RESUMEN
The Sodium Glucose Cotransporter Isoform 1 (Sglt-1) is a symporter that moves Na+ and glucose into the cell. While most studies have focused on the role of Sglt-1 in the small intestine and kidney, little is known about this transporter's expression and function in other tissues. We have previously shown that Sglt-1 is expressed in the mouse sperm flagellum and that its inhibition interferes with sperm metabolism and function. Here, we further investigated the importance of Sglt-1 in sperm, using a Sglt-1 knockout mouse (Sglt-1 KO). RNA, immunocytochemistry, and glucose uptake analysis confirmed the ablation of Sglt-1 in sperm. Sglt-1 KO male mice are fertile and exhibit normal sperm counts and morphology. However, Sglt-1 null sperm displayed a significant reduction in total, progressive and other parameters of sperm motility compared to wild type (WT) sperm. The reduction in motility was exacerbated when sperm were challenged to swim in media with higher viscosity. Parameters of capacitation, namely protein tyrosine phosphorylation and acrosomal reaction, were similar in Sglt-1 KO and WT sperm. However, Sglt-1 KO sperm displayed a significant decrease in hyperactivation. The impaired motility of Sglt-1 null sperm was observed in media containing glucose as the only energy substrate. Interestingly, the addition of pyruvate and lactate to the media partially recovered sperm motility of Sglt-1 KO sperm, both in the low and high viscosity media. Altogether, these results support an important role for Sglt-1 in sperm energetics and function, providing sperm with a higher capacity for glucose uptake.
Asunto(s)
Transportador 1 de Sodio-Glucosa , Motilidad Espermática , Animales , Masculino , Ratones , Glucosa/metabolismo , Ratones Noqueados , Semen/metabolismo , Transportador 1 de Sodio-Glucosa/genética , Transportador 1 de Sodio-Glucosa/metabolismo , Capacitación Espermática/fisiología , Motilidad Espermática/fisiología , Espermatozoides/metabolismoRESUMEN
The organic cation transporters (OCTs) OCT1, OCT2, OCT3, novel OCT (OCTN)1, OCTN2, multidrug and toxin exclusion (MATE)1, and MATE kidney-specific 2 are polyspecific transporters exhibiting broadly overlapping substrate selectivities. They transport organic cations, zwitterions, and some uncharged compounds and operate as facilitated diffusion systems and/or antiporters. OCTs are critically involved in intestinal absorption, hepatic uptake, and renal excretion of hydrophilic drugs. They modulate the distribution of endogenous compounds such as thiamine, L-carnitine, and neurotransmitters. Sites of expression and functions of OCTs have important impact on energy metabolism, pharmacokinetics, and toxicity of drugs, and on drug-drug interactions. In this work, an overview about the human OCTs is presented. Functional properties of human OCTs, including identified substrates and inhibitors of the individual transporters, are described. Sites of expression are compiled, and data on regulation of OCTs are presented. In addition, genetic variations of OCTs are listed, and data on their impact on transport, drug treatment, and diseases are reported. Moreover, recent data are summarized that indicate complex drug-drug interaction at OCTs, such as allosteric high-affinity inhibition of transport and substrate dependence of inhibitor efficacies. A hypothesis about the molecular mechanism of polyspecific substrate recognition by OCTs is presented that is based on functional studies and mutagenesis experiments in OCT1 and OCT2. This hypothesis provides a framework to imagine how observed complex drug-drug interactions at OCTs arise. Finally, preclinical in vitro tests that are performed by pharmaceutical companies to identify interaction of novel drugs with OCTs are discussed. Optimized experimental procedures are proposed that allow a gapless detection of inhibitory and transported drugs.
Asunto(s)
Proteínas de Transporte de Catión Orgánico/metabolismo , Animales , Humanos , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Especificidad por SustratoRESUMEN
An increase of glomerular filtration rate (GFR) is a common observation in early diabetes and is considered a key risk factor for subsequent kidney injury. However, the mechanisms underlying diabetic hyperfiltration have not been fully clarified. Here, we tested the hypothesis that macula densa neuronal nitric oxide synthase (NOS1) is upregulated via sodium glucose cotransporter type 1 (SGLT1) in diabetes, which then inhibits tubuloglomerular feedback (TGF) promoting glomerular hyperfiltration. Therefore, we examined changes in cortical NOS1 expression and phosphorylation, nitric oxide production in the macula densa, TGF response, and GFR during the early stage of insulin-deficient (Akita) diabetes in wild-type and macula densa-specific NOS1 knockout mice. A set of sophisticated techniques including microperfusion of juxtaglomerular apparatus in vitro, micropuncture of kidney tubules in vivo, and clearance kinetics of plasma fluorescent-sinistrin were employed. Complementary studies tested the role of SGLT1 in SGLT1 knockout mice and explored NOS1 expression and phosphorylation in kidney biopsies of cadaveric donors. Diabetic mice had upregulated macula densa NOS1, inhibited TGF and elevated GFR. Macula densa-selective NOS1 knockout attenuated the diabetes-induced TGF inhibition and GFR elevation. Additionally, deletion of SGLT1 prevented the upregulation of macula densa NOS1 and attenuated inhibition of TGF in diabetic mice. Furthermore, the expression and phosphorylation levels of NOS1 were increased in cadaveric kidneys of diabetics and positively correlated with blood glucose as well as estimated GFR in the donors. Thus, our findings demonstrate that the macula densa SGLT1-NOS1-TGF pathway plays a crucial role in the control of GFR in diabetes.
Asunto(s)
Diabetes Mellitus Experimental , Transportador 1 de Sodio-Glucosa/metabolismo , Animales , Diabetes Mellitus Experimental/metabolismo , Retroalimentación , Tasa de Filtración Glomerular/fisiología , Glomérulos Renales/metabolismo , Túbulos Renales/metabolismo , Ratones , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo I/metabolismoRESUMEN
Although sodium glucose cotransporter 1 (SGLT1) has been identified as one of the major SGLT isoforms expressed in the heart, its exact role remains elusive. Evidence using phlorizin, the most common inhibitor of SGLTs, has suggested its role in glucose transport. However, phlorizin could also affect classical facilitated diffusion via glucose transporters (GLUTs), bringing into question the relevance of SGLT1 in overall cardiac glucose uptake. Accordingly, we assessed the contribution of SGLT1 in cardiac glucose uptake using the SGLT1 knockout mouse model, which lacks exon 1. Glucose uptake was similar in cardiomyocytes isolated from SGLT1-knockout (Δex1KO) and control littermate (WT) mice either under basal state, insulin, or hyperglycemia. Similarly, in vivo basal and insulin-stimulated cardiac glucose transport measured by micro-PET scan technology did not differ between WT and Δex1KO mice. Micromolar concentrations of phlorizin had no impact on glucose uptake in either isolated WT or Δex1KO-derived cardiomyocytes. However, higher concentrations (1 mM) completely inhibited insulin-stimulated glucose transport without affecting insulin signaling nor GLUT4 translocation independently from cardiomyocyte genotype. Interestingly, we discovered that mouse and human hearts expressed a shorter slc5a1 transcript, leading to SGLT1 protein lacking transmembrane domains and residues involved in glucose and sodium bindings. In conclusion, cardiac SGLT1 does not contribute to overall glucose uptake, probably due to the expression of slc5a1 transcript variant. The inhibitory effect of phlorizin on cardiac glucose uptake is SGLT1-independent and can be explained by GLUT transporter inhibition. These data open new perspectives in understanding the role of SGLT1 in the heart.NEW & NOTEWORTHY Ever since the discovery of its expression in the heart, SGLT1 has been considered as similar as the intestine and a potential contributor to cardiac glucose transport. For the first time, we have demonstrated that a slc5a1 transcript variant is present in the heart that has no significant impact on cardiac glucose handling.
Asunto(s)
Glucosa/metabolismo , Miocitos Cardíacos/metabolismo , Transportador 1 de Sodio-Glucosa/metabolismo , Animales , Transporte Biológico , Células Cultivadas , Transportador de Glucosa de Tipo 4/antagonistas & inhibidores , Transportador de Glucosa de Tipo 4/metabolismo , Hipoglucemiantes/farmacología , Insulina/farmacología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Florizina/farmacología , Isoformas de Proteínas , Ratas Wistar , Transportador 1 de Sodio-Glucosa/antagonistas & inhibidores , Transportador 1 de Sodio-Glucosa/genéticaRESUMEN
Inhibitors of Na+/Cl- dependent high affinity transporters for norepinephrine (NE), serotonin (5-HT), and/or dopamine (DA) represent frequently used drugs for treatment of psychological disorders such as depression, anxiety, obsessive-compulsive disorder, attention deficit hyperactivity disorder, and addiction. These transporters remove NE, 5-HT, and/or DA after neuronal excitation from the interstitial space close to the synapses. Thereby they terminate transmission and modulate neuronal behavioral circuits. Therapeutic failure and undesired central nervous system side effects of these drugs have been partially assigned to neurotransmitter removal by low affinity transport. Cloning and functional characterization of the polyspecific organic cation transporters OCT1 (SLC22A1), OCT2 (SLC22A2), OCT3 (SLC22A3) and the plasma membrane monoamine transporter PMAT (SLC29A4) revealed that every single transporter mediates low affinity uptake of NE, 5-HT, and DA. Whereas the organic transporters are all located in the blood brain barrier, OCT2, OCT3, and PMAT are expressed in neurons or in neurons and astrocytes within brain areas that are involved in behavioral regulation. Areas of expression include the dorsal raphe, medullary motoric nuclei, hypothalamic nuclei, and/or the nucleus accumbens. Current knowledge of the transport of monoamine neurotransmitters by the organic cation transporters, their interactions with psychotropic drugs, and their locations in the brain is reported in detail. In addition, animal experiments including behavior tests in wildtype and knockout animals are reported in which the impact of OCT2, OCT3, and/or PMAT on regulation of salt intake, depression, mood control, locomotion, and/or stress effect on addiction is suggested.
Asunto(s)
Encéfalo , Proteínas de Transporte de Catión Orgánico , Animales , Transporte Biológico , Encéfalo/metabolismo , Cationes , Norepinefrina , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismoRESUMEN
Absorption of monosaccharides is mainly mediated by Na+-D-glucose cotransporter SGLT1 and the facititative transporters GLUT2 and GLUT5. SGLT1 and GLUT2 are relevant for absorption of D-glucose and D-galactose while GLUT5 is relevant for D-fructose absorption. SGLT1 and GLUT5 are constantly localized in the brush border membrane (BBM) of enterocytes, whereas GLUT2 is localized in the basolateral membrane (BLM) or the BBM plus BLM at low and high luminal D-glucose concentrations, respectively. At high luminal D-glucose, the abundance SGLT1 in the BBM is increased. Hence, D-glucose absorption at low luminal glucose is mediated via SGLT1 in the BBM and GLUT2 in the BLM whereas high-capacity D-glucose absorption at high luminal glucose is mediated by SGLT1 plus GLUT2 in the BBM and GLUT2 in the BLM. The review describes functions and regulations of SGLT1, GLUT2, and GLUT5 in the small intestine including diurnal variations and carbohydrate-dependent regulations. Also, the roles of SGLT1 and GLUT2 for secretion of enterohormones are discussed. Furthermore, diseases are described that are caused by malfunctions of small intestinal monosaccharide transporters, such as glucose-galactose malabsorption, Fanconi syndrome, and fructose intolerance. Moreover, it is reported how diabetes, small intestinal inflammation, parental nutrition, bariatric surgery, and metformin treatment affect expression of monosaccharide transporters in the small intestine. Finally, food components that decrease D-glucose absorption and drugs in development that inhibit or downregulate SGLT1 in the small intestine are compiled. Models for regulations and combined functions of glucose transporters, and for interplay between D-fructose transport and metabolism, are discussed.
Asunto(s)
Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Enfermedades Intestinales/metabolismo , Intestino Delgado/metabolismo , Transportador 1 de Sodio-Glucosa/metabolismo , Animales , Proteínas Facilitadoras del Transporte de la Glucosa/química , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Humanos , Absorción Intestinal , Transportador 1 de Sodio-Glucosa/química , Transportador 1 de Sodio-Glucosa/genéticaRESUMEN
Energy demand of neurons in brain that is covered by glucose supply from the blood is ensured by glucose transporters in capillaries and brain cells. In brain, the facilitative diffusion glucose transporters GLUT1-6 and GLUT8, and the Na+-D-glucose cotransporters SGLT1 are expressed. The glucose transporters mediate uptake of D-glucose across the blood-brain barrier and delivery of D-glucose to astrocytes and neurons. They are critically involved in regulatory adaptations to varying energy demands in response to differing neuronal activities and glucose supply. In this review, a comprehensive overview about verified and proposed roles of cerebral glucose transporters during health and diseases is presented. Our current knowledge is mainly based on experiments performed in rodents. First, the functional properties of human glucose transporters expressed in brain and their cerebral locations are described. Thereafter, proposed physiological functions of GLUT1, GLUT2, GLUT3, GLUT4, and SGLT1 for energy supply to neurons, glucose sensing, central regulation of glucohomeostasis, and feeding behavior are compiled, and their roles in learning and memory formation are discussed. In addition, diseases are described in which functional changes of cerebral glucose transporters are relevant. These are GLUT1 deficiency syndrome (GLUT1-SD), diabetes mellitus, Alzheimer's disease (AD), stroke, and traumatic brain injury (TBI). GLUT1-SD is caused by defect mutations in GLUT1. Diabetes and AD are associated with changed expression of glucose transporters in brain, and transporter-related energy deficiency of neurons may contribute to pathogenesis of AD. Stroke and TBI are associated with changes of glucose transporter expression that influence clinical outcome.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Errores Innatos del Metabolismo de los Carbohidratos/metabolismo , Diabetes Mellitus/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Proteínas de Transporte de Monosacáridos/deficiencia , Animales , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Humanos , Proteínas de Transporte de Monosacáridos/metabolismoRESUMEN
