Sodium-myoinositol cotransporter-1, SMIT1, mediates the production of reactive oxygen species induced by hyperglycemia in the heart.
ABSTRACT: Hyperglycemia (HG) stimulates the production of reactive oxygen species in the heart through activation of NADPH oxidase 2 (NOX2). This production is independent of glucose metabolism but requires sodium/glucose cotransporters (SGLT). Seven SGLT isoforms (SGLT1 to 6 and sodium-myoinositol cotransporter-1, SMIT1) are known, although their expression and function in the heart remain elusive. We investigated these 7 isoforms and found that only SGLT1 and SMIT1 were expressed in mouse, rat and human hearts. In cardiomyocytes, galactose (transported through SGLT1) did not activate NOX2. Accordingly, SGLT1 deficiency did not prevent HG-induced NOX2 activation, ruling it out in the cellular response to HG. In contrast, myo-inositol (transported through SMIT1) reproduced the toxic effects of HG. SMIT1 overexpression exacerbated glucotoxicity and sensitized cardiomyocytes to HG, whereas its deletion prevented HG-induced NOX2 activation. In conclusion, our results show that heart SMIT1 senses HG and triggers NOX2 activation. This could participate in the redox signaling in hyperglycemic heart and contribute to the pathophysiology of diabetic cardiomyopathy.
Project description:Empagliflozin, a sodium-glucose co-transporter (SGLT) inhibitor, reduces heart failure and sudden cardiac death but the underlying mechanisms remain elusive. In cardiomyocytes, SGLT1 and SGLT2 expression is upregulated in diabetes mellitus, heart failure, and myocardial infarction. We hypothesise that empagliflozin exerts direct effects on cardiomyocytes that attenuate diabetic cardiomyopathy. To test this hypothesis, cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) were used to test the potential effects of empagliflozin on neutralization of cardiac dysfunction induced by diabetic-like cultures. Our results indicated that insulin-free high glucose culture significantly increased the size of and NPPB, SGLT1 and SGLT2 expression of hiPSC-derived cardiomyocytes. In addition, high glucose-treated hiPSC-derived cardiomyocytes exhibited reduced contractility regardless of the increased calcium transient capacity. Interestingly, application of empagliflozin before or after high glucose treatment effectively reduced the high glucose-induced cardiac abnormalities. Since application of empagliflozin did not significantly alter viability or glycolytic capacity of the hiPSC-derived cardiomyocytes, it is plausible that empagliflozin exerts its effects via the down-regulation of SGLT1, SGLT2 and GLUT1 expression. These observations provide supportive evidence that may help explain its unexpected benefit observed in the EMPA-REG trial.
Project description:AIMS:We previously reported that sodium-dependent glucose cotransporter 1 (SGLT1) is highly expressed in cardiomyocytes and is further up-regulated in ischaemia. This study aimed to determine the mechanisms by which SGLT1 contributes to ischaemia/reperfusion (I/R) injury. METHODS AND RESULTS:Mice with cardiomyocyte-specific knockdown of SGLT1 (TGSGLT1-DOWN) and wild-type controls were studied. In vivo, the left anterior descending coronary artery was ligated for 30?min and reperfused for 48?h. Ex vivo, isolated perfused hearts were exposed to 20?min no-flow and up to 2?h reperfusion. In vitro, HL-1 cells and isolated adult murine ventricular cardiomyocytes were exposed to 1?h hypoxia and 24?h reoxygenation (H/R). We found that TGSGLT1-DOWN hearts were protected from I/R injury in vivo and ex vivo, with decreased infarct size, necrosis, dysfunction, and oxidative stress. 5'-AMP-activated protein kinase (AMPK) activation increased SGLT1 expression, which was abolished by extracellular signal-related kinase (ERK) inhibition. Co-immunoprecipitation studies showed that ERK, but not AMPK, interacts directly with SGLT1. AMPK activation increased binding of the hepatocyte nuclear factor 1 and specificity protein 1 transcription factors to the SGLT1 gene, and HuR to SGLT1 mRNA. In cells, up-regulation of SGLT1 during H/R was abrogated by AMPK inhibition. Co-immunoprecipitation studies showed that SGLT1 interacts with epidermal growth factor receptor (EGFR), and EGFR interacts with protein kinase C (PKC). SGLT1 overexpression activated PKC and NADPH oxidase 2 (Nox2), which was attenuated by PKC inhibition, EGFR inhibition, and/or disruption of the interaction between EGFR and SGLT1. CONCLUSION:During ischaemia, AMPK up-regulates SGLT1 through ERK, and SGLT1 interacts with EGFR, which in turn increases PKC and Nox2 activity and oxidative stress. SGLT1 may represent a novel therapeutic target for mitigating I/R injury.
