Angiotensin II-induced redox-sensitive SGLT1 and 2 expression promotes high glucose-induced endothelial cell senescence.

High glucose (HG)-induced endothelial senescence and dysfunction contribute to the increased cardiovascular risk in diabetes. Empagliflozin, a selective sodium glucose co-transporter2 (SGLT2) inhibitor, reduced the risk of cardiovascular mortality in type 2 diabetic patients but the protective mechanism remains unclear. This study examines the role of SGLT2 in HG-induced endothelial senescence and dysfunction. Porcine coronary artery cultured endothelial cells (ECs) or segments were exposed to HG (25 mmol/L) before determination of senescence-associated beta-galactosidase activity, protein level by Western blot and immunofluorescence staining, mRNA by RT-PCR, nitric oxide (NO) by electron paramagnetic resonance, oxidative stress using dihydroethidium and glucose uptake using 2-NBD-glucose. HG increased ECs senescence markers and oxidative stress, down-regulated eNOS expression and NO formation, and induced the expression of VCAM-1, tissue factor, and the local angiotensin system, all these effects were prevented by empagliflozin. Empagliflozin and LX-4211 (dual SGLT1/2 inhibitor) reduced glucose uptake stimulated by HG and H2 O2 in ECs. HG increased SGLT1 and 2 protein levels in cultured ECs and native endothelium. Inhibition of the angiotensin system prevented HG-induced ECs senescence and SGLT1 and 2 expression. Thus, HG-induced ECs ageing is driven by the local angiotensin system via the redox-sensitive up-regulation of SGLT1 and 2, and, in turn, enhanced glucotoxicity.

Empagliflozin improved systolic blood pressure, endothelial dysfunction and heart remodeling in the metabolic syndrome ZSF1 rat.

BACKGROUND: Empagliflozin (empa), a selective sodium-glucose cotransporter (SGLT)2 inhibitor, reduced cardiovascular mortality and hospitalization for heart failure in patients with type 2 diabetes at high cardiovascular risk independent of glycemic control. The cardiovascular protective effect of empa was evaluated in an experimental model of metabolic syndrome, the obese ZSF1 rat, and its' lean control. METHODS: Lean and obese ZSF1 rats were either non-treated or treated with empa (30 mg/kg/day) for 6 weeks. Vascular reactivity was assessed using mesenteric artery rings, systolic blood pressure by tail-cuff sphygmomanometry, heart function and structural changes by echocardiography, and protein expression levels by Western blot analysis. RESULTS: Empa treatment reduced blood glucose levels from 275 to 196 mg/dl in obese ZSF1 rats whereas normoglycemia (134 mg/dl) was present in control lean ZSF1 rats and was unaffected by empa. Obese ZSF1 rats showed increased systolic blood pressure, and blunted endothelium-dependent relaxations associated with the appearance of endothelium-dependent contractile responses (EDCFs) compared to control lean rats. These effects were prevented by the empa treatment. Obese ZSF1 rats showed increased weight of the heart and of the left ventricle volume without the presence of diastolic or systolic dysfunction, which were improved by the empa treatment. An increased expression level of senescence markers (p53, p21, p16), tissue factor, VCAM-1, SGLT1 and SGLT2 and a down-regulation of eNOS were observed in the aortic inner curvature compared to the outer one in the control lean rats, which were prevented by the empa treatment. In the obese ZSF1 rats, no such effects were observed. The empa treatment reduced the increased body weight and weight of lungs, spleen, liver and perirenal fat, hyperglycemia and the increased levels of total cholesterol and triglycerides in obese ZSF1 rats, and increased blood ketone levels and urinary glucose excretion in control lean and obese ZSF1 rats. CONCLUSION: Empa reduced glucose levels by 28% and improved both endothelial function and cardiac remodeling in the obese ZSF1 rat. Empa also reduced the increased expression level of senescence, and atherothrombotic markers at arterial sites at risk in the control lean, but not obese, ZSF1 rat.

Organic anion transporter OAT3 enhances the glucosuric effect of the SGLT2 inhibitor empagliflozin.

