Sodium-glucose Cotransporter 2 Expression Research Articles

Empagliflozin prevents oxidative stress induced by acute hyperglycemia in cardiac myocytes.

Empagliflozin, a sodium-glucose cotransporter-2 (SGLT-2) inhibitor, has shown to improve cardiovascular outcomes in patients with diabetes and heart failure. Empagliflozin has also been shown to provide advantageous effects in excised hearts and isolated cardiomyocytes despite SGLT-2 expression lacking in cardiac myocytes. This suggests empagliflozin may have important off-target effects. Previous studies suggested that empagliflozin effects may include attenuation of both reactive oxygen species (ROS) and Ca/calmodulin-dependent kinase II activation, but exact signaling details remained unclear. Using transgenic mice expressing glutaredoxin 1-coupled redox-sensitive green fluorescent protein 2 (Grx1-roGFP2) sensor localized to either the cytosol or mitochondria, we tested for empagliflozin effect on oxidative stress in myocytes bathed in high-glucose (30 mmol/L) containing Tyrode's solution. To assess the changes in redox potential, we exposed each cell to H2O2 and dithiothreitol to calibrate the sensor. We found acute hyperglycemia induced significant increases in ROS over a 3-5min of perfusion, specifically in the cytosolic compartment. This is in line with previous report showing that hyperglycemia activates CaMKII by intracellular O-GlcNAcylation, then the active CaMKII promotes cytosolic ROS production via NAPDH oxidase 2 (NOX2). Importantly, cells preincubated with empagliflozin were fully protected against the hyperglycemia-induced increase in ROS. We also extended our studies on angiotensin II (AngII)-dependent ROS production because AngII activates NOX2, and the excess ROS promotes oxidation- dependent CaMKII activation. Myocytes pretreated with the selective CaMKII inhibitor autocamtide-2-relative inhibitory peptide (cell-permeable myristoylated form) were used to test the role of CaMKII in ROS production by either hyperglycemia or Ang-II. These studies could reveal whether empagliflozin acts on glucose uptake, CaMKII activation, or via direct antioxidant mechanisms. Our findings may provide a mechanistic basis for the positive effects of empagliflozin in myocytes under conditions relevant to heart disease.

Canagliflozin primes antitumor immunity by triggering PD-L1 degradation in endocytic recycling.

Understanding the regulatory mechanisms of PD-L1 expression in tumors provides key clues for improving immune checkpoint blockade efficacy or developing novel oncoimmunotherapy. Here, we showed that the FDA-approved sodium-glucose cotransporter-2 (SGLT2) inhibitor canagliflozin dramatically suppressed PD-L1 expression and enhanced T cell-mediated cytotoxicity. Mechanistic study revealed that SGLT2 colocalized with PD-L1 at the plasma membrane and recycling endosomes and thereby prevented PD-L1 from proteasome-mediated degradation. Canagliflozin disturbed the physical interaction between SGLT2 and PD-L1 and subsequently allowed the recognition of PD-L1 by Cullin3SPOP E3 ligase, which triggered the ubiquitination and proteasome-mediated degradation of PD-L1. In mouse models and humanized immune-transformation models, either canagliflozin treatment or SGLT2 silencing significantly reduced PD-L1 expression and limited tumor progression - to a level equal to the PD-1 mAb - which was correlated with an increase in the activity of antitumor cytotoxic T cells. Notably, prolonged progression-free survival and overall survival curves were observed in the group of PD-1 mAb-treated patients with non-small cell lung cancer with high expression of SGLT2. Therefore, our study identifies a regulator of cell surface PD-L1, provides a ready-to-use small-molecule drug for PD-L1 degradation, and highlights a potential therapeutic target to overcome immune evasion by tumor cells.