Project description:Sodium-glucose cotransporter proteins (SGLT) belong to the SLC5A family, characterized by the cotransport of Na(+) with solute. SGLT1 is responsible for intestinal glucose absorption. Until recently the only role described for SGLT proteins was to transport sugar with Na(+). However, human SGLT3 (hSGLT3) does not transport sugar but causes depolarization of the plasma membrane when expressed in Xenopus oocytes. For this reason SGLT3 was suggested to be a sugar sensor rather than a transporter. Despite 70% amino acid identity between hSGLT3 and hSGLT1, their sugar transport, apparent sugar affinities, and sugar specificity differ greatly. Residue 457 is important for the function of SGLT1 and mutation at this position in hSGLT1 causes glucose-galactose malabsorption. Moreover, the crystal structure of vibrio SGLT reveals that the residue corresponding to 457 interacts directly with the sugar molecule. We thus wondered if this residue could account for some of the functional differences between SGLT1 and SGLT3.We mutated the glutamate at position 457 in hSGLT3 to glutamine, the amino acid present in all SGLT1 proteins, and characterized the mutant. Surprisingly, we found that E457Q-hSGLT3 transported sugar, had the same stoichiometry as SGLT1, and that the sugar specificity and apparent affinities for most sugars were similar to hSGLT1. We also show that SGLT3 functions as a sugar sensor in a living organism. We expressed hSGLT3 and E457Q-hSGLT3 in C. elegans sensory neurons and found that animals sensed glucose in an hSGLT3-dependent manner.In summary, we demonstrate that hSGLT3 functions as a sugar sensor in vivo and that mutating a single amino acid converts this sugar sensor into a sugar transporter similar to SGLT1.
Project description:The sodium-glucose cotransporter (SGLT) inhibitors represent a new alternative for treating patients with diabetes mellitus. They act primarily by inhibiting glucose reabsorption in the renal tubule and therefore, decreasing blood glucose levels. While little is yet known about SGLT subtype 1, SGLT2 inhibitors have demonstrated to significantly reduce cardiovascular mortality and heart failure hospitalizations. This cardioprotective benefit seems to be independent of their glucose-lowering properties; however, the underlying mechanism(s) remains still unclear and numerous hypotheses have been postulated to date. Moreover, preclinical research has suggested an important role of SGLT1 receptors on myocardial ischemia. Following acute phase of cardiac injury there is an increased activity of SGLT1 cotransport that ensures adequate energy supply to the cardiac cells. Nonetheless, a long-term upregulation of this receptor may not be that beneficial and whether its inhibition is positive or not should be further addressed. This review aims to present the most cutting-edge insights into SGLT receptors.
Project description:Obesity and high saturated fat intake increase the risk of heart failure and arrhythmias. The molecular mechanisms are poorly understood. We hypothesized that physiologic levels of saturated fat could increase mitochondrial reactive oxygen species (ROS) in cardiomyocytes, leading to abnormalities of calcium homeostasis and mitochondrial function. We investigated the effect of saturated fat on mitochondrial function and calcium homeostasis in isolated ventricular myocytes. The saturated fatty acid palmitate causes a decrease in mitochondrial respiration in cardiomyocytes. Palmitate, but not the monounsaturated fatty acid oleate, causes an increase in both total cellular ROS and mitochondrial ROS. Palmitate depolarizes the mitochondrial inner membrane and causes mitochondrial calcium overload by increasing sarcoplasmic reticulum calcium leak. Inhibitors of PKC or NOX2 prevent mitochondrial dysfunction and the increase in ROS, demonstrating that PKC-NOX2 activation is also required for amplification of palmitate induced-ROS. Cardiomyocytes from mice with genetic deletion of NOX2 do not have palmitate-induced ROS or mitochondrial dysfunction. We conclude that palmitate induces mitochondrial ROS that is amplified by NOX2, causing greater mitochondrial ROS generation and partial depolarization of the mitochondrial inner membrane. The abnormal sarcoplasmic reticulum calcium leak caused by palmitate could promote arrhythmia and heart failure. NOX2 inhibition is a potential therapy for heart disease caused by diabetes or obesity.
