SGLT1 participates in the development of vascular cognitive impairment in a mouse model of small vessel disease.

Sodium/glucose cotransporter 1 (SGLT1) participates in ischemia-reperfusion-induced cerebral injury. However, whether SGLT1 participates in the development of small vessel disease induced-vascular cognitive impairment is unknown. We examined the roles of SGLT1 in the development of vascular cognitive impairment in a mouse model of small vessel disease. Small vessel disease was created by placement of an ameroid constrictor around the right common carotid artery (CCA) and placement of a microcoil around the left CCA (ACAS) in wild-type (WT) and SGLT1-knock out (KO) mice. Two and/or 4 weeks after ACAS, all experiments were performed. Hematoxylin/eosin staining demonstrated that the number of pyknotic cell deaths was greater in the ACAS WT than ACAS SGLT1-KO hippocampus. The latency to fall in a wire hang test was significantly shorter in ACAS than sham-operated WT mice, whereas it was similar between ACAS and sham-operated SGLT1-KO mice. The Morris water maze test revealed that ACAS WT mice exhibited longer escape latencies than ACAS SGLT1-KO mice. ACAS significantly increased SGLT1 gene expression in WT mouse brains. Gene expressions of MCP-1, IL-1ß, TNF-α, and IL-6 were increased in ACAS WT compared with sham-operated WT mouse brains. Their increased gene expressions were significantly decreased in ACAS SGLT1-KO compared with ACAS WT mice. These results suggest that SGLT1 plays important roles in the development of small vessel dementia.

Knockout of Na+-glucose cotransporter SGLT1 mitigates diabetes-induced upregulation of nitric oxide synthase NOS1 in the macula densa and glomerular hyperfiltration.

Na+-glucose cotransporter (SGLT)1 mediates glucose reabsorption in late proximal tubules. SGLT1 also mediates macula densa (MD) sensing of an increase in luminal glucose, which increases nitric oxide (NO) synthase 1 (MD-NOS1)-mediated NO formation and potentially glomerular filtratrion rate (GFR). Here, the contribution of SGLT1 was tested by gene knockout (-/-) in type 1 diabetic Akita mice. A low-glucose diet was used to prevent intestinal malabsorption in Sglt1-/- mice and minimize the contribution of intestinal SGLT1. Hyperglycemia was modestly reduced in Sglt1-/- versus littermate wild-type Akita mice (480 vs. 550 mg/dl), associated with reduced diabetes-induced increases in GFR, kidney weight, glomerular size, and albuminuria. Blunted hyperfiltration was confirmed in streptozotocin-induced diabetic Sglt1-/- mice, associated with similar hyperglycemia versus wild-type mice (350 vs. 385 mg/dl). Absence of SGLT1 attenuated upregulation of MD-NOS1 protein expression in diabetic Akita mice and in response to SGLT2 inhibition in nondiabetic mice. During SGLT2 inhibition in Akita mice, Sglt1-/- mice had likewise reduced blood glucose (200 vs. 300 mg/dl), associated with lesser MD-NOS1 expression, GFR, kidney weight, glomerular size, and albuminuria. Absence of Sglt1 in Akita mice increased systolic blood pressure, associated with suppressed renal renin mRNA expression. This may reflect fluid retention due to blunted hyperfiltration. SGLT2 inhibition prevented the blood pressure increase in Sglt1-/- Akita mice, possibly due to additive glucosuric/diuretic effects. The data indicate that SGLT1 contributes to diabetic hyperfiltration and limits diabetic hypertension. Potential mechanisms include its role in glucose-driven upregulation of MD-NOS1 expression. This pathway may increase GFR to maintain volume balance when enhanced MD glucose delivery indicates upstream saturation of SGLTs and thus hyperreabsorption.

Gene deletion of the Na+-glucose cotransporter SGLT1 ameliorates kidney recovery in a murine model of acute kidney injury induced by ischemia-reperfusion.

Renal Na+-glucose cotransporter SGLT1 mediates glucose reabsorption in the late proximal tubule, a hypoxia-sensitive tubular segment that enters the outer medulla. Gene deletion in mice (Sglt1-/-) was used to determine the role of the cotransporter in acute kidney injury induced by ischemia-reperfusion (IR), including the initial injury and subsequent recovery phase. On days 1 and 16 after IR, absolute and fractional urinary glucose excretion remained greater in Sglt1-/- mice versus wild-type (WT) littermates, consistent with a sustained contribution of SGLT1 to tubular glucose reabsorption in WT mice. Absence of SGLT1 did not affect the initial kidney impairment versus WT mice, as indicated by similar increases on day 1 in plasma concentrations of creatinine and urinary excretion of the tubular injury marker kidney injury molecule-1 as well as a similar rise in plasma osmolality and fall in urine osmolality as indicators of impaired urine concentration. Recovery of kidney function on days 14/16, however, was improved in Sglt1-/- versus WT mice, as indicated by lower plasma creatinine, higher glomerula filtration rate (by FITC-sinistrin in awake mice), and more completely restored urine and plasma osmolality. This was associated with a reduced tubular injury score in the cortex and outer medulla, better preserved renal mRNA expression of tubular transporters (Sglt2 and Na+-K+-2Cl- cotransporter Nkcc2), and a lesser rise in renal mRNA expression of markers of injury, inflammation, and fibrosis [kidney injury molecule-1, chemokine (C-C motif) ligand 2, fibronectin 1, and collagen type I-α1] in Sglt1-/- versus WT mice. These results suggest that SGLT1 activity in the late proximal tubule may have deleterious effects during recovery of IR-induced acute kidney injury and identify SGLT1 as a potential therapeutic target.