S-51-4: RENAL DENERVATION IMPROVES FLUID RETENTION BY MODULATING RENAL SGLT2 FUNCTION IN RATS WITH HEART FAILURE

Objective: Renal denervation (RDN) has been developed as a neuromodulation therapy for patients with treatment-resistant hypertension. RDN is expected to prevent not only hypertension but also heart failure (HF). Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been shown to ameliorate HF and widely used for HF patients. The present study was conducted to assess the effect of RDN on HF and the interaction between renal sympathetic nerves and SGLT2 function in HF. Design and method: HF was induced by coronary artery ligation in rats. Four weeks after coronary artery ligation surgery, surgical bilateral RDN was performed. Western blot analysis was performed to assess the expression levels of SGLT2. One week after RDN surgery, as a functional test for SGLT2 activation, natriuretic response to intravenous injection of SGLT2 inhibitor dapagliflozin (0.5 mg/kg, i.v.) was investigated. Furthermore, the changes in expression and localization of SGLT2 were assessed in human embryonic kidney cells treated with norepinephrine directly, in vitro. Results: Western blot analysis indicated that renal SGLT2 expression is increased in rats with HF. RDN significantly reduced enhanced renal SGLT2 expression in HF. Natriuretic response to dapagliflozin were greater in HF than Sham (Cumulative sodium excretion at 120 min, 227 ± 35 vs. 133 ± 13 μEq/gkw, P < 0.05, n = 6). RDN significantly attenuated natriuretic response to dapagliflozin in HF (Cumulative sodium excretion at 120 min, 138 ± 19 vs. 227 ± 35 μEq/gkw, P < 0.05, n = 6). Direct application of norepinephrine on human embryonic kidney cells, in vitro, resulted in an increased expression of SGLT2 and specifically promoted the translocation of SGLT2 from the cytosol to the cell surface (Cytosolic SGLT2/GAPDH 0.51 ± 0.05 vs. 0.86 ± 0.08, Membranous SGLT2/Na-K-ATPase 1.06 ± 0.10 vs. 0.54 ± 0.03, P < 0.01, n = 6). Moreover, RDN increased the cytosolic SGLT2 while decreasing membranous SGLT2 in the kidneys of rats with HF (Cytosolic SGLT2/GAPDH 1.30 ± 0.11 vs. 0.58 ± 0.09, Membranous SGLT2/Na-K-ATPase 0.51 ± 0.10 vs. 2.08 ± 0.08, P < 0.01, n = 6). Conclusions: These findings suggest that the excessive activation of renal sympathetic nerves in HF increases the expression of SGLT2 protein trafficked by norepinephrine to cell membrane in proximal tubules resulting in greater sodium retention associated with HF condition. RDN mitigates the enhanced expression and activity of SGLT2 and ameliorates the subsequent sodium and water retention associated with HF, suggesting the potential therapeutic use of RDN for HF.

Dapagliflozin Prevents High-Glucose-Induced Cellular Senescence in Renal Tubular Epithelial Cells.

Gliflozins are a new class of antidiabetic drugs with renoprotective properties. In cultures of primary human renal tubular epithelial cells (RPTECs) subjected to high-glucose conditions in the presence or absence of dapagliflozin, we evaluated cellular senescence pathways. High glucose increased sodium-glucose cotransporter-2 (SGLT-2) expression and glucose consumption, enhancing reactive oxygen species production. The latter induced DNA damage, ataxia telangiectasia mutated kinase (ATM), and p53 phosphorylation. Stabilized p53 increased the cell cycle inhibitor p21, resulting in cell cycle arrest and increasing the cellular senescence marker beta-galactosidase (GLB-1). RPTECs under high glucose acquired a senescence-associated secretory phenotype, which was detected by the production of IL-1β, IL-8, and TGF-β1. By decreasing SGLT-2 expression and glucose consumption, dapagliflozin inhibited the above pathway and prevented RPTEC senescence. In addition, dapagliflozin reduced the cell cycle inhibitor p16 independently of the glucose conditions. Neither glucose concentration nor dapagliflozin affected the epithelial-to-mesenchymal transition when assessed with α-smooth muscle actin (α-SMA). Thus, high glucose induces p21-dependent RPTEC senescence, whereas dapagliflozin prevents it. Since cellular senescence contributes to the pathogenesis of diabetic nephropathy, delineating the related molecular mechanisms and the effects of the widely used gliflozins on them is of particular interest and may lead to novel therapeutic approaches.

Impact of Lycium barbarum polysaccharide on the expression of glucagon-like peptide 1 in vitro and in vivo

Several studies showed the efficacy of Lycium barbarum polysaccharide (LBP) in diabetic animals and patients with type 2 diabetes mellitus (T2DM). However, the mechanism of LBP in alleviating T2DM based on glucagon-like peptide 1 (GLP1) has not been suitably elucidated. GLP1 is an important peptide that plays a role in blood glucose homeostasis. Inhibition of sodium/glucose cotransporter 1 (SGLT1) can result in a net increase in GLP1 release. We found that LBP could reduce SGLT1 expression. Thus, the effects of LBP on the first- and second-phase secretion of GLP1 were systematically assessed in vitro using STC1 cells and in vivo using diabetic KKAy mice. LBP could induce the first-phase secretion of GLP1 by stimulating calcium ion influx in vitro and by inhibiting alpha-glucosidase activity in vivo. Regulation of Gcg gene expression by modulating the Wnt/β-catenin and cAMP/Epac pathways, as well as inhibition of alpha-glucosidase activity, was responsible for the second-phase secretion of GLP1. LBP could stimulate GLP1 secretion; however, dipeptidyl peptidase 4 (DPP4) activated by LBP might offset the second-phase secretion of GLP1. Thus, we suggest considering the simultaneous use of LBP and a DPP4 inhibitor to stimulate slow, continuous GLP1 secretion. Further studies are warranted for in-depth mechanistic information.

Hedgehog interacting protein activates sodium-glucose cotransporter 2 expression and promotes renal tubular epithelial cell senescence in a mouse model of type 1 diabetes.