The sodium-glucose cotransporter SGLT2 inhibitor empagliflozin (plasma protein binding ~88%) may reach its target in the brush border of the early proximal tubule by glomerular filtration and tubular secretion. Here we determined whether empagliflozin is secreted by renal tubules in mice and whether genetic knockout of the basolateral organic anion transporter 3 ( Oat3-/-) affects its tubular secretion or glucosuric effect. Renal clearance studies in wild-type (WT) mice showed that tubular secretion accounted for 50-70% of empagliflozin urinary excretion. Immunostaining indicated that SGLT2 and OAT3 localization partially overlapped in proximal tubule S1 and S2 segments. Glucosuria in metabolic cage studies was reduced in Oat3-/- vs. WT mice for acute empagliflozin doses of 1, 3, and 10 mg/kg, whereas 30 mg/kg induced similar maximal glucosuria in both genotypes. Chronic application of empagliflozin (~25 mg·kg-1 ·day-1) in Oat3-/- mice was associated with lower urinary glucose-to-creatinine ratios despite maintaining slightly higher blood glucose levels than WT. On a whole kidney level, renal secretion of empagliflozin was largely unchanged in Oat3-/- mice. However, the absence of OAT3 attenuated the influence of empagliflozin on fractional glucose excretion; higher levels of plasma or filtered empagliflozin were needed to induce similar increases in fractional renal glucose excretion. We conclude that empagliflozin is excreted into the urine to similar extent by glomerular filtration and tubular secretion. The latter can occur largely independent of OAT3. However, OAT3 increases the glucosuric effect of empagliflozin, which may relate to the partial overlap of its localization with SGLT2 and thus OAT3-mediated tubular secretion of empagliflozin in the early proximal tubule.

Glycemic control by the SGLT2 inhibitor empagliflozin decreases aortic stiffness, renal resistivity index and kidney injury.

BACKGROUND: Arterial stiffness is emerging as an independent risk factor for the development of chronic kidney disease. The sodium glucose co-transporter 2 (SGLT2) inhibitors, which lower serum glucose by inhibiting SGLT2-mediated glucose reabsorption in renal proximal tubules, have shown promise in reducing arterial stiffness and the risk of cardiovascular and kidney disease in individuals with type 2 diabetes mellitus. Since hyperglycemia contributes to arterial stiffness, we hypothesized that the SGLT2 inhibitor empagliflozin (EMPA) would improve endothelial function, reduce aortic stiffness, and attenuate kidney disease by lowering hyperglycemia in type 2 diabetic female mice (db/db). MATERIALS/METHODS: Ten-week-old female wild-type control (C57BLKS/J) and db/db (BKS.Cg-Dock7m+/+Leprdb/J) mice were divided into three groups: lean untreated controls (CkC, n = 17), untreated db/db (DbC, n = 19) and EMPA-treated db/db mice (DbE, n = 19). EMPA was mixed with normal mouse chow at a concentration to deliver 10 mg kg-1 day-1, and fed for 5 weeks, initiated at 11 weeks of age. RESULTS: Compared to CkC, DbC showed increased glucose levels, blood pressure, aortic and endothelial cell stiffness, and impaired endothelium-dependent vasorelaxation. Furthermore, DbC exhibited impaired activation of endothelial nitric oxide synthase, increased renal resistivity and pulsatility indexes, enhanced renal expression of advanced glycation end products, and periarterial and tubulointerstitial fibrosis. EMPA promoted glycosuria and blunted these vascular and renal impairments, without affecting increases in blood pressure. In addition, expression of "reversion inducing cysteine rich protein with Kazal motifs" (RECK), an anti-fibrotic mediator, was significantly suppressed in DbC kidneys and partially restored by EMPA. Confirming the in vivo data, EMPA reversed high glucose-induced RECK suppression in human proximal tubule cells. CONCLUSIONS: Empagliflozin ameliorates kidney injury in type 2 diabetic female mice by promoting glycosuria, and possibly by reducing systemic and renal artery stiffness, and reversing RECK suppression.

The Favorable Effect of Empagliflozin on Erectile Function in an Experimental Model of Type 2 Diabetes.