Cardiac sodium-dependent glucose cotransporter 1 is a novel mediator of ischaemia/reperfusion injury.

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.

Altered pancreatic islet morphology and function in SGLT1 knockout mice on a glucose-deficient, fat-enriched diet.

OBJECTIVES: Glycemic control by medical treatment represents one therapeutic strategy for diabetic patients. The Na+-d-glucose cotransporter 1 (SGLT1) is currently of high interest in this context. SGLT1 is known to mediate glucose absorption and incretin secretion in the small intestine. Recently, inhibition of SGLT1 function was shown to improve postprandial hyperglycemia. In view of the lately demonstrated SGLT1 expression in pancreatic islets, we investigated if loss of SGLT1 affects islet morphology and function. METHODS: Effects associated with the loss of SGLT1 on pancreatic islet (cyto) morphology and function were investigated by analyzing islets of a SGLT1 knockout mouse model, that were fed a glucose-deficient, fat-enriched diet (SGLT1-/--GDFE) to circumvent the glucose-galactose malabsorption syndrome. To distinguish diet- and Sglt1-/--dependent effects, wildtype mice on either standard chow (WT-SC) or the glucose-free, fat-enriched diet (WT-GDFE) were used as controls. Feeding a glucose-deficient, fat-enriched diet further required the analysis of intestinal SGLT1 expression and function under diet-conditions. RESULTS: Consistent with literature, our data provide evidence that small intestinal SGLT1 mRNA expression and function is regulated by nutrition. In contrast, pancreatic SGLT1 mRNA levels were not affected by the applied diet, suggesting different regulatory mechanisms for SGLT1 in diverse tissues. Morphological changes such as increased islet sizes and cell numbers associated with changes in proliferation and apoptosis and alterations of the ß- and α-cell population are specifically observed for pancreatic islets of SGLT1-/--GDFE mice. Glucose stimulation revealed no insulin response in SGLT1-/--GDFE mice while WT-GDFE mice displayed only a minor increase of blood insulin. Irregular glucagon responses were observed for both, SGLT1-/--GDFE and WT-GDFE mice. Further, both animal groups showed a sustained release of GLP-1 compared to WT-SC controls. CONCLUSION: Loss or impairment of SGLT1 results in abnormal pancreatic islet (cyto)morphology and disturbed islet function regarding the insulin or glucagon release capacity from ß- or α-cells, respectively. Consequently, our findings propose a new, additional role for SGLT1 maintaining proper islet structure and function.

SGLT1 Deficiency Turns Listeria Infection into a Lethal Disease in Mice.

BACKGROUND: Cellular glucose uptake may involve either non-concentrative glucose carriers of the GLUT family or Na+-coupled glucose-carrier SGLT1, which accumulates glucose against glucose gradients and may thus accomplish cellular glucose uptake even at dramatically decreased extracellular glucose concentrations. SGLT1 is not only expressed in epithelia but as well in tumour cells and immune cells. Immune cell functions strongly depend on their metabolism, therefore we hypothesized that deficiency of SGLT1 modulates the defence against bacterial infection. To test this hypothesis, we infected wild type mice and gene targeted mice lacking functional SGLT1 with Listeria monocytogenes. METHODS: SGLT1 deficient mice and wild type littermates were infected with 1x104 CFU Listeria monocytogenes intravenously. Bacterial titers were determined by colony forming assay, SGLT1, TNF-α, IL-6 and IL-12a transcript levels were determined by qRT-PCR, as well as SGLT1 protein abundance and localization by immunohistochemistry. RESULTS: Genetic knockout of SGLT1 (Slc5a1-/- mice) significantly compromised bacterial clearance following Listeria monocytogenes infection with significantly enhanced bacterial load in liver, spleen, kidney and lung, and significantly augmented hepatic expression of TNF-α and IL-12a. While all wild type mice survived, all SGLT1 deficient mice died from the infection. CONCLUSIONS: SGLT1 is required for bacterial clearance and host survival following murine Listeria infection.

Sodium influx through cerebral sodium-glucose transporter type 1 exacerbates the development of cerebral ischemic neuronal damage.