BACKGROUND: Glomerular hyperfiltration is common in early diabetes and is considered a risk factor for later diabetic nephropathy. We propose that sodium-glucose cotransporter 1 (SGLT1) senses increases in luminal glucose at the macula densa, enhancing generation of neuronal nitric oxide synthase 1 (NOS1)-dependent nitric oxide (NO) in the macula densa and blunting the tubuloglomerular feedback (TGF) response, thereby promoting the rise in GFR. METHODS: We used microperfusion, micropuncture, and renal clearance of FITC-inulin to examine the effects of tubular glucose on NO generation at the macula densa, TGF, and GFR in wild-type and macula densa-specific NOS1 knockout mice. RESULTS: Acute intravenous injection of glucose induced hyperglycemia and glucosuria with increased GFR in mice. We found that tubular glucose blunts the TGF response in vivo and in vitro and stimulates NO generation at the macula densa. We also showed that SGLT1 is expressed at the macula densa; in the presence of tubular glucose, SGLT1 inhibits TGF and NO generation, but this action is blocked when the SGLT1 inhibitor KGA-2727 is present. In addition, we demonstrated that glucose increases NOS1 expression and NOS1 phosphorylation at Ser1417 in mouse renal cortex and cultured human kidney tissue. In macula densa-specific NOS1 knockout mice, glucose had no effect on NO generation, TGF, and GFR. CONCLUSIONS: We identified a novel mechanism of acute hyperglycemia-induced hyperfiltration wherein increases in luminal glucose at the macula densa upregulate the expression and activity of NOS1 via SGLT1, blunting the TGF response and promoting glomerular hyperfiltration.
Asunto(s)
Glucosa/metabolismo , Hiperglucemia/fisiopatología , Glomérulos Renales/fisiopatología , Túbulos Renales Distales/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Transportador 1 de Sodio-Glucosa/metabolismo , Animales , Retroalimentación Fisiológica , Tasa de Filtración Glomerular , Glucósidos/farmacología , Humanos , Inulina/metabolismo , Glomérulos Renales/metabolismo , Túbulos Renales Distales/fisiopatología , Masculino , Ratones , Ratones Noqueados , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo I/genética , Fosforilación , Pirazoles/farmacología , Transducción de Señal , Transportador 1 de Sodio-Glucosa/antagonistas & inhibidoresRESUMEN
Organic cation transporters OCT1 (SLC22A1) and OCT2 (SLC22A2) are critically involved in absorption and excretion of diverse cationic drugs. Because drug-drug interactions at these transporters may induce adverse drug effects in patients, in vitro testing during drug development for interaction with the human transporters is mandatory. Recent data performed with rat OCT1 (rOCT1) suggest that currently performed in vitro tests assuming one polyspecific binding site are insufficient. Here we measured the binding and transport of model substrate 1-methyl-4-phenylpyridinium+ (MPP+) by cell-free-expressed fusion proteins of rOCT1 and rOCT1 mutants with green fluorescent protein that had been reconstituted into nanodiscs or proteoliposomes. The nanodiscs were formed with major scaffold protein (MSP) and different phospholipids, whereas the proteoliposomes were formed with a mixture of cholesterol, phosphatidylserine, and phosphatidylcholine. In nanodiscs formed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or cholesterol, phosphatidylserine, and phosphatidylcholine, two low-affinity MPP+ binding sites and one high-affinity MPP+ binding site per transporter monomer were determined. Mutagenesis revealed that tryptophan 218 and aspartate 475 in neighboring positions in the modeled outward-open cleft contribute to one low-affinity binding site, whereas arginine 440 located distantly in the cleft is critical for MPP+ binding to another low-affinity site. Comparing MPP+ binding with MPP+ transport suggests that the low-affinity sites are involved in MPP+ transport, whereas high-affinity MPP+ binding influences transport allosterically. The data will be helpful in the interpretation of future crystal structures and provides a rationale for future in vitro testing that is more sophisticated and reliable, leading to the generation of pharmacophore models with high predictive power.