Project description:Na(+)-glucose cotransporter 1 (SGLT1)-mediated glucose uptake leads to activation of Na(+)-H(+) exchanger 3 (NHE3) in the intestine by a process that is not dependent on glucose metabolism. This coactivation may be important for postprandial nutrient uptake. However, it remains to be determined whether SGLT-mediated glucose uptake regulates NHE3-mediated NaHCO3 reabsorption in the renal proximal tubule. Considering that this nephron segment also expresses SGLT2 and that the kidneys and intestine show significant variations in daily glucose availability, the goal of this study was to determine the effect of SGLT-mediated glucose uptake on NHE3 activity in the renal proximal tubule. Stationary in vivo microperfusion experiments showed that luminal perfusion with 5 mM glucose stimulates NHE3-mediated bicarbonate reabsorption. This stimulatory effect was mediated by glycolytic metabolism but not through ATP production. Conversely, luminal perfusion with 40 mM glucose inhibited NHE3 because of cell swelling. Notably, pharmacologic inhibition of SGLT activity by Phlorizin produced a marked inhibition of NHE3, even in the absence of glucose. Furthermore, immunofluorescence experiments showed that NHE3 colocalizes with SGLT2 but not SGLT1 in the rat renal proximal tubule. Collectively, these findings show that glucose exerts a bimodal effect on NHE3. The physiologic metabolism of glucose stimulates NHE3 transport activity, whereas, supraphysiologic glucose concentrations inhibit this exchanger. Additionally, Phlorizin-sensitive SGLT transporters and NHE3 interact functionally in the proximal tubule.
Project description:The highly expressed cell adhesion receptor CD29 (?(1)-integrin) is essential for cardiomyocyte growth and survival, and its loss of function causes severe heart disease. However, CD29-induced signalling in cardiomyocytes is ill defined and may involve reactive oxygen species (ROS). A decisive source of cardiac ROS is the abundant NADPH oxidase (NOX) isoform NOX2. Because understanding of NOX-derived ROS in the heart is still poor, we sought to test the role of ROS and NOX in CD29-induced survival signalling in cardiomyocytes.In neonatal rat ventricular myocytes, CD29 activation induced intracellular ROS formation (oxidative burst) as assessed by flow cytometry using the redox-sensitive fluorescent dye dichlorodihydrofluorescein diacetate. This burst was inhibited by apocynin and diphenylene iodonium. Further, activation of CD29 enhanced NOX activity (lucigenin-enhanced chemiluminescence) and activated the MEK/ERK and PI3K/Akt survival pathways. CD29 also induced phosphorylation of the inhibitory Ser9 on the pro-apoptotic kinase glycogen synthase kinase-3? in a PI3K/Akt- and MEK-dependent manner, and improved cardiomyocyte viability under conditions of oxidative stress. The ROS scavenger MnTMPyP or adenoviral co-overexpression of the antioxidant enzymes superoxide dismutase and catalase inhibited CD29-induced pro-survival signalling. Further, CD29-induced protective pathways were lost in mouse cardiomyocytes deficient for NOX2 or functional p47(phox), a regulatory subunit of NOX.p47(phox)-dependent, NOX2-derived ROS are mandatory for CD29-induced pro-survival signalling in cardiomyocytes. These findings go in line with a growing body of evidence suggesting that ROS can be beneficial to the cell and support a crucial role for NOX2-derived ROS in cell survival in the heart.
