Estrogen receptor 1 (ESR1) regulates VEGFA in adipose tissue.

Vascular endothelial growth factor A (VEGFA) is a key factor in the regulation of angiogenesis in adipose tissue. Poor vascularization during adipose tissue proliferation causes fibrosis and local inflammation, and is associated with insulin resistance. It is known that 17-beta estradiol (E2) regulates adipose tissue function and VEGFA expression in other tissues; however, the ability of E2 to regulate VEGFA in adipose tissue is currently unknown. In this study, we showed that, in 3T3-L1 cells, E2 and the estrogen receptor 1 (ESR1) agonist PPT induced VEGFA expression, while ESR1 antagonist (MPP), and selective knockdown of ESR1 using siRNA decreased VEGFA and prevented the ability of E2 to modulate its expression. Additionally, we found that E2 and PPT induced the binding of hypoxia inducible factor 1 alpha subunit (HIF1A) in the VEGFA gene promoter. We further found that VEGFA expression was lower in inguinal and gonadal white adipose tissues of ESR1 total body knockout female mice compared to wild type mice. In conclusion, our data provide evidence of an important role for E2/ESR1 in modulating adipose tissue VEGFA, which is potentially important to enhance angiogenesis, reduce inflammation and improve adipose tissue function.

Identification of nuclear factor-κB sites in the Slc2a4 gene promoter.

Glucose transporter GLUT4 protein, codified by Slc2a4 gene plays a key role in glycemic homeostasis. Insulin resistance, as in obesity, has been associated to inflammatory state, in which decreased GLUT4 is a feature. Inflammatory NF-κB transcriptional factor has been proposed as a repressor of Slc2a4; although, the binding site(s) in Slc2a4 promoter and the direct repressor effect have never been reported yet. A motif-based sequence analysis of mouse Slc2a4 promoter revealed two putative κB sites located inside -83/-62 and -134/-113 bp. Eletrophoretic mobility assay showed that p50 and p65 NF-κB subunits bind to both putative κB sites. Chromatin immunoprecipitation assay using genomic DNA from adipocytes confirmed p50- and p65-binding to Slc2a4 promoter. Moreover, transfection experiments revealed that NF-κB binds to the -134/-113bp region of the mouse Slc2a4 gene promoter, inhibiting the Slc2a4 gene transcription. The current findings demonstrate the existence of two κB sites in Slc2a4 gene promote, and that NF-κB has a direct repressor effect upon the Slc2a4 gene, providing an important link between insulin resistance and inflammation.

Inhibition of cannabinoid CB1 receptor upregulates Slc2a4 expression via nuclear factor-κB and sterol regulatory element-binding protein-1 in adipocytes.

Evidences have suggested that the endocannabinoid system is overactive in obesity, resulting in enhanced endocannabinoid levels in both circulation and visceral adipose tissue. The blockade of cannabinoid receptor type 1 (CB1) has been proposed for the treatment of obesity. Besides loss of body weight, CB1 antagonism improves insulin sensitivity, in which the glucose transporter type 4 (GLUT4) plays a key role. The aim of this study was to investigate the modulation of GLUT4-encoded gene (Slc2a4 gene) expression by CB1 receptor. For this, 3T3-L1 adipocytes were incubated in the presence of a highly selective CB1 receptor agonist (1 µM arachidonyl-2'-chloroethylamide) and/or a CB1 receptor antagonist/inverse agonist (0.1, 0.5, or 1 µM AM251, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide). After acute (2 and 4 h) and chronic (24 h) treatments, cells were harvested to evaluate: i) Slc2a4, Cnr1 (CB1 receptor-encoded gene), and Srebf1 type a (SREBP-1a type-encoded gene) mRNAs (real-time PCR); ii) GLUT4 protein (western blotting); and iii) binding activity of nuclear factor (NF)-κB and sterol regulatory element-binding protein (SREBP)-1 specifically in the promoter of Slc2a4 gene (electrophoretic mobility shift assay). Results revealed that both acute and chronic CB1 receptor antagonism greatly increased (∼2.5-fold) Slc2a4 mRNA and protein content. Additionally, CB1-induced upregulation of Slc2a4 was accompanied by decreased binding activity of NF-κB at 2 and 24 h, and by increased binding activity of the SREBP-1 at 24 h. In conclusion, these findings reveal that the blockade of CB1 receptor markedly increases Slc2a4/GLUT4 expression in adipocytes, a feature that involves NF-κB and SREBP-1 transcriptional regulation.

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.

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.

SLC2A2 gene expression in kidney of diabetic rats is regulated by HNF-1alpha and HNF-3beta.

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.

Na+-glucose cotransporter SGLT1 protein in salivary glands: potential involvement in the diabetes-induced decrease in salivary flow.