Asunto(s)
1-Metil-4-fenilpiridinio/metabolismo , Proteínas de Transporte de Catecolaminas en la Membrana Plasmática/metabolismo , Animales , Sitios de Unión , Proteínas Fluorescentes Verdes/metabolismo , Mutagénesis/fisiología , Fosfolípidos/metabolismo , Proteolípidos/metabolismo , RatasRESUMEN
A domain of protein RS1 (RSC1A1) called RS1-Reg down-regulates the plasma membrane abundance of Na+-d-glucose cotransporter SGLT1 by blocking the exocytotic pathway at the trans-Golgi. This effect is blunted by intracellular glucose but prevails when serine in a QSP (Gln-Ser-Pro) motif is replaced by glutamate [RS1-Reg(S20E)]. RS1-Reg binds to ornithine decarboxylase (ODC) and inhibits ODC in a glucose-dependent manner. Because the ODC inhibitor difluoromethylornithine (DFMO) acts like RS1-Reg(S20E), and DFMO and RS1-Reg(S20E) are not cumulative, we raised the hypothesis that RS1-Reg(S20E) down-regulates the exocytotic pathway of SGLT1 at the trans-Golgi by inhibiting ODC. We investigated whether QEP down-regulates human SGLT1 (hSGLT1) like hRS1-Reg(S20E) and whether human Na+-d-glucose cotransporter hSGLT2 and the human glucose sensor hSGLT3 are also addressed. We expressed hSGLT1, hSGLT1 linked to yellow fluorescent protein (hSGLT1-YFP), hSGLT2-YFP and hSGLT3-YFP in oocytes of Xenopus laevis, injected hRS1-Reg(S20E), QEP, DFMO, and/or α-methyl-d-glucopyranoside (AMG), and measured AMG uptake, glucose-induced currents, and plasma membrane-associated fluorescence after 1 hour. We also performed in vitro AMG uptake measurements into small intestinal mucosa of mice and human. The data indicate that QEP down-regulates the exocytotic pathway of SGLT1 similar to hRS1-Reg(S20E). Our results suggests that both peptides also down-regulate hSGLT2 and hSGLT3 via the same pathway. Thirty minutes after application of 5 mM QEP in the presence of 5 mM d-glucose, hSGLT1-mediated AMG uptake into small intestinal mucosa was decreased by 40% to 50%. Thus oral application of QEP in a formulation that optimizes uptake into enterocytes but prevents entry into the blood is proposed as novel antidiabetic therapy.
Asunto(s)
Regulación hacia Abajo/fisiología , Exocitosis/fisiología , Glucosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Péptidos/metabolismo , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Adulto , Animales , Transporte Biológico/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Eflornitina/farmacología , Exocitosis/efectos de los fármacos , Femenino , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Oocitos/metabolismo , Ornitina Descarboxilasa/metabolismo , Xenopus laevisRESUMEN
Olfactory receptors are G protein-coupled receptors that serve to detect odorants in the nose. Additionally, these receptors are expressed in other tissues, where they have functions outside the canonical smell response. Olfactory receptor 1393 (Olfr1393) was recently identified as a novel regulator of Na+-glucose cotransporter 1 (Sglt1) localization in the renal proximal tubule. Glucose reabsorption in the proximal tubule (via Sglt1 and Sglt2) has emerged as an important contributor to the development of diabetes. Inhibition of Sglt2 is accepted as a viable therapeutic treatment option for patients with type 2 diabetes and has been shown to delay development of diabetic kidney disease. We hypothesized that Olfr1393 may contribute to the progression of type 2 diabetes, particularly the development of hyperfiltration, which has been linked to increased Na+ reabsorption in the proximal tubule via the Sglts. To test this hypothesis, Olfr1393 wild-type (WT) and knockout (KO) mice were challenged with a high-fat diet to induce early-stage type 2 diabetes. After 16 wk on the high-fat diet, fasting blood glucose values were increased and glucose tolerance was impaired in the male WT mice. Both of these effects were significantly blunted in the male KO mice. In addition, male and female WT mice developed diabetes-induced hyperfiltration, which was attenuated in the Olfr1393 KO mice and corresponded with a reduction in luminal expression of Sglt2. Collectively, these data indicate that renal Olfr1393 can contribute to the progression of type 2 diabetes, likely as a regulator of Na+-glucose cotransport in the proximal tubule.
Asunto(s)
Glucemia/metabolismo , Diabetes Mellitus Tipo 2/etiología , Dieta Alta en Grasa , Túbulos Renales Proximales/metabolismo , Obesidad/complicaciones , Receptores Odorantes/metabolismo , Reabsorción Renal , Animales , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Tasa de Filtración Glomerular , Resistencia a la Insulina , Túbulos Renales Proximales/fisiopatología , Masculino , Ratones Noqueados , Obesidad/genética , Obesidad/metabolismo , Receptores Odorantes/genética , Receptores Odorantes/fisiología , Factores de TiempoRESUMEN
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.
