Transporter GLT-1 Research Articles

The flavanone homoeriodictyol increases SGLT-1-mediated glucose uptake but decreases serotonin release in differentiated Caco-2 cells.

Flavanoids and related polyphenols, among them hesperitin, have been shown to modulate cellular glucose transport by targeting SGLT-1 and GLUT-2 transport proteins. We aimed to investigate whether homoeriodictyol, which is structurally related to hesperitin, affects glucose uptake in differentiