well as hepatic inflammation markers, and induced histological signals of non-alcoholic steatohepatitis (NASH, according to NASH-CRN Pathology Committee System). Phlorizin treatment restored all these parameters (mean NASH score reduced from 5.25 to 2.75 P<0.001); however, plasma transaminase concentration was partially reverted and some hepatic inflammation markers remained unaltered. CONCLUSION: NAFLD accompanies altered hepatic glucose metabolism in T2D mice and that greatly ameliorated through short-term treatment with the dual SGLT1/2 inhibitor. This suggests that altered hepatic glucose metabolism participates in T2D-related NAFLD and highlights the pharmacological inhibition of SGLTs as a useful approach not only for controlling glycemia but also for mitigating development and/or progression of NAFLD.

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.

Resveratrol improves glycemic control in insulin-treated diabetic rats: participation of the hepatic territory.

BACKGROUND: Resveratrol is a natural polyphenol that has been proposed to improve glycemic control in diabetes, by mechanisms that involve improvement in insulin secretion and activity. In type 1 diabetes (T1D), in which insulin therapy is obligatory, resveratrol treatment has never been investigated. The present study aimed to evaluate resveratrol as an adjunctive agent to insulin therapy in a T1D-like experimental model. METHODS: Rats were rendered diabetic by streptozotocin (STZ) treatment. Twenty days later, four groups of animals were studied: non-diabetic (ND); diabetic treated with placebo (DP); diabetic treated with insulin (DI) and diabetic treated with insulin plus resveratrol (DIR). After 30 days of treatment, 24-hour urine was collected; then, blood, soleus muscle, proximal small intestine, renal cortex and liver were sampled. Specific glucose transporter proteins were analyzed (Western blotting) in each territory of interest. Solute carrier family 2 member 2 (Slc2a2), phosphoenolpyruvate carboxykinase (Pck1) and glucose-6-phosphatase catalytic subunit (G6pc) mRNAs (qPCR), glycogen storage and sirtuin 1 (SIRT1) activity were analyzed in liver. RESULTS: Diabetes induction increased blood glucose, plasma fructosamine concentrations, and glycosuria. Insulin therapy partially recovered the glycemic control; however, resveratrol as adjunctive therapy additionally improved glycemic control and restored plasma fructosamine concentration to values of non-diabetic rats. Resveratrol did not alter the expression of the glucose transporters GLUT2 and SGLT1 in the intestine, GLUT2 and SGLT2 in kidney and GLUT4 in soleus, suggesting that fluxes of glucose in these territories were unaltered. Differently, in liver, resveratrol promoted a reduction in Slc2a2, Pck1, and G6pc mRNAs, as well as in GLUT2 protein (P < 0.05, DIR vs. DI); besides, it increased (P < 0.01, DIR vs. DI) the hepatic glycogen content, and SIRT1 protein. CONCLUSIONS: Resveratrol is able to improve glycemic control in insulin-treated T1D-like rats. This effect seems not to involve changes in glucose fluxes in the small intestine, renal proximal tubule, and soleus skeletal muscle; but to be related to several changes in the liver, where downregulation of Slc2a2/GLUT2, Pck1, and G6pc expression was observed, favoring reduction of glucose production and efflux. Besides, resveratrol increased SIRT1 nuclear protein content in liver, which may be related to the observed gene expression regulations.

Increased SGLT1 expression in salivary gland ductal cells correlates with hyposalivation in diabetic and hypertensive rats.