Asunto(s)
Lesión Renal Aguda/metabolismo , Tasa de Filtración Glomerular , Glucosa/metabolismo , Túbulos Renales Proximales/metabolismo , Reabsorción Renal , Daño por Reperfusión/metabolismo , Transportador 1 de Sodio-Glucosa/deficiencia , Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Lesión Renal Aguda/fisiopatología , Animales , Modelos Animales de Enfermedad , Eliminación de Gen , Túbulos Renales Proximales/patología , Túbulos Renales Proximales/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Recuperación de la Función , Daño por Reperfusión/genética , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatología , Transportador 1 de Sodio-Glucosa/genética , Transportador 2 de Sodio-Glucosa/genética , Transportador 2 de Sodio-Glucosa/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12/genética , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismo , Factores de TiempoRESUMEN
Inhibitors of the Na+-glucose cotransporter SGLT2 enhance urinary glucose and urate excretion and lower plasma urate levels. The mechanisms remain unclear, but a role for enhanced glucose in the tubular fluid, which may interact with tubular urate transporters, such as the glucose transporter GLUT9 or the urate transporter URAT1, has been proposed. Studies were performed in nondiabetic mice treated with the SGLT2 inhibitor canagliflozin and in gene-targeted mice lacking the urate transporter Glut9 in the tubule or in mice with whole body knockout of Sglt2, Sglt1, or Urat1. Renal urate handling was assessed by analysis of urate in spontaneous plasma and urine samples and normalization to creatinine concentrations or by renal clearance studies with assessment of glomerular filtration rate by FITC-sinistrin. The experiments confirmed the contribution of URAT1 and GLUT9 to renal urate reabsorption, showing a greater contribution of the latter and additive effects. Genetic and pharmacological inhibition of SGLT2 enhanced fractional renal urate excretion (FE-urate), indicating that a direct effect of the SGLT2 inhibitor on urate transporters is not absolutely necessary. Consistent with a proposed role of increased luminal glucose delivery, the absence of Sglt1, which by itself had no effect on FE-urate, enhanced the glycosuric and uricosuric effects of the SGLT2 inhibitor. The SGLT2 inhibitor enhanced renal mRNA expression of Glut9 in wild-type mice, but tubular GLUT9 seemed dispensable for the increase in FE-urate in response to canagliflozin. First evidence is presented that URAT1 is required for the acute uricosuric effect of the SGLT2 inhibitor in mice.
Asunto(s)
Canagliflozina/farmacología , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Transportadores de Anión Orgánico/metabolismo , Eliminación Renal/efectos de los fármacos , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Transportador 2 de Sodio-Glucosa/efectos de los fármacos , Ácido Úrico/orina , Uricosúricos/farmacología , Animales , Genotipo , Proteínas Facilitadoras del Transporte de la Glucosa/deficiencia , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Túbulos Renales Proximales/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Transportadores de Anión Orgánico/deficiencia , Transportadores de Anión Orgánico/genética , Fenotipo , Reabsorción Renal/efectos de los fármacos , Transportador 2 de Sodio-Glucosa/deficiencia , Transportador 2 de Sodio-Glucosa/genética , Transportador 2 de Sodio-Glucosa/metabolismoRESUMEN
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.
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Glucemia/metabolismo , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Tipo 1/enzimología , Nefropatías Diabéticas/enzimología , Tasa de Filtración Glomerular , Riñón/enzimología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Transportador 1 de Sodio-Glucosa/deficiencia , Albuminuria/enzimología , Albuminuria/genética , Albuminuria/fisiopatología , Animales , Biomarcadores/sangre , Glucemia/efectos de los fármacos , Presión Sanguínea , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/fisiopatología , Nefropatías Diabéticas/sangre , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/fisiopatología , Tasa de Filtración Glomerular/efectos de los fármacos , Riñón/efectos de los fármacos , Riñón/fisiopatología , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo I/genética , Reabsorción Renal , Renina/sangre , Renina/genética , Transducción de Señal , Transportador 1 de Sodio-Glucosa/genética , Transportador 2 de Sodio-Glucosa , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Regulación hacia ArribaRESUMEN
BACKGROUND & AIMS: Inactivating mutations in MYO5B cause microvillus inclusion disease (MVID), but the physiological cause of the diarrhea associated with this disease is unclear. We investigated whether loss of MYO5B results in aberrant expression of apical enterocyte transporters. METHODS: We studied alterations in apical membrane transporters in MYO5B-knockout mice, as well as mice with tamoxifen-inducible, intestine-specific disruption of Myo5b (VilCreERT2;Myo5bflox/flox mice) or those not given tamoxifen (controls). Intestinal tissues were collected from mice and analyzed by immunostaining, immunoelectron microscopy, or cultured enteroids were derived. Functions of brush border transporters in intestinal mucosa were measured in Ussing chambers. We obtained duodenal biopsy specimens from individuals with MVID and individuals without MVID (controls) and compared transporter distribution by immunocytochemistry. RESULTS: Compared to intestinal tissues from