Senescent renal tubular cells may be linked to diabetic kidney disease (DKD)-related tubulopathy. We studied mice with or without diabetes in which hedgehog interacting protein (HHIP) was present or specifically knocked out in renal tubules (HhipRT-KO), hypothesising that local deficiency of HHIP in the renal tubules would attenuate tubular cell senescence, thereby preventing DKD tubulopathy. Low-dose streptozotocin was employed to induce diabetes in both HhipRT-KO and control (Hhipfl/fl) mice. Transgenic mice overexpressing Hhip in renal proximal tubular cells (RPTC) (HhipRPTC-Tg) were used for validation, and primary RPTCs and human RPTCs (HK2) were used for in vitro studies. Kidney morphology/function, tubular senescence and the relevant molecular measurements were assessed. Compared with Hhipfl/fl mice with diabetes, HhipRT-KO mice with diabetes displayed lower blood glucose levels, normalised GFR, ameliorated urinary albumin/creatinine ratio and less severe DKD, including tubulopathy. Sodium-glucose cotransporter 2 (SGLT2) expression was attenuated in RPTCs of HhipRT-KO mice with diabetes compared with Hhipfl/fl mice with diabetes. In parallel, an increased tubular senescence-associated secretory phenotype involving release of inflammatory cytokines (IL-1β, IL-6 and monocyte chemoattractant protein-1) and activation of senescence markers (p16, p21, p53) in Hhipfl/fl mice with diabetes was attenuated in HhipRT-KO mice with diabetes. In contrast, HhipRPTC-Tg mice had increased tubular senescence, which was inhibited by canagliflozin in primary RPTCs. In HK2 cells, HHIP overexpression or recombinant HHIP increased SGLT2 protein expression and promoted cellular senescence by targeting both ataxia-telangiectasia mutated and ataxia-telangiectasia and Rad3-related-mediated cell arrest. Tubular HHIP deficiency prevented DKD-related tubulopathy, possibly via the inhibition of SGLT2 expression and cellular senescence.

Monocyte and endothelial dysfunction induced by hyperglycemia is reversed by Empagliflozin mainly through glucose-transport independent mechanisms

Abstract Type II Diabetes mellitus (T2DM) leads via hyperglycemia (HG)-associated inflammation and oxidative stress to vascular cell dysfunction. The EMPA-REG trial revealed a reduction of cardiovascular mortality in T2DM patients by Empagliflozin, a sodium-glucose co-transporter-2 (SGLT-2) inhibitor. SGLT-2 is expressed on monocytes and endothelial cells (EC), therefore the aim of this study was to investigate the potential role of Empagliflozin for glucose transport regulation and improvement of HG-induced dysfunction of monocytes and EC. HUVEC and HCAEC were used as EC model cells. Primary human monocytes were isolated from T2DM patients and healthy controls. The cells were exposed to HG conditions in vitro in presence of 40 or 100 ng/ml Empagliflozin. Using RT-qPCR and FACS, expression levels of SGLT-2 were analysed. Glucose uptake assays were performed with a fluorescent derivative of glucose, 2-NBDG. To measure reactive oxygen species (ROS) accumulation, we used the H2DFFDA method via FACS and fluorescence microscopy. Monocyte and EC chemotaxis was analysed using modified Boyden chamber assays. We could prove an expression of SGLT-2 in primary human monocytes and EC. The transcripts of SGLT-2 in T2DM monocytes did not show a difference. SGLT2-levels in monocytes and EC were not altered by HG. In presence of GLUT inhibitor in vitro glucose uptake assays revealed the ability of SGLT-2 inhibition to mildly supress the glucose uptake, whereas uptake by monocytes and EC remained comparable. Nevertheless, in presence of Empagliflozin HG-induced ROS accumulation in monocytes (1.3-fold, p=0.021) and ECs (1.5-fold, p=0.003) was reduced. HG monocytes and EC readily exhibited an impaired chemotaxis behaviour. Treating the cells in HG with Empagliflozin reversed the PlGF-1 resistance phenotype of HG monocytes (1.4-fold, p=0.002). Comparably, Empagliflozin was able to rescued the blunted VEGF-A responses of HG ECs (1.35-fold, p=0.028), which could be ascribed to restoration of VEGFR-2 receptor levels on the EC surface. Most of the aberrant phenotypes exhibited by HG monocytes and EC were entirely recapitulated by induction of oxidative stress. We could mimic these effects of Empagliflozin using the general antioxidant N-acetyl-L-cysteine (NAC). Our study reveals a beneficial role of Empagliflozin for reversing HG-induced vascular cell dysfunction. The expressed SGLT-2 on monocytes and EC does not serve as primary glucose transporter. So it is highly to speculate that HG-mediated enhanced glucotoxicity in those cells is not prevented by Empagliflozin through inhibition of glucose uptake itself. Reduction of oxidative stress by Empagliflozin was revealed as central mechanism for an improved function of HG monocytes and EC in vitro. Finally, we conclude that antioxidant properties of Empagliflozin reverse vascular cell dysfunction independently of glucose transport. This is likely to support the beneficial effect of Empagliflozin in cardiovascular disease. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Medical Faculty, University of Münster, Germany

Hepatic expression of sodium–glucose cotransporter 2 (SGLT2) in patients with chronic liver disease