INTRODUCTION: Following the results of the EMPA-REG Outcome trial, we hypothesized that empagliflozin, a highly potent and specific sodium/glucose cotransporteur 2 inhibitor, could improve type 2 diabetes mellitus (T2DM)-associated erectile dysfunction (ED), a highly prevalent complication of T2DM, very often coexisting with cardiovascular complications and considered as a prognostic factor of cardiovascular disease in men with diabetes. AIM: To investigate the effects of chronic treatment with empagliflozin on ED in a T2DM rat model in the presence or absence of sildenafil. METHODS: Male Goto-Kakizaki (GK), a model of T2DM, and age-matched Wistar rats received placebo or empagliflozin treatment at 25.3 ± 0.9 mg/kg/d for 4 weeks. Then, the in vivo effect of empagliflozin on erectile function was assessed by electrical stimulation of the cavernous nerve at different frequencies under anesthesia in the presence or absence of acute intravenous injection of sildenafil. Endothelium-dependent, -independent, and nitrergic relaxations of cavernosal strips from the rats were studied. MAIN OUTCOME MEASURES: Body weight, food consumption, metabolic parameters, plasma inflammation biomarkers, and in vivo erectile responses elicited by electrical stimulation of the cavernous nerve in empagliflozin-treated and untreated GK rats and control Wistar rats were assessed and followed by concentration or frequency response curves to endothelium-dependent, -independent, and nitrergic relaxations of cavernosal strips from these rats. RESULTS: Chronic empagliflozin followed by acute sildenafil significantly improved erectile responses in adult GK rats (n = 12-15/group). Ratios of intracavernous pressure and area under the curve/mean arterial pressure during the electrical stimulation were significantly increased in empagliflozin-treated vs untreated GK rats. Nitrergic relaxations of cavernosal strips from GK rats were significantly increased with empagliflozin compared with placebo. Moreover, the effect of sildenafil on erectile function was not altered by empagliflozin treatment. CLINICAL IMPLICATIONS: Empagliflozin may benefit T2DM patient with ED. STRENGTHS & LIMITATIONS: The mechanism(s) by which empagliflozin shows favorable effect on erectile function in GK rats needs to be further elucidated. CONCLUSION: Empagliflozin shows favorable effect on erectile function in diabetic GK rats mediated by an improvement of nitrergic relaxation of erectile tissue. Whether this favorable effect on ED in the experimental context of T2DM is due to better glycemic control or to another effect of empagliflozin deserves further investigation. Assaly R, Gorny D, Compagnie S, et al. The Favorable Effect of Empagliflozin on Erectile Function in an Experimental Model of Type 2 Diabetes. J Sex Med 2018;15:1224-1234.

Sodium glucose transporter 2 (SGLT2) inhibition with empagliflozin improves cardiac diastolic function in a female rodent model of diabetes.

Obese and diabetic individuals are at increased risk for impairments in diastolic relaxation and heart failure with preserved ejection fraction. The impairments in diastolic relaxation are especially pronounced in obese and diabetic women and predict future cardiovascular disease (CVD) events in this population. Recent clinical data suggest sodium glucose transporter-2 (SGLT2) inhibition reduces CVD events in diabetic individuals, but the mechanisms of this CVD protection are unknown. To determine whether targeting SGLT2 improves diastolic relaxation, we utilized empagliflozin (EMPA) in female db/db mice. Eleven week old female db/db mice were fed normal mouse chow, with or without EMPA, for 5 weeks. Blood pressure (BP), HbA1c and fasting glucose were significantly increased in untreated db/db mice (DbC) (P < 0.01). EMPA treatment (DbE) improved glycemic indices (P < 0.05), but not BP (P > 0.05). At baseline, DbC and DbE had already established impaired diastolic relaxation as indicated by impaired septal wall motion (>tissue Doppler derived E'/A' ratio) and increased left ventricular (LV) filling pressure (

Empagliflozin Improves Left Ventricular Diastolic Dysfunction in a Genetic Model of Type 2 Diabetes.

PURPOSE: Cardiovascular (CV) diseases in type 2 diabetes (T2DM) represent an enormous burden with high mortality and morbidity. Sodium-glucose cotransporter 2 (SGLT2) inhibitors have recently emerged as a new antidiabetic class that improves glucose control, as well as body weight and blood pressure with no increased risk of hypoglycemia. The first CV outcome study terminated with empagliflozin, a specific SGLT2 inhibitor, has shown a reduction in CV mortality and in heart failure hospitalization, suggesting a beneficial impact on cardiac function which remains to be demonstrated. This study was designed to examine the chronic effect of empagliflozin on left ventricular (LV) systolic and diastolic functions in a genetic model of T2DM, ob/ob mice. METHODS AND RESULTS: Cardiac phenotype was characterized by echocardiography, in vivo hemodynamics, histology, and molecular profiling. Our results demonstrate that empagliflozin significantly lowered HbA1c and slightly reduced body weight compared to vehicle treatment with no obvious changes in insulin levels. Empagliflozin also improved LV maximum pressure and in vivo indices of diastolic function. While systolic function was grossly not affected in both groups at steady state, response to dobutamine stimulation was significantly improved in the empagliflozin-treated group, suggesting amelioration of contractile reserve. This was paralleled by an increase in phospholamban (PLN) phosphorylation and increased SERCA2a/PLN ratio, indicative of enhanced SERCA2a function, further supporting improved cardiac relaxation and diastolic function. In addition, empagliflozin reconciled diabetes-associated increase in MAPKs and dysregulated phosphorylation of IRS1 and Akt, leading to improvement in myocardial insulin sensitivity and glucose utilization. CONCLUSION: The data show that chronic treatment with empagliflozin improves diastolic function, preserves calcium handling and growth signaling pathways and attenuates myocardial insulin resistance in ob/ob mice, findings suggestive of a potential clinical utility for empagliflozin in the treatment of diastolic dysfunction.