littermate controls, intestinal tissues from MYO5B-knockout mice had decreased apical localization of SLC9A3 (also called NHE3), SLC5A1 (also called SGLT1), aquaporin (AQP) 7, and sucrase isomaltase, and subapical localization of intestinal alkaline phosphatase and CDC42. However, CFTR was present on apical membranes of enterocytes from MYO5B knockout and control mice. Intestinal biopsies from patients with MVID had subapical localization of NHE3, SGLT1, and AQP7, but maintained apical CFTR. After tamoxifen administration, VilCreERT2;Myo5bflox/flox mice lost apical NHE3, SGLT1, DRA, and AQP7, similar to germline MYO5B knockout mice. Intestinal tissues from VilCreERT2;Myo5bflox/flox mice had increased CFTR in crypts and CFTR localized to the apical membranes of enterocytes. Intestinal mucosa from VilCreERT2;Myo5bflox/flox mice given tamoxifen did not have an intestinal barrier defect, based on Ussing chamber analysis, but did have decreased SGLT1 activity and increased CFTR activity. CONCLUSIONS: Although trafficking of many apical transporters is regulated by MYO5B, trafficking of CFTR is largely independent of MYO5B. Decreased apical localization of NHE3, SGLT1, DRA, and AQP7 might be responsible for dysfunctional water absorption in enterocytes of patients with MVID. Maintenance of apical CFTR might exacerbate water loss by active secretion of chloride into the intestinal lumen.
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Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Enterocitos/metabolismo , Síndromes de Malabsorción/genética , Microvellosidades/patología , Mucolipidosis/genética , Miosina Tipo V/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Acuaporinas/metabolismo , Duodeno/metabolismo , Duodeno/patología , Silenciador del Gen , Humanos , Mucosa Intestinal , Intestinos/citología , Intestinos/patología , Síndromes de Malabsorción/patología , Ratones , Ratones Noqueados , Microvellosidades/genética , Mucolipidosis/patología , Transporte de Proteínas , Transportador 1 de Sodio-Glucosa/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Complejo Sacarasa-Isomaltasa/metabolismo , Tamoxifeno/administración & dosificaciónRESUMEN
In vitro evaluation of drugs for interaction with transporters is essential during drug development. As polyspecific organic cation transporters (OCTs) are critical for pharmacokinetics of many cationic drugs, in vitro testing of human OCT1 and human OCT2 is recommended. In the currently applied tests it is determined whether uptake of one model cation in stably transfected epithelial cells is inhibited using a substrate concentration in the micromolar range. In this review experimental evidence for the existence of low- and high-affinity cation binding sites in OCTs that may interact with drugs is compiled. Most data were obtained from studies performed with rat Oct1. Whereas overlapping low-affinity cation binding sites are directly involved in transport, the high-affinity cation binding sites may induce allosteric inhibition of transport. Remarkably, high-affinity inhibition is only observed when uptake is measured using nanomolar substrate concentrations far below the respective Km values. Affinities of inhibitors are dependent on molecular structure and concentration of the employed substrate. Because the currently applied in vitro tests for identification of interaction of novel drugs with OCTs do not consider the influence of substrate structure and are not capable of identifying high-affinity inhibition, more sophisticated testing protocols are proposed.
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Proteínas de Transporte de Catión Orgánico/efectos de los fármacos , Proteínas de Transporte de Catión Orgánico/metabolismo , Animales , Sitios de Unión , Humanos , Transporte de ProteínasRESUMEN
The effects of mutations in the modeled outward-open cleft of rat organic cation transporter 1 (rOCT1) on affinities of substrates and inhibitors were investigated. Human embryonic kidney 293 cells were stably transfected with rOCT1 or rOCT1 mutants, and uptake of the substrates 1-methyl-4-phenylpyridinium+ (MPP+) and tetraethylammonium+ (TEA+) or inhibition of MPP+ uptake by the nontransported inhibitors tetrabutylammonium+ (TBuA+), tetrapentylammonium+ (TPeA+), and corticosterone was measured. Uptake measurements were performed on confluent cell layers using a 2-minute incubation or in dissociated cells using incubation times of 1, 5, or 10 seconds. With both methods, different apparent Michaelis-Menten constant (Km) values, different IC50 values, and varying effects of mutations were determined. In addition, varying IC50 values for the inhibition of MPP+ uptake and varying effects of mutations were obtained when different MPP+ concentrations far below the apparent Km value were used for uptake measurements. Eleven mutations were investigated by measuring initial uptake in dissociated cells and employing 0.1 µM MPP+ for uptake during inhibition experiments. Altered affinities for substrates and/or inhibitors were observed when Phe160, Trp218, Arg440, Leu447, and Asp475 were mutated. The mutations resulted in changes of apparent Km values for TEA+ and/or MPP+ Mutation of Trp218 and Asp475 led to altered IC50 values for TBuA+, TPeA+, and corticosterone, whereas the mutation of Phe160 and Leu447 changed the IC50 values for two inhibitors. Thereby amino acids in the outward-facing conformation of rOCT1 could be identified that interact with structurally different inhibitors and probably also with different substrates.