Sodium–glucose cotransporter 2 (SGLT2) occurs in the proximal renal tubule cells. We investigate the hepatic expression of SGLT2 and its related factors in patients with chronic liver disease. This is a retrospective human study. The liver tissues were biopsied from patients with chronic liver disease (n = 30). The expression levels of SGLT2 were evaluated by immunostaining. Furthermore, the undirected graphical model was used to identify factors associated with hepatic expression levels of SGLT2. The SGLT2 expression was observed in not only the kidney, but also the liver in immunostaining (SGLT2 intensity: kidney 165.8 ± 15.6, liver 114.4 ± 49.0 arbitrary units, P < 0.01) and immunoblotting. There was no significant difference in hepatic expression of SGLT2 in the stratified analysis according to age, sex, BMI, and the severity of the liver disease. In the undirected graphical model, SGLT2 directly interacted with various factors such as sex, fatty change, neutrophil-to-lymphocyte ratio, triglyceride, hemoglobin A1c, creatinine, and albumin (partial correlation coefficient 0.4–0.6 for sex and 0.2–0.4 for others). The expression of SGLT2 was observed in the hepatocytes of patients with chronic liver disease. The undirected graphical model demonstrated the complex interaction of hepatic expression levels of SGLT2 with gender, inflammation, renal function, and lipid/glucose/protein metabolisms.

Sodium-glucose cotransporter-2 (SGLT2) expression in diabetic and non-diabetic failing human cardiomyocytes

This study aimed at investigating the SGLT2 expression in human cardiomyocytes. Human studies evaluating cardiomyocyte SGLT2s expression are limited. To better clarify this issue, SGLT2 protein expression was assessed in human hearts of diabetic and non-diabetic patients, and in AC16 human cardiomyocyte cell line. A prospective study with a follow-up of patients who underwent their first heart transplant (HTX) was performed. Explanted heart, basal (1 week after HTX), and final (48 weeks after HTX) endomyocardial biopsies (EMBs) from patients were evaluated for SGLT2 occurrence in cardiomyocyte with immunohistochemistry, immunofluorescence and SGLT2 quantization with both real-time reverse transcription-polymerase chain reaction and Western blot analysis. The immunofluorescence co-localization of SGLT2 in cardiomyocyte evidenced that an increased expression in the explanted heart from diabetic patients compared to non-diabetic (p < 0.001). In all final EMBs from diabetic patients, the expression of SGLT2 in cardiomyocyte was increased compared to non-diabetic (p < 0.01). This evidence was confirmed by Western blot analysis of SGLT2 protein. In addition, PCR analysis revealed very low mRNA levels in basal EMBs from diabetic and non-diabetic patients (p = NS), whereas final EMBs from diabetic patients showed higher SGLT2 mRNA levels in diabetic compared to non-diabetic patients (p < 0.05). Cultured human cardiomyocytes exposed to high-glucose showed increased expression of SGLT2 protein compared to cells exposed to normal glucose (p < 0.05). The presence of SGLT2 in cardiomyocytes supports the hypothesis of SGLT2i-mediated impact on metabolic pathways within cardiomyocytes. Moreover, metabolic disorders linked to diabetes may lead promptly to upregulation of SGLT2 levels in human cardiomyocytes.

Comparative study on the gastrointestinal- and immune- regulation functions of Hedysari Radix Praeparata Cum Melle and Astragali Radix Praeparata cum Melle in rats with spleen-qi deficiency, based on fuzzy matter-element analysis

Context Hedysari Radix Praeparata Cum Melle (HRPCM) and Astragali Radix Praeparata Cum Melle (ARPCM) are used interchangeably in clinics to treat spleen-qi deficiency (SQD) symptom mainly including gastrointestinal dysfunction and decreased immunity, which has unknown differences in efficacy. Objective To investigate the differences between HRPCM and ARPCM on intervening gastrointestinal- and immune-function with SQD syndrome. Materials and methods After the SQD model was established, the Sprague–Dawley (SD) rats were randomly divided into nine groups (n = 10): normal; model; Bu-Zhong-Yi-Qi Pills; 18.9, 12.6 and 6.3 g/kg dose groups of HRPCM and ARPCM. Gastrointestinal function including d-xylose, gastrin, amylase vasoactive intestinal peptide, motilin, pepsin, H+/K+-ATPase, Na+/K+-ATPase, sodium-glucose cotransporter 1 (SGLT1), glucose transporter 2 (GLUT2) and immune function including spleen and thymus index, blood routine, interleukin (IL)-2, IL-6, interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α), immunoglobulin (Ig) M, IgA, IgG and delayed-type hypersensitivity (DTH) were detected. Finally, the efficacy differences were analysed comprehensively by the fuzzy matter-element method. Results In regulating immune, the doses differences in efficacy between HRPCM and ARPCM showed in the high-dose (18.9 g/kg), but there were no differences in the middle- and low- dose (12.6 and 6.3 g/kg); the efficacy differences were primarily reflected in levels of IL-6, IFN-γ, TNF-α and IgM in serum, and the mRNA expression of IL-6 and IFN-γ in the spleen. In regulating gastrointestinal, the efficacy differences were primarily reflected in the levels of D-xylose, MTL, and GAS in serum, and the mRNA and protein expression of SGLT1 and GLUT2 in jejunum and ileum. Discussion and conclusions HRPCM is more effective than ARPCM on regulating gastrointestinal function and immune function with SQD syndrome. Therefore, we propose that HRPCM should be mainly used to treat SQD syndrome in the future.