Oral health complications in diabetes include decreased salivary secretion. The SLC5A1 gene encodes the Na(+)-glucose cotransporter SGLT1 protein, which not only transports glucose, but also acts as a water channel. Since SLC5A1 expression is altered in kidneys of diabetic subjects, we hypothesize that it could also be altered in salivary glands, contributing to diabetic dysfunction. The present study shows a diabetes-induced decrease (p < 0.001) in salivary secretion, which was accompanied by enhanced (p < 0.05) SGLT1 mRNA expression in parotid (50%) and submandibular (30%) glands. Immunohistochemical analysis of parotid gland of diabetic rats revealed that SGLT1 protein expression increased in the luminal membrane of ductal cells, which can stimulate water reabsorption from primary saliva. Furthermore, SGLT1 protein was reduced in myoepithelial cells of the parotid from diabetic animals, and that, by reducing cellular contractile activity, might also be related to reduced salivary flux. Six-day insulin-treated diabetic rats reversed all alterations. In conclusion, diabetes increases SLC5A1 gene expression in salivary glands, increasing the SGLT1 protein content in the luminal membrane of ductal cells, which, by increasing water reabsorption, might explain the diabetes-induced decrease in salivary secretion.

Na(+) -glucose transporter-2 messenger ribonucleic acid expression in kidney of diabetic rats correlates with glycemic levels: involvement of hepatocyte nuclear factor-1alpha expression and activity.

Mutations in Na(+)-glucose transporters (SGLT)-2 and hepatocyte nuclear factor (HNF)-1alpha genes have been related to renal glycosuria and maturity-onset diabetes of the young 3, respectively. However, the expression of these genes have not been investigated in type 1 and type 2 diabetes. Here in kidney of diabetic rats, we tested the hypotheses that SGLT2 mRNA expression is altered; HNF-1alpha is involved in this regulation; and glycemic homeostasis is a related mechanism. The in vivo binding of HNF-1alpha into the SGLT2 promoter region in renal cortex was confirmed by chromatin immunoprecipitation assay. SGLT2 and HNF-1alpha mRNA expression (by Northern and RT-PCR analysis) and HNF-1 binding activity of nuclear proteins (by EMSA) were investigated in diabetic rats and treated or not with insulin or phlorizin (an inhibitor of SGLT2). Results showed that diabetes increases SGLT2 and HNF-1alpha mRNA expression (~50%) and binding of nuclear proteins to a HNF-1 consensus motif (~100%). Six days of insulin or phlorizin treatment restores these parameters to nondiabetic-rat levels. Moreover, both treatments similarly reduced glycemia, despite the differences in plasma insulin and urinary glucose concentrations, highlighting the plasma glucose levels as involved in the observed modulations. This study shows that SGLT2 mRNA expression and HNF-1alpha expression and activity correlate positively in kidney of diabetic rats. It also shows that diabetes-induced changes are reversed by lowering glycemia, independently of insulinemia. Our demonstration that HNF-1alpha binds DNA that encodes SGLT2 supports the hypothesis that HNF-1alpha, as a modulator of SGLT2 expression, may be involved in diabetic kidney disease.

Changes in sodium or glucose filtration rate modulate expression of glucose transporters in renal proximal tubular cells of rat.

Renal glucose reabsorption is mediated by luminal sodium-glucose cotransporters (SGLTs) and basolateral facilitative glucose transporters (GLUTs). The modulators of these transporters are not known, and their substrates glucose and Na+ are potential candidates. In this study we examined the role of glucose and Na+ filtration rate on gene expression of glucose transporters in renal proximal tubule. SGLT1, SGLT2, GLUT1 and GLUT2 mRNAs were assessed by Northern blotting; and GLUT1 and GLUT2 proteins were assessed by Western blotting. Renal cortex and medulla samples from control rats (C), diabetic rats (D) with glycosuria, and insulin-resistant 15-month old rats (I) without glycosuria; and from normal (NS), low (LS), and high (HS) Na+-diet fed rats were studied. Compared to C and I rats, D rats increased (P < 0.05) gene expression of SGLT2 by approximately 36%, SGLT1 by approximately 20%, and GLUT2 by approximately 100%, and reduced (P < 0.05) gene expression of GLUT1 by more than 50%. Compared to NS rats, HS rats increased (P < 0.05) SGLT2, GLUT2, and GLUT1 expression by approximately 100%, with no change in SGLT1 mRNA expression, and LS rats increased (P < 0.05) GLUT1 gene expression by approximately 150%, with no changes in other transporters. In summary, the results showed that changes in glucose or Na+ filtrated rate modulate the glucose transporters gene expression in epithelial cells of the renal proximal tubule.