Empagliflozin Protects against Diet-Induced NLRP-3 Inflammasome Activation and Lipid Accumulation.

The aim of this study was to evaluate the effects of chronic treatment with empagliflozin, a potent and selective sodium glucose cotransporter-2 inhibitor, in a murine model of diet-induced obesity and insulin resistance, focusing on drug effects on body weight reduction and nucleotide-binding domain, leucine-rich repeat containing protein (NLRP)-3 inflammasome activation, which have never been investigated to date. Male C57BL/6 mice were fed control or a high fat-high sugar (HFHS) diet for 4 months. Over the last 2 months, subsets of animals were treated with empagliflozin (1-10 mg/kg) added to the diet. Empagliflozin evoked body weight reduction (P < 0.001 for the highest dose) and positive effects on fasting glycemia and homeostasis model assessment of insulin resistance. In addition, the drug was able to reduce renal tubular damage and liver triglycerides level in a dose-dependent manner. Interestingly, empagliflozin also decreased cardiac lipid accumulation. Moreover, diet-induced activation of NLRP-3 in kidney and liver (not observed in the heart) was dose-dependently attenuated by empagliflozin. Our results clearly demonstrate the ability of empagliflozin to counteract the deleterious effects evoked by chronic exposure to HFHS diet. Most notably, empagliflozin treatment was associated with NLRP-3 inflammasome signaling modulation, suggesting that this inhibition may contribute to the drug therapeutic effects.

Characterization of Micro-RNA Changes during the Progression of Type 2 Diabetes in Zucker Diabetic Fatty Rats.

The aim of the present pilot study was the identification of micro-RNA changes over time during the development and progression of type 2 diabetes (T2D) in Zucker diabetic fatty rats (ZDF rats). T2D is a complex metabolic disorder that is characterized, inter alia, by progressive failure of pancreatic ß cells to produce insulin, but also by functional or morphological modifications of others organ, such as liver, adipose tissue and the cardiovascular system. Micro-RNAs are a novel class of biomarkers that have the potential to represent biomarkers of disease progression. In this study, the onset and progression of diabetes was followed in ZDF rats from six weeks until 17 weeks of age. After an initial phase of hyperinsulinemia, the animals developed T2D and lost the capacity to produce sufficient insulin. Circulating miRNAs were measured from plasma samples at four time points: pre-diabetes (six weeks of age), hyperinsulinemia (eight weeks), ß cell failure (11 weeks) and late-stage diabetes (17 weeks) using TaqMan miRNA arrays. Bioinformatic analysis revealed distinct changes of circulating miRNAs over time. Several miRNAs were found to be increased over the course of the disease progression, such as miR-122, miR-133, miR-210 and miR-375. The most significantly decreased miRNAs were miR-140, miR-151-3p, miR-185, miR-203, miR-434-3p and miR-450a. Some of the miRNAs have also been identified in type 2 diabetic patients recently and, therefore, may have the potential to be useful biomarkers for the disease progression of T2D and/or the treatment response for anti-diabetic medications.

Fingerprints of hSGLT5 sugar and cation selectivity.

Sodium glucose cotransporters (SGLTs) mediate the translocation of carbohydrates across the brush border membrane of different organs such as intestine, kidney, and brain. The human SGLT5 (hSGLT5), in particular, is localized in the kidney were it is responsible for mannose and fructose reabsorption from the glomerular filtrate as confirmed by more recent studies on hSGLT5 knockout mice. Here we characterize the functional properties of hSGLT5 expressed in a stable T-Rex-HEK-293 cell line using biochemical and electrophysiological assays. We confirmed that hSGLT5 is a sodium/mannose transporter that is blocked by phlorizin. Li(+) and H(+) ions were also able to drive mannose transport, and transport was electrogenic. Our results moreover indicate that substrates require a pyranose ring with an axial hydroxyl group (-OH) on carbon 2 (C-2). Compared with Na(+)/glucose cotransport, the level of function of Na(+)/mannose cotransport in rat kidney slices was low.

The SGLT2 inhibitor empagliflozin ameliorates early features of diabetic nephropathy in BTBR ob/ob type 2 diabetic mice with and without hypertension.