BACKGROUND: Oral health complications in diabetes and hypertension include decreased salivary secretion. The sodium-glucose cotransporter 1 (SGLT1) protein, which transports 1 glucose/2 Na+/264 H2O molecules, is described in salivary glands. We hypothesized that changes in SGLT1 expression in the luminal membrane of ductal cell may be related to an altered salivary flow. FINDINGS: By immunohistochemistry, we investigated SGLT1 expression in ductal cells of parotid and submandibular glands from Wistar Kyoto rats (WKY), diabetic WKY (WKY-D), spontaneously hypertensive rats (SHR) and diabetic SHR (SHR-D), as well as in parotid glands from WKY subjected to sympathetic stimulation, with or without previous propranolol blockade. Diabetes and hypertension decreased the salivary secretion and increased SGLT1 expression in the luminal membrane of ductal cells, and their association exacerbated the regulations observed. After 30 min of sympathetic stimulation, SGLT1 increased in the luminal membrane of ductal cells, and that was blocked by previous injection of propranolol. CONCLUSIONS: SGLT1 expression increases in the luminal membrane of salivary gland ductal cells and the salivary flow decreases in diabetic and hypertensive rats, which may be related to sympathetic activity. This study highlights the water transporter role of SGLT1 in salivary glands, which, by increasing ductal water reabsorption, may explain the hyposalivation of diabetic and hypertensive subjects.

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.

Beta-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 beta-adrenergic-dependent way to ensure energy disposal for contractile function. Herein we investigate beta-blockade or beta-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%). beta1-beta2-, and beta1-beta2-beta3-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 beta-adrenergic activity regulates GLUT4 gene expression in skeletal muscle during fasting, highlighting its participation in preservation of GLUT4 protein in glycolytic muscle.

Insulin but not phlorizin treatment induces a transient increase in GLUT2 gene expression in the kidney of diabetic rats.

BACKGROUND/AIMS: Increases in the renal glucose transporter gene expression are involved in renal tubule-glomerular diseases. Here we investigate the GLUT2 gene expression changes in the kidney of diabetic rats, by using insulin or phlorizin treatment. METHODS: Rats were rendered diabetic and studied 20 days later: 4-12 h after one single injection of insulin or phlorizin, and 1-6 days after insulin or phlorizin injection twice a day, comparing with diabetic rats injected with placebo. GLUT2 was investigated by Northern and Western analysis. RESULTS: In 20-day diabetic rats, acute treatment with insulin lowered the plasma glucose and increased the GLUT2 mRNA ( approximately 100%, p < 0.001) without changes in the protein content, while phlorizin lowered the plasma glucose, but changed neither the GLUT2 mRNA nor the protein expression. Twenty-four hours of insulin treatment increased both GLUT2 mRNA ( approximately 100%, p < 0.001) and protein ( approximately 50%, p < 0.01), but no effects of phlorizin were observed. After 6 days, insulin and phlorizin similarly reduced glycemia, with opposite effects upon plasma insulin and urinary glucose, and both treatments decreased GLUT2 mRNA and protein (p < 0.05). CONCLUSION: In kidney of diabetic rats, an initial and transient upregulation of GLUT2 was induced specifically by insulin only. The 6-day normalization of GLUT2, however, was induced by both insulin and phlorizin treatment, which seems to be related to the plasma glucose lowering.

Acute and short-term insulin-induced molecular adaptations of GLUT2 gene expression in the renal cortex of diabetic rats.

Increased GLUT2 gene expression in the renal proximal tubule of diabetic rats is an adaptive condition, which may be important in the diabetic nephropathy development. We investigated the effects of insulin treatment upon the renal GLUT2 overexpression of diabetic rats. Acute treatment, surprisingly, induced a rapid further increase in GLUT2 mRNA content. Twelve hours after insulin injection, GLUT2 mRNA was twice the value of saline-injected rats (P<0.001), when GLUT2 protein remained unchanged. In response to short-term treatment, both GLUT2 mRNA and protein were increased in 1-day treated rats (P<0.05 versus saline-injected), decreasing after that, and reaching, within 6 days, values close to those of non-diabetic rats. Concluding, insulin treatment induced: initially, an additional upregulation of GLUT2 gene expression, involving posttranscriptional modulation; thereafter, downregulation of GLUT2 expression, which returns to non-diabetic levels. The former may be related to increased insulin concentration, the latter may be due to glycemic control.