We recently reported that cerebral sodium-glucose transporter type 1 (SGLT-1) plays a role in exacerbation of cerebral ischemia. However, the mechanism by which cerebral SGLT-1 acts remains unclear. Here we demonstrated that sodium influx through cerebral SGLT-1 exacerbates cerebral ischemic neuronal damage. SGLT-specific sodium ion influx was induced using α-methyl-D-glucopyranoside (α-MG). Intracellular sodium concentrations in primary cortical neurons were estimated using sodium-binding benzofuran isophthalate fluorescence. SGLT-1 knockdown in primary cortical neurons and mice was achieved using SGLT-1 siRNA. The survival rates of primary cultured cortical neurons were assessed using biochemical assays 1 day after treatment. Middle cerebral artery occlusion (MCAO) was used to generate a focal cerebral ischemic model in SGLT-1 knockdown mice. The change in fasting blood glucose levels, infarction development, and behavioral abnormalities were assessed 1 day after MCAO. Treatment with 200mM α-MG induced a continuous increase in the intracellular sodium concentration, and this increase was normalized after α-MG removal. Neuronal SGLT-1 knockdown had no effect on 100µM H2O2-induced neuronal cell death; however, the knockdown prevented the neuronal cell death induced by 17.5mM glucose and the co-treatment of 100µM H2O2/8.75mM glucose. Neuronal SGLT-1 knockdown also suppressed the cell death induced by α-MG alone and the co-treatment of 100µM H2O2/0.01mM α-MG. Our in vivo results showed that the exacerbation of cerebral ischemic neuronal damage induced by the intracerebroventricular administration of 5.0µg α-MG/mouse was ameliorated in cerebral SGLT-1 knockdown mice. Thus, sodium influx through cerebral SGLT-1 may exacerbate cerebral ischemia-induced neuronal damage.

The role of SGLT1 and GLUT2 in intestinal glucose transport and sensing.

Intestinal glucose absorption is mediated by SGLT1 whereas GLUT2 is considered to provide basolateral exit. Recently, it was proposed that GLUT2 can be recruited into the apical membrane after a high luminal glucose bolus allowing bulk absorption of glucose by facilitated diffusion. Moreover, SGLT1 and GLUT2 are suggested to play an important role in intestinal glucose sensing and incretin secretion. In mice that lack either SGLT1 or GLUT2 we re-assessed the role of these transporters in intestinal glucose uptake after radiotracer glucose gavage and performed Western blot analysis for transporter abundance in apical membrane fractions in a comparative approach. Moreover, we examined the contribution of these transporters to glucose-induced changes in plasma GIP, GLP-1 and insulin levels. In mice lacking SGLT1, tissue retention of tracer glucose was drastically reduced throughout the entire small intestine whereas GLUT2-deficient animals exhibited higher tracer contents in tissue samples than wild type animals. Deletion of SGLT1 resulted also in reduced blood glucose elevations and abolished GIP and GLP-1 secretion in response to glucose. In mice lacking GLUT2, glucose-induced insulin but not incretin secretion was impaired. Western blot analysis revealed unchanged protein levels of SGLT1 after glucose gavage. GLUT2 detected in apical membrane fractions mainly resulted from contamination with basolateral membranes but did not change in density after glucose administration. SGLT1 is unequivocally the prime intestinal glucose transporter even at high luminal glucose concentrations. Moreover, SGLT1 mediates glucose-induced incretin secretion. Our studies do not provide evidence for GLUT2 playing any role in either apical glucose influx or incretin secretion.

Homozygote inositol transporter knockout mice show a lithium-like phenotype.

OBJECTIVE: Lithium inhibits inositol monophosphatase and also reduces inositol transporter function. To determine if one or more of these mechanisms might underlie the behavioral effects of lithium, we studied inositol transporter knockout mice. We previously reported that heterozygous knockout mice with reduction of 15-37% in brain inositol had no abnormalities of pilocarpine sensitivity or antidepressant-like behavior in the Porsolt forced swim test. We now report on studies of homozygous inositol transporter knockout mice. METHODS: Homozygote knockout mice were rescued by 2% inositol supplementation to the drinking water of the dam mice through pregnancy and lactation. Genotyping was carried out by polymerase chain reaction followed by agarose electrophoresis. Brain free myo-inositol levels were determined gas-chromatographically. Motor activity and coordination were assessed by the rotarod test. Behavior of the mice was studied in lithium-pilocarpine seizure models for lithium action and in the Porsolt forced swim test model for depression. RESULTS: In homozygote knockout mice, free inositol levels were reduced by 55% in the frontal cortex and by 60% in the hippocampus. There were no differences in weight or motor coordination by the rotarod test. They behaved similarly to lithium-treated animals in the model of pilocarpine seizures and in the Porsolt forced swimming test model of depression. CONCLUSIONS: Reduction of brain inositol more than 15-37% may be required to elicit lithium-like neurobehavioral effects.