Diabetic nephropathy is the leading cause of end-stage renal disease in humans in the Western world. The recent development of Na+-glucose cotransporter 2 (SGLT2) inhibitors offers a new antidiabetic therapy via enhanced glucose excretion. Whether this strategy exerts beneficial effects on the development of type 2 diabetic nephropathy is still largely unclear. We investigated the effects of the specific SGLT2 inhibitor empagliflozin in BTBR.Cg-Lep/WiscJ (BTBR ob/ob) mice, which spontaneously develop type 2 diabetic nephropathy. In the first experiment, BTBR ob/ob mice received either a diet containing 300 ppm empagliflozin or equicaloric placebo chow for 12 wk. In the second experiment, BTBR ob/ob mice received 1 µg·kg body wt(-1)·day(-1) ANG II to induce arterial hypertension and were separated into the same two diet groups for 6 wk. In both experiments, empagliflozin treatment enhanced glucosuria, thereby lowering blood glucose. Independently of hypertension, empagliflozin reduced albuminuria in diabetic mice. However, empagliflozin treatment affected diabetes-related glomerular hypertrophy, markers of renal inflammation, and mesangial matrix expansion only in BTBR ob/ob mice without hypertension. In summary, empagliflozin demonstrated significant antihyperglycemic effects, differentially ameliorating early features of diabetic nephropathy in BTBR ob/ob mice with and without hypertension.

Increase in SGLT1-mediated transport explains renal glucose reabsorption during genetic and pharmacological SGLT2 inhibition in euglycemia.

In the kidney, the sodium-glucose cotransporters SGLT2 and SGLT1 are thought to account for >90 and ∼3% of fractional glucose reabsorption (FGR), respectively. However, euglycemic humans treated with an SGLT2 inhibitor maintain an FGR of 40-50%, mimicking values in Sglt2 knockout mice. Here, we show that oral gavage with a selective SGLT2 inhibitor (SGLT2-I) dose dependently increased urinary glucose excretion (UGE) in wild-type (WT) mice. The dose-response curve was shifted leftward and the maximum response doubled in Sglt1 knockout (Sglt1-/-) mice. Treatment in diet with the SGLT2-I for 3 wk maintained 1.5- to 2-fold higher urine glucose/creatinine ratios in Sglt1-/- vs. WT mice, associated with a temporarily greater reduction in blood glucose in Sglt1-/- vs. WT after 24 h (-33 vs. -11%). Subsequent inulin clearance studies under anesthesia revealed free plasma concentrations of the SGLT2-I (corresponding to early proximal concentration) close to the reported IC50 for SGLT2 in mice, which were associated with FGR of 64 ± 2% in WT and 17 ± 2% in Sglt1-/-. Additional intraperitoneal application of the SGLT2-I (maximum effective dose in metabolic cages) increased free plasma concentrations ∼10-fold and reduced FGR to 44 ± 3% in WT and to -1 ± 3% in Sglt1-/-. The absence of renal glucose reabsorption was confirmed in male and female Sglt1/Sglt2 double knockout mice. In conclusion, SGLT2 and SGLT1 account for renal glucose reabsorption in euglycemia, with 97 and 3% being reabsorbed by SGLT2 and SGLT1, respectively. When SGLT2 is fully inhibited by SGLT2-I, the increase in SGLT1-mediated glucose reabsorption explains why only 50-60% of filtered glucose is excreted.

SGLT2 inhibitor empagliflozin reduces renal growth and albuminuria in proportion to hyperglycemia and prevents glomerular hyperfiltration in diabetic Akita mice.

Our previous work has shown that gene knockout of the sodium-glucose cotransporter SGLT2 modestly lowered blood glucose in streptozotocin-diabetic mice (BG; from 470 to 300 mg/dl) and prevented glomerular hyperfiltration but did not attenuate albuminuria or renal growth and inflammation. Here we determined effects of the SGLT2 inhibitor empagliflozin (300 mg/kg of diet for 15 wk; corresponding to 60-80 mg·kg(-1)·day(-1)) in type 1 diabetic Akita mice that, opposite to streptozotocin-diabetes, upregulate renal SGLT2 expression. Akita diabetes, empagliflozin, and Akita + empagliflozin similarly increased renal membrane SGLT2 expression (by 38-56%) and reduced the expression of SGLT1 (by 33-37%) vs. vehicle-treated wild-type controls (WT). The diabetes-induced changes in SGLT2/SGLT1 protein expression are expected to enhance the BG-lowering potential of SGLT2 inhibition, and empagliflozin strongly lowered BG in Akita (means of 187-237 vs. 517-535 mg/dl in vehicle group; 100-140 mg/dl in WT). Empagliflozin modestly reduced GFR in WT (250 vs. 306 µl/min) and completely prevented the diabetes-induced increase in glomerular filtration rate (GFR) (255 vs. 397 µl/min). Empagliflozin attenuated increases in kidney weight and urinary albumin/creatinine ratio in Akita in proportion to hyperglycemia. Empagliflozin did not increase urinary glucose/creatinine ratios in Akita, indicating the reduction in filtered glucose balanced the inhibition of glucose reabsorption. Empagliflozin attenuated/prevented the increase in systolic blood pressure, glomerular size, and molecular markers of kidney growth, inflammation, and gluconeogenesis in Akita. We propose that SGLT2 inhibition can lower GFR independent of reducing BG (consistent with the tubular hypothesis of diabetic glomerular hyperfiltration), while attenuation of albuminuria, kidney growth, and inflammation in the early diabetic kidney may mostly be secondary to lower BG.