Abstract 6402: Associations of sodium glucose cotransporter-2 (SGLT2), metabolic disorders, and survival following incident epithelial ovarian cancer

Abstract Background: Up to 75% of ovarian cancers will become platinum resistant and survival following the development of resistance is typically less than one year. New therapies with higher curative rates are needed to improve prognosis. There has been increasing interest in inhibitors of sodium glucose cotransporter-2 (SGLT2) in suppressing tumor growth. Use of SGLT2 inhibitors show promise not only due to their ability to inhibit glucose uptake, but also from the subsequent improvements in other metabolic disorders that may affect cancer prognosis. Purpose: We evaluated the associations of tumor SGLT2 protein expression with survival following diagnosis of incident epithelial ovarian cancer (EOC). Methods: Tumor samples were obtained from 492 patients undergoing debulking surgery for primary EOC at Roswell Park Comprehensive Cancer Center between 1995 and 2002. Only incident cases and patients who did not receive neo-adjuvant chemotherapy were included in analyses (n=396). Tissue samples were compiled into tissue microarray blocks that were sectioned, stained, imaged, and analyzed for SGLT2 expression. Data on patient, clinical, and tumor characteristics, along with available information on components of metabolic disorders, were linked to each patient. Multivariate Cox proportional hazards models were performed to determine hazards ratios (HR) and 95% confidence intervals (CI) for EOC-specific and all-cause mortality. Results: In our analysis of 396 EOC patients seen at Roswell Park, we observed significant associations between high SGLT2 expression and decreased risk of both EOC-specific (HR: 0.68; 95% CI: 0.49-0.94) and all-cause mortality (HR: 0.71; 95% CI: 0.53-0.95). In particular, among those with advanced-stage tumors, there was a significant association between high SGLT2 expression and lower risk of EOC-specific (HR: 0.61; 95% CI: 0.42-0.91) and all-cause mortality (HR: 0.64; 95% CI: 0.45-0.90). A lower risk of all-cause mortality was observed among those with high-grade tumors (HR: 0.69; 95% CI: 0.48-0.98). Trends of increasing SGLT2 associated with decreased risk of mortality varied by histological subtype. The inverse association between high SGLT2 and EOC-death was stronger among those with a history of hyperlipidemia (HR: 0.46; 95% CI: 0.19-0.99) and obesity (HR: 0.43; 95% CI: 0.23-0.82) compared to other metabolic syndrome components. These associations were less robust for all-cause mortality. Conclusion: Contrary to our initial hypothesis, higher SGLT2 expression among EOC patients may be associated with more favorable prognosis. This association may be stronger for high grade and advanced stage tumors as well as non-serous subtypes. Identifying other factors associated with levels of expression could aid in the development of potential screening interventions and more personalized treatment. Citation Format: Jennifer M. Mongiovi, Jo L. Freudenheim, Jeff C. Miecznikowski, Susan E. McCann, Ashley E. Stenzel, Wiam Bshara, Agnieszka Witkiewicz, Leighton Stein, Lisa D. Kellenberger, Ahmad Al Sulimani, Kirsten B. Moysich. Associations of sodium glucose cotransporter-2 (SGLT2), metabolic disorders, and survival following incident epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6402.

SGLT2 inhibitor activates the STING/IRF3/IFN-β pathway and induces immune infiltration in osteosarcoma

SGLT2 (sodium-glucose cotransporter 2) is an important mediator of epithelial glucose transport and has been reported that SGLT2, robustly and diffusely expressed in malignant cancer cells, was overexpressed in various tumors, and inhibiting the SGLT2 expression significantly inhibited tumor progression. By blocking the functional activity of SGLT2, SGLT2 inhibitors have shown anticancer effects in several malignant cancers, including breast cancer, cervical cancer, hepatocellular cancer, prostate cancer, and lung cancer. However, the anticancer effect of SGLT2 inhibitors in osteosarcoma and the specific mechanism are still unclear. In the present study, we found that SGLT2 was overexpressed at the protein level in osteosarcoma. Furthermore, our results showed that the SGLT2 inhibitor significantly inhibited osteosarcoma tumor growth and induced infiltration of immune cells in vivo by upregulating STING expression and activating the IRF3/IFN-β pathway, which could attribute to the suppression of AKT phosphorylation. In addition, the combined treatment with SGLT2 inhibitor and STING agonist 2’3’-cGAMP exerted synergistic antitumor effects in osteosarcoma. Furthermore, the overexpression of SGLT2 at the protein level was correlated with the degradation of SGLT2 induced by TRIM21. This result demonstrated that SGLT2 is a novel therapeutic target of osteosarcoma, and that the SGLT2 inhibitor, especially in combination with 2’3’-cGAMP, is a potential therapeutic drug.

Sodium-glucose co-transporter 1 (SGLT1) differentially regulates gluconeogenesis and GLP-1 receptor (GLP-1R) expression in different diabetic rats: a preliminary validation of the hypothesis of "SGLT1 bridge" as an indication for "surgical diabetes".