Project description:Glucose is a major metabolic substrate required for cancer cell survival and growth. It is mainly imported into cells by facilitated glucose transporters (GLUTs). Here we demonstrate the importance of another glucose import system, the sodium-dependent glucose transporters (SGLTs), in pancreatic and prostate adenocarcinomas, and investigate their role in cancer cell survival. Three experimental approaches were used: (i) immunohistochemical mapping of SGLT1 and SGLT2 distribution in tumors; (ii) measurement of glucose uptake in fresh isolated tumors using an SGLT-specific radioactive glucose analog, ?-methyl-4-deoxy-4-[(18)F]fluoro-D-glucopyranoside (Me4FDG), which is not transported by GLUTs; and (iii) measurement of in vivo SGLT activity in mouse models of pancreatic and prostate cancer using Me4FDG-PET imaging. We found that SGLT2 is functionally expressed in pancreatic and prostate adenocarcinomas, and provide evidence that SGLT2 inhibitors block glucose uptake and reduce tumor growth and survival in a xenograft model of pancreatic cancer. We suggest that Me4FDG-PET imaging may be used to diagnose and stage pancreatic and prostate cancers, and that SGLT2 inhibitors, currently in use for treating diabetes, may be useful for cancer therapy.
Project description:Salusin-? accelerates inflammatory responses in vascular endothelial cells, and increases oxidative stress in vascular smooth muscle cells. Plasma salusin-? levels were increased in diabetic patients. This study was designed to determine whether salusin-? is involved in the pathogenesis of diabetic cardiomyopathy (DCM), and whether knockdown of salusin-? attenuates cardiac inflammation and oxidative stress in rats with DCM. H9c2 or neonatal rat cardiomyocytes were incubated with 33.3?mM of glucose to mimic the high glucose (HG) in diabetes. Streptozotocin and high-fat diet were used to induce type 2 diabetes in rats. HG induced salusin-? expression in H9c2 cells. Salusin-? caused greater responses of oxidative stress, NF?B activation and inflammation in HG-treated H9c2 cells than these in control H9c2 cells. Diphenyleneiodonium (a NAD(P)H oxidase inhibitor) or N-acetylcysteine (an antioxidant) inhibited the salusin-?-induced NF?B activation and inflammation. Bay11-7082 (a NF?B inhibitor) attenuated salusin-?-induced inflammation but not oxidative stress. Knockdown of salusin-? prevented the HG-induced oxidative stress, NF?B activation and inflammation in neonatal rat cardiomyocytes. Silencing salusin-? with adenoviruse-mediated shRNA had no significant effects on blood glucose and insulin resistance, but attenuated ventricular dysfunction in diabetic rats. Oxidative stress, NF?B activation, inflammation, salusin-? upregulation in myocardium of diabetic rats were prevented by knockdown of salusin-?. These results indicate that salusin-? contributes to inflammation in DCM via NOX2/ROS/NF?B signaling, and that knockdown of salusin-? attenuates cardiac dysfunction, oxidative stress and inflammation in DCM.
Project description:Renin-angiotensin system activation is a feature of many cardiovascular conditions. Activity of myocardial reduced nicotinamide adenine dinucleotide phosphate oxidase 2 (NADPH oxidase 2 or Nox2) is enhanced by angiotensin II (Ang II) and contributes to increased hypertrophy, fibrosis, and adverse remodeling. Recent studies found that Nox2-mediated reactive oxygen species production modulates physiological cardiomyocyte function.This study sought to investigate the effects of cardiomyocyte Nox2 on contractile function during increased Ang II activation.We generated a cardiomyocyte-targeted Nox2-transgenic mouse model and studied the effects of in vivo and ex vivo Ang II stimulation, as well as chronic aortic banding.Chronic subpressor Ang II infusion induced greater cardiac hypertrophy in transgenic than wild-type mice but unexpectedly enhanced contractile function. Acute Ang II treatment also enhanced contractile function in transgenic hearts in vivo and transgenic cardiomyocytes ex vivo. Ang II-stimulated Nox2 activity increased sarcoplasmic reticulum (SR) Ca(2+) uptake in transgenic mice, increased the Ca(2+) transient and contractile amplitude, and accelerated cardiomyocyte contraction and relaxation. Elevated Nox2 activity increased phospholamban phosphorylation in both hearts and cardiomyocytes, related to inhibition of protein phosphatase 1 activity. In a model of aortic banding-induced chronic pressure overload, heart function was similarly depressed in transgenic and wild-type mice.We identified a novel mechanism in which Nox2 modulates cardiomyocyte SR Ca(2+) uptake and contractile function through redox-regulated changes in phospholamban phosphorylation. This mechanism can drive increased contractility in the short term in disease states characterized by enhanced renin-angiotensin system activation.