The sodium glucose cotransporter type 2 inhibitor empagliflozin preserves ß-cell mass and restores glucose homeostasis in the male zucker diabetic fatty rat.

Type 2 diabetes is characterized by impaired ß-cell function associated with progressive reduction of insulin secretion and ß-cell mass. Evidently, there is an unmet need for treatments with greater sustainability in ß-cell protection and antidiabetic efficacy. Through an insulin and ß cell-independent mechanism, empagliflozin, a specific sodium glucose cotransporter type 2 (SGLT-2) inhibitor, may potentially provide longer efficacy. This study compared the antidiabetic durability of empagliflozin treatment (10 mg/kg p.o.) against glibenclamide (3 mg/kg p.o.) and liraglutide (0.2 mg/kg s.c.) on deficient glucose homeostasis and ß-cell function in Zucker diabetic fatty (ZDF) rats. Empagliflozin and liraglutide led to marked improvements in fed glucose and hemoglobin A1c levels, as well as impeding a progressive decline in insulin levels. In contrast, glibenclamide was ineffective. Whereas the effects of liraglutide were less pronounced at week 8 of treatment compared with week 4, those of empagliflozin remained stable throughout the study period. Similarly, empagliflozin improved glucose tolerance and preserved insulin secretion after both 4 and 8 weeks of treatment. These effects were reflected by less reduction in ß-cell mass with empagliflozin or liraglutide at week 4, whereas only empagliflozin showed ß-cell sparing effects also at week 8. Although this study cannot be used to dissociate the absolute antidiabetic efficacy among the different mechanisms of drug action, the study demonstrates that empagliflozin exerts a more sustained improvement of glucose homeostasis and ß-cell protection in the ZDF rat. In comparison with other type 2 diabetic treatments, SGLT-2 inhibitors may through insulin-independent pathways thus enhance durability of ß-cell protection and antidiabetic efficacy.

SGLT2 Inhibition Mediates Protection from Diabetic Kidney Disease by Promoting Ketone Body-Induced mTORC1 Inhibition.

SGLT2 inhibitors offer strong renoprotection in subjects with diabetic kidney disease (DKD). But the mechanism for such protection is not clear. Here, we report that in damaged proximal tubules of high-fat diet-fed ApoE-knockout mice, a model of non-proteinuric DKD, ATP production shifted from lipolysis to ketolysis dependent due to hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). We further found that empagliflozin raised endogenous ketone body (KB) levels, and thus its use or treatment with 1,3-butanediol, a KB precursor, prevented decreases in renal ATP levels and organ damage in the mice. The renoprotective effect of empagliflozin was abolished by gene deletion of Hmgcs2, a rate-limiting enzyme of ketogenesis. Furthermore, KBs attenuated mTORC1-associated podocyte damage and proteinuria in diabetic db/db mice. Our findings show that SGLT2 inhibition-associated renoprotection is mediated by an elevation of KBs that in turn corrects mTORC1 hyperactivation that occurs in non-proteinuric and proteinuric DKD.

SGLT2 is not expressed in pancreatic α- and ß-cells, and its inhibition does not directly affect glucagon and insulin secretion in rodents and humans.