BackgroundSodium-glucose co-transporter 1 (SGLT1) may play a synergistic role in gluconeogenesis (GNG) and glucagon-like peptide-1 (GLP-1) expression. We proposed the hypothesis of a “SGLT1 bridge” as an indication for “surgical diabetes” that was preliminary validated in the present study.MethodsWe selected nonobese diabetic Goto-Kakizaki (GK) rats and Zuker diabetic fat (ZDF) rats to represent advanced and early diabetes, respectively. Based on glucose gavage with or without SGLT1 inhibitor phlorizin, the rats were divided into 4 groups: Gk-Glu, GK-P, ZDF-Glu, and ZDF-P. The expressions of SGLT1, GLP-1 receptor (GLP-1R), glucose-6 phosphatase (G6Pase), and phosphoenolpyruvate carboxykinase-1 (Pck1) were determined by immunohistochemistry (IHC) or quantitative reverse transcription polymerase chain reaction (RT-qPCR), and the effects of phlorizin were analyzed.ResultsGlucose tolerance was worse in GK rats and the homeostasis model assessment-insulin resistance (HOMA-IR) was higher in ZDF rats, indicating different pathophysiological conditions between the different diabetic rats. GK rats showed higher activity of duodenal SGLT1 (P=0.022) and jejunal SGLT1 mRNA expression (P=0.000) and lower SGLT1 mRNA expression in the liver (P=0.000) and pancreas (P=0.000). Phlorizin effectively inhibited the activity of duodenal SGLT1 in both GK rats (P=0.000) and ZDF rats (P=0.000). In ZDF rats, the expression of GLP-1R mRNA was downregulated in the jejunum (P=0.001) and upregulated in the pancreas (P=0.021) by phlorizin, but there were no regulatory effects on GLP-1R mRNA in the jejunum and pancreas of GK rats. As for the regulatory effects on GNG, phlorizin upregulated Pck1 mRNA in the duodenum (P=0.000) and the jejunum (P=0.038), whereas it downregulated hepatic G6Pase mRNA in ZDF rats (P=0.005) and Pck1 mRNA expression in GK rats (P=0.001), suggesting that SGLT1 inhibitor may have upregulated intestinal GNG in ZDF rats and downregulated hepatic GNG in both ZDF and GK rats.ConclusionsSGLT1 showed synergistic regulatory effects on the entero-insular axis (EIA) and the gut-brain-liver axis (GBLA), preliminarily validating the hypothesis of a “SGLT1 bridge”. The distinct expression of SGLT1 and its differentially regulatory effects on diabetic rats with different pathophysiological conditions may provide probable potential indications involved in the “Surgical Diabetes” that is supposed as the inclusion for diabetic surgery.

Empagliflozin Ameliorates Preeclampsia and Reduces Postpartum Susceptibility to Adriamycin in a Mouse Model Induced by Angiotensin Receptor Agonistic Autoantibodies.

Preeclampsia (PE) is the leading cause of maternal and perinatal morbidity and mortality and also is a risk factor for cardiovascular and kidney disease later in life. PE is associated with oversecretion of autoantibodies against angiotensin II type 1 receptor (AT1-AA) by the placenta into the maternal circulation. Here, we sought to determine the therapeutic value of the sodium-glucose co-transporter 2 (SGLT2) inhibitor empagliflozin (EMPA) in mice with AT1-AA-induced preeclampsia. Pregnant mice were injected with AT1-AA at gestation day (GD) 13 and treated daily with EMPA until GD 19, at which point some of the maternal mice were sacrificed and assessed. The other maternal mice were labored on time and challenged with adriamycin (ADR) at 12 weeks postpartum; their offspring were assessed for fetal outcomes. We showed that EMPA treatment significantly relieved high systolic blood pressure and proteinuria and ameliorated kidney injury in PE mice without affecting fetal outcomes. EMPA also ameliorated podocyte injury and oxidative stress, reduced the expression of SGLT2 and activated the AMPK/SIRT1 signaling pathway in vivo and in vitro. Remarkably, EMPA treatment during pregnancy reduced ADR-induced kidney and podocyte injury postpartum. These findings suggest that EMPA could be a potential pharmacological agent for PE.

Empagliflozin protects glomerular endothelial cell architecture in experimental diabetes through the VEGF-A/caveolin-1/PV-1 signaling pathway.