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Proteínas de Transporte de Catecolaminas en la Membrana Plasmática/antagonistas & inhibidores , Proteínas de Transporte de Catecolaminas en la Membrana Plasmática/metabolismo , Mutagénesis/efectos de los fármacos , 1-Metil-4-fenilpiridinio/metabolismo , 1-Metil-4-fenilpiridinio/farmacología , Animales , Relación Dosis-Respuesta a Droga , Femenino , Células HEK293 , Humanos , Mutagénesis/fisiología , Compuestos de Amonio Cuaternario/metabolismo , Compuestos de Amonio Cuaternario/farmacología , Ratas , Especificidad por Sustrato/efectos de los fármacos , Especificidad por Sustrato/fisiología , Xenopus laevisRESUMEN
There is very limited human renal sodium gradient-dependent glucose transporter protein (SGLT2) mRNA and protein expression data reported in the literature. The first aim of this study was to determine SGLT2 mRNA and protein levels in human and animal models of diabetic nephropathy. We have found that the expression of SGLT2 mRNA and protein is increased in renal biopsies from human subjects with diabetic nephropathy. This is in contrast to db-db mice that had no changes in renal SGLT2 protein expression. Furthermore, the effect of SGLT2 inhibition on renal lipid content and inflammation is not known. The second aim of this study was to determine the potential mechanisms of beneficial effects of SGLT2 inhibition in the progression of diabetic renal disease. We treated db/db mice with a selective SGLT2 inhibitor JNJ 39933673. We found that SGLT2 inhibition caused marked decreases in systolic blood pressure, kidney weight/body weight ratio, urinary albumin, and urinary thiobarbituric acid-reacting substances. SGLT2 inhibition prevented renal lipid accumulation via inhibition of carbohydrate-responsive element-binding protein-ß, pyruvate kinase L, SCD-1, and DGAT1, key transcriptional factors and enzymes that mediate fatty acid and triglyceride synthesis. SGLT2 inhibition also prevented inflammation via inhibition of CD68 macrophage accumulation and expression of p65, TLR4, MCP-1, and osteopontin. These effects were associated with reduced mesangial expansion, accumulation of the extracellular matrix proteins fibronectin and type IV collagen, and loss of podocyte markers WT1 and synaptopodin, as determined by immunofluorescence microscopy. In summary, our study showed that SGLT2 inhibition modulates renal lipid metabolism and inflammation and prevents the development of nephropathy in db/db mice.
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Nefropatías Diabéticas/metabolismo , Enfermedades Renales/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Animales , Diabetes Mellitus Experimental , Nefropatías Diabéticas/prevención & control , Humanos , Inflamación/prevención & control , Ratones , ARN Mensajero/análisis , Transportador 2 de Sodio-Glucosa/análisis , Transportador 2 de Sodio-Glucosa/genéticaRESUMEN
The chronic intrinsic diuretic and natriuretic tone of sodium-glucose cotransporter 2 (SGLT2) inhibitors is incompletely understood because their effect on body fluid volume (BFV) has not been fully evaluated and because they often increase food and fluid intake at the same time. Here we first compared the effect of the SGLT2 inhibitor ipragliflozin (Ipra, 0.01% in diet for 8 wk) and vehicle (Veh) in Spontaneously Diabetic Torii rat, a nonobese type 2 diabetic model, and nondiabetic Sprague-Dawley rats. In nondiabetic rats, Ipra increased urinary excretion of Na+ (UNaV) and fluid (UV) associated with increased food and fluid intake. Diabetes increased these four parameters, but Ipra had no further effect, probably because of its antihyperglycemic effect, such that glucosuria and, as a consequence, food and fluid intake were unchanged. Fluid balance and BFV, determined by bioimpedance spectroscopy, were similar among the four groups. To study the impact of food and fluid intake, nondiabetic rats were treated for 7 days with Veh, Ipra, or Ipra+pair feeding+pair drinking (Pair-Ipra). Pair-Ipra maintained a small increase in UV and UNaV versus Veh despite similar food and fluid intake. Pair-Ipra induced a negative fluid balance and decreased BFV, whereas Ipra or Veh had no significant effect compared with basal values. In conclusion, SGLT2 inhibition induces a sustained diuretic and natriuretic tone. Homeostatic mechanisms are activated to stabilize BFV, including compensatory increases in fluid and food intake.