OBJECTIVE: Sodium-glucose cotransporter 2 (SGLT2) inhibitors (SGLT2i), or gliflozins, are anti-diabetic drugs that lower glycemia by promoting glucosuria, but they also stimulate endogenous glucose and ketone body production. The likely causes of these metabolic responses are increased blood glucagon levels, and decreased blood insulin levels, but the mechanisms involved are hotly debated. This study verified whether or not SGLT2i affect glucagon and insulin secretion by a direct action on islet cells in three species, using multiple approaches. METHODS: We tested the in vivo effects of two selective SGLT2i (dapagliflozin, empagliflozin) and a SGLT1/2i (sotagliflozin) on various biological parameters (glucosuria, glycemia, glucagonemia, insulinemia) in mice. mRNA expression of SGLT2 and other glucose transporters was assessed in rat, mouse, and human FACS-purified α- and ß-cells, and by analysis of two human islet cell transcriptomic datasets. Immunodetection of SGLT2 in pancreatic tissues was performed with a validated antibody. The effects of dapagliflozin, empagliflozin, and sotagliflozin on glucagon and insulin secretion were assessed using isolated rat, mouse and human islets and the in situ perfused mouse pancreas. Finally, we tested the long-term effect of SGLT2i on glucagon gene expression. RESULTS: SGLT2 inhibition in mice increased the plasma glucagon/insulin ratio in the fasted state, an effect correlated with a decline in glycemia. Gene expression analyses and immunodetections showed no SGLT2 mRNA or protein expression in rodent and human islet cells, but moderate SGLT1 mRNA expression in human α-cells. However, functional experiments on rat, mouse, and human (29 donors) islets and the in situ perfused mouse pancreas did not identify any direct effect of dapagliflozin, empagliflozin or sotagliflozin on glucagon and insulin secretion. SGLT2i did not affect glucagon gene expression in rat and human islets. CONCLUSIONS: The data indicate that the SGLT2i-induced increase of the plasma glucagon/insulin ratio in vivo does not result from a direct action of the gliflozins on islet cells.

Empagliflozin Increases Cardiac Energy Production in Diabetes: Novel Translational Insights Into the Heart Failure Benefits of SGLT2 Inhibitors.

SGLT2 inhibitors have profound benefits on reducing heart failure and cardiovascular mortality in individuals with type 2 diabetes, although the mechanism(s) of this benefit remain poorly understood. Because changes in cardiac bioenergetics play a critical role in the pathophysiology of heart failure, the authors evaluated cardiac energy production and substrate use in diabetic mice treated with the SGTL2 inhibitor, empagliflozin. Empagliflozin treatment of diabetic db/db mice prevented the development of cardiac failure. Glycolysis, and the oxidation of glucose, fatty acids and ketones were measured in the isolated working heart perfused with 5 mmol/l glucose, 0.8 mmol/l palmitate, 0.5 mmol/l ß-hydroxybutyrate (ßOHB), and 500 µU/ml insulin. In vehicle-treated db/db mice, cardiac glucose oxidation rates were decreased by 61%, compared with control mice, but only by 43% in empagliflozin-treated diabetic mice. Interestingly, cardiac ketone oxidation rates in db/db mice decreased to 45% of the rates seen in control mice, whereas a similar decrease (43%) was seen in empagliflozin-treated db/db mice. Overall cardiac adenosine triphosphate (ATP) production rates decreased by 36% in db/db vehicle-treated hearts compared with control mice, with fatty acid oxidation providing 42%, glucose oxidation 26%, ketone oxidation 10%, and glycolysis 22% of ATP production in db/db mouse hearts. In empagliflozin-treated db/db mice, cardiac ATP production rates increased by 31% compared with db/db vehicle-treated mice, primarily due to a 61% increase in the contribution of glucose oxidation to energy production. Cardiac efficiency (cardiac work/O2 consumed) decreased by 28% in db/db vehicle-treated hearts, compared with control hearts, and empagliflozin did not increase cardiac efficiency per se. Because ketone oxidation was impaired in db/db mouse hearts, the authors determined whether this contributed to the decrease in cardiac efficiency seen in the db/db mouse hearts. Addition of 600 µmol/l ßOHB to db/db mouse hearts perfused with 5 mmol/l glucose, 0.8 mmol/l palmitate, and 100 µU/ml insulin increased ketone oxidation rates, but did not decrease either glucose oxidation or fatty acid oxidation rates. The presence of ketones did not increase cardiac efficiency, but did increase ATP production rates, due to the additional contribution of ketone oxidation to energy production. The authors conclude that empagliflozin treatment is associated with an increase in ATP production, resulting in an enhanced energy status of the heart.

SGLT2 Inhibition by Empagliflozin Promotes Fat Utilization and Browning and Attenuates Inflammation and Insulin Resistance by Polarizing M2 Macrophages in Diet-induced Obese Mice.