In addition to having blood glucose-lowering effects, inhibitors of sodium glucose cotransporter 2 (SGLT2) afford renoprotection in diabetes. We sought to investigate which components of the glomerular filtration barrier could be involved in the antiproteinuric and renoprotective effects of SGLT2 inhibition in diabetes. BTBR (black and tan, brachyuric) ob/ob mice that develop a type 2 diabetic nephropathy received a standard diet with or without empagliflozin for 10 weeks, starting at 8 weeks of age, when animals had developed albuminuria. Empagliflozin caused marked decreases in blood glucose levels and albuminuria but did not correct glomerular hyperfiltration. The protective effect of empagliflozin against albuminuria was not due to a reduction in podocyte damage as empagliflozin did not affect the larger podocyte filtration slit pore size nor the defective expression of nephrin and nestin. Empagliflozin did not reduce the thickening of the glomerular basement membrane. In BTBR ob/ob mice, the most profound abnormality seen using electron microscopy was in the endothelial aspect of the glomerular capillary, with significant loss of endothelial fenestrations. Remarkably, empagliflozin ameliorated the subverted microvascular endothelial ultrastructure. Caveolae and bridging diaphragms between adjacent endothelial fenestrae were seen in diabetic mice and associated with increased expression of caveolin-1 and the appearance of PV-1. These endothelial abnormalities were limited by the SGLT2 inhibitor. Although no expression of SGLT2 was found in glomerular endothelial cells, SGLT2 was expressed in the podocytes of diabetic mice. VEGF-A, which is a known stimulus for endothelial caveolin-1 and PV-1, was increased in podocytes of BTBR ob/ob mice and normalized by SGLT2 inhibitor treatment. Thus, empagliflozin's protective effect on the glomerular endothelium of diabetic mice could be due to a limitation of the paracrine signaling of podocyte-derived VEGF-A that resulted in a reduction of the abnormal endothelial caveolin-1 and PV-1, with the consequent preservation of glomerular endothelial function and permeability. © 2022 The Pathological Society of Great Britain and Ireland.

Glycaemia and body weight are regulated by sodium-glucose cotransporter 1 (SGLT1) expression via O-GlcNAcylation in the intestine

ObjectiveThe intestine is an important organ for nutrient metabolism via absorption and endocrine systems. Nutrients regulate O-GlcNAcylation, a post-translational modification of various proteins by O-GlcNAc transferase (OGT). We have previously shown that general OGT knockout induced severe weight loss and hypoglycaemia in mice, but little is known about how O-GlcNAcylation in the intestine modulates nutrient metabolism, especially glucose metabolism, through absorption. We aimed to reveal the roles of O-GlcNAcylation in glucose absorption by the small intestine and elucidate the mechanism by which O-GlcNAcylation regulates sodium-glucose cotransporter 1 (SGLT1) expression. MethodsFirst, we fasted normal mice and examined the changes in glucose transporters and O-GlcNAcylation in the intestine. Then, we generated two lines of small intestine-specific OGT-deficient mice (congenital: Ogt-VKO, tamoxifen-inducible: Ogt-iVKO) and observed the changes in body weight and in glucose and lipid metabolism. Finally, we investigated Sglt1 gene regulation by O-GlcNAcylation using enteroendocrine STC-1 cells. ResultsFasting decreased O-GlcNAcylation in the intestinal epithelium of normal mice. The Ogt-VKO mice showed significantly lower non-fasted blood glucose levels and were underweight compared with litter matched controls. Glycaemic excursion in the Ogt-VKO mice was significantly lower during the oral glucose tolerance test but comparable during the intraperitoneal glucose tolerance test. Furthermore, the Ogt-VKO mice exhibited lower Sglt1 expression in the small intestine compared with the control mice. We obtained similar results using the Ogt-iVKO mice only after tamoxifen administration. The oral d-xylose administration test revealed that the intestinal sugar absorption was diminished in the Ogt-iVKO mice and that GLP-1 secretion did not sufficiently increase after glucose gavage in the Ogt-iVKO mice. When using STC-1 cells, O-GlcNAcylation increased Sglt1 mRNA via a PKA/CREB-dependent pathway. ConclusionCollectively, loss of O-GlcNAcylation in the intestine reduced glucose absorption via suppression of SGLT1 expression; this may lead to new treatments for malabsorption, obesity and diabetes.

Sodium, Glucose and Dysregulated Glucagon Secretion: The Potential of Sodium Glucose Transporters.

Diabetes is defined by hyperglycaemia due to progressive insulin resistance and compromised insulin release. In parallel, alpha cells develop dysregulation of glucagon secretion. Diabetic patients have insufficient glucagon secretion during hypoglycaemia and a lack of inhibition of glucagon secretion at higher blood glucose levels resulting in postprandial hyperglucagonaemia, which contributes to the development of hyperglycaemia. Sodium-glucose co-transporter 2 (SGLT2) inhibitors are an efficient pharmacologic approach for the treatment of hyperglycaemia in type 2 diabetes. While SGLT2 inhibitors aim at increasing glycosuria to decrease blood glucose levels, these inhibitors also increase circulating glucagon concentrations. Here, we review recent advances in our understanding of how SGLTs are involved in the regulation of glucagon secretion. Sodium plays an important role for alpha cell function, and a tight regulation of intracellular sodium levels is important for maintaining plasma membrane potential and intracellular pH. This involves the sodium-potassium pump, sodium-proton exchangers and SGLTs. While the expression of SGLT2 in alpha cells remains controversial, SGLT1 seems to play a central role for alpha cell function. Under hyperglycaemic conditions, SGLT1 mediated accumulation of sodium results in alpha cell dysregulation due to altered cellular acidification and ATP production. Taken together, this suggests that SGLT1 could be a promising, yet highly underappreciated drug target to restore alpha cell function and improve treatment of both type 1 and 2 diabetes.