Sodium-glucose cotransporter (SGLT) 2 inhibitors increase urinary glucose excretion (UGE), leading to blood glucose reductions and weight loss. However, the impacts of SGLT2 inhibition on energy homeostasis and obesity-induced insulin resistance are less well known. Here, we show that empagliflozin, a SGLT2 inhibitor, enhanced energy expenditure and attenuated inflammation and insulin resistance in high-fat-diet-induced obese (DIO) mice. C57BL/6J mice were pair-fed a high-fat diet (HFD) or a HFD with empagliflozin for 16weeks. Empagliflozin administration increased UGE in the DIO mice, whereas it suppressed HFD-induced weight gain, insulin resistance, and hepatic steatosis. Moreover, empagliflozin shifted energy metabolism towards fat utilization, elevated AMP-activated protein kinase and acetyl-CoA carbolxylase phosphorylation in skeletal muscle, and increased hepatic and plasma fibroblast growth factor 21 levels. Importantly, empagliflozin increased energy expenditure, heat production, and the expression of uncoupling protein 1 in brown fat and in inguinal and epididymal white adipose tissue (WAT). Furthermore, empagliflozin reduced M1-polarized macrophage accumulation while inducing the anti-inflammatory M2 phenotype of macrophages within WAT and liver, lowering plasma TNFα levels and attenuating obesity-related chronic inflammation. Thus, empagliflozin suppressed weight gain by enhancing fat utilization and browning and attenuated obesity-induced inflammation and insulin resistance by polarizing M2 macrophages in WAT and liver.

The SGLT2 inhibitor empagliflozin improves the primary diabetic complications in ZDF rats.

Hyperglycemia associated with inflammation and oxidative stress is a major cause of vascular dysfunction and cardiovascular disease in diabetes. Recent data reports that a selective sodium-glucose co-transporter 2 inhibitor (SGLT2i), empagliflozin (Jardiance®), ameliorates glucotoxicity via excretion of excess glucose in urine (glucosuria) and significantly improves cardiovascular mortality in type 2 diabetes mellitus (T2DM). The overarching hypothesis is that hyperglycemia and glucotoxicity are upstream of all other complications seen in diabetes. The aim of this study was to investigate effects of empagliflozin on glucotoxicity, ß-cell function, inflammation, oxidative stress and endothelial dysfunction in Zucker diabetic fatty (ZDF) rats. Male ZDF rats were used as a model of T2DM (35 diabetic ZDF-Leprfa/fa and 16 ZDF-Lepr+/+ controls). Empagliflozin (10 and 30mg/kg/d) was administered via drinking water for 6 weeks. Treatment with empagliflozin restored glycemic control. Empagliflozin improved endothelial function (thoracic aorta) and reduced oxidative stress in the aorta and in blood of diabetic rats. Inflammation and glucotoxicity (AGE/RAGE signaling) were epigenetically prevented by SGLT2i treatment (ChIP). Linear regression analysis revealed a significant inverse correlation of endothelial function with HbA1c, whereas leukocyte-dependent oxidative burst and C-reactive protein (CRP) were positively correlated with HbA1c. Viability of hyperglycemic endothelial cells was pleiotropically improved by SGLT2i. Empagliflozin reduces glucotoxicity and thereby prevents the development of endothelial dysfunction, reduces oxidative stress and exhibits anti-inflammatory effects in ZDF rats, despite persisting hyperlipidemia and hyperinsulinemia. Our preclinical observations provide insights into the mechanisms by which empagliflozin reduces cardiovascular mortality in humans (EMPA-REG trial).

CV Protection in the EMPA-REG OUTCOME Trial: A "Thrifty Substrate" Hypothesis.

The striking and unexpected relative risk reductions in cardiovascular (CV) mortality (38%), hospitalization for heart failure (35%), and death from any cause (32%) observed in the EMPA-REG OUTCOME trial using an inhibitor of sodium-glucose cotransporter 2 (SGLT2) in patients with type 2 diabetes and high CV risk have raised the possibility that mechanisms other than those observed in the trial-modest improvement in glycemic control, small decrease in body weight, and persistent reductions in blood pressure and uric acid level-may be at play. We hypothesize that under conditions of mild, persistent hyperketonemia, such as those that prevail during treatment with SGLT2 inhibitors, ß-hydroxybutyrate is freely taken up by the heart (among other organs) and oxidized in preference to fatty acids. This fuel selection improves the transduction of oxygen consumption into work efficiency at the mitochondrial level. In addition, the hemoconcentration that typically follows SGLT2 inhibition enhances oxygen release to the tissues, thereby establishing a powerful synergy with the metabolic substrate shift. These mechanisms would cooperate with other SGLT2 inhibition-induced changes (chiefly, enhanced diuresis and reduced blood pressure) to achieve the degree of cardioprotection revealed in the EMPA-REG OUTCOME trial. This hypothesis opens up new lines of investigation into the pathogenesis and treatment of diabetic and nondiabetic heart disease.