Enhanced Expression and Function of Renal SGLT2 (Sodium-Glucose Cotransporter 2) in Heart Failure: Role of Renal Nerves.

Recent clinical studies demonstrate that SGLT2 (sodium-glucose cotransporter 2) inhibitors ameliorate heart failure (HF). The present study was conducted to assess the expression and function of renal SGLT2 and the influence of enhanced renal sympathetic tone in HF. Four weeks after coronary artery ligation surgery to induce HF, surgical bilateral renal denervation (RDN) was performed in rats. Four groups of rats (Sham-operated control [Sham], Sham+RDN, HF and HF+RDN; n=6/group) were used. Immunohistochemistry and Western blot analysis were performed to evaluate the renal SGLT2 expression. One week after RDN (5 weeks after induction of HF), intravenous injection of SGLT2 inhibitor dapagliflozin were performed to assess renal excretory responses. In vitro, human embryonic kidney cells were used to investigate the fractionation of SGLT2 after norepinephrine treatment. In rats with HF, (1) SGLT2 expression in the proximal tubule of the kidney was increased; (2) the response of increases in urine flow, sodium excretion, and glucose excretion to dapagliflozin were greater; and (3) RDN attenuated renal SGLT2 expression and normalized renal functional responses to dapagliflozin. In vitro, norepinephrine promoted translocation of SGLT2 to the cell membrane. These results indicate that the enhanced tonic renal sympathetic nerve activation in HF increases the expression and functional activity of renal SGLT2. Potentiated trafficking of SGLT2 to cell surface in renal proximal tubules mediated by norepinephrine may contribute to this functional activation of SGLT2 in HF. These findings provide critical insight into the underlying mechanisms for the beneficial effects of SGLT2 inhibitors on HF reported in the clinical studies.

The use of SGLT2 inhibitors in achieving glycaemic control in maturity-onset diabetes of the young type 3.

SummaryMaturity-onset diabetes of the young type 3 (MODY3) accounts for approximately 50% of cases of MODY. First-line treatment with sulfonylureas has been well established for individuals with MODY3. In contrast, the use of sodium-glucose co-transporter 2 (SGLT2) inhibitors in the treatment of individuals with MODY3 remains unclear. This case illustrates the in vivo effect of an SGLT2 inhibitor in a 30-year-old woman with MODY3 with poor glycaemic control despite the treatment with supramaximal doses of sulfonylurea and metformin. The addition of a SGLT2 inhibitor resulted in a rapid improvement in glycaemic control without any hypoglycaemic episodes. This case suggests that SGLT2 inhibitors may be an effective and potent treatment option in addition to sulfonylureas for individuals with MODY3.Learning pointsMaturity-onset diabetes of the young type 3 (MODY3) arises from mutations in the hepatocyte nuclear factor-1alpha gene, which controls the expression of sodium-glucose co-transporter 2 (SGLT2) in the kidneys.Paradoxically, despite individuals with MODY3 having reduced expression of SGLT2, SGLT2 inhibitors induce higher glycosuria in individuals with MODY3 compared to individuals with type 2 diabetes mellitus.SGLT2 inhibitors may be an effective treatment for achieving glycaemic control in individuals with MODY3.

Mizagliflozin, a selective SGLT1 inhibitor, improves vascular cognitive impairment in a mouse model of small vessel disease.

Previously, we showed that sodium/glucose cotransporter 1 (SGLT1) participates in vascular cognitive impairment in small vessel disease. We hypothesized that SGLT1 inhibitors can improve the small vessel disease induced‐vascular cognitive impairment. We examined the effects of mizagliflozin, a selective SGLT1 inhibitor, and phlorizin, a non‐selective SGLT inhibitor, on vascular cognitive impairment in a mouse model of small vessel disease. Small vessel disease was created using a mouse model of asymmetric common carotid artery surgery (ACAS). Two and/or 4 weeks after ACAS, all experiments were performed. Cerebral blood flow (CBF) was decreased in ACAS compared with sham‐operated mice. Phlorizin but not mizagliflozin reversed the decreased CBF of ACAS mice. Both mizagliflozin and phlorizin reversed the ACAS‐induced decrease in the latency to fall in a wire hang test of ACAS mice. Moreover, they reversed the ACAS‐induced longer escape latencies in the Morris water maze test of ACAS mice. ACAS increased SGLT1 and proinflammatory cytokine gene expressions in mouse brains and phlorizin but not mizagliflozin normalized all gene expressions in ACAS mice. Hematoxylin/eosin staining demonstrated that they inhibited pyknotic cell death in the ACAS mouse hippocampus. In PC12HS cells, IL‐1β increased SGLT1 expression and decreased survival rates of cells. Both mizagliflozin and phlorizin increased the survival rates of IL‐1β‐treated PC12HS cells. These results suggest that mizagliflozin and phlorizin can improve vascular cognitive impairment through the inhibition of neural SGLT1 and phlorizin also does so through the improvement of CBF in a mouse model of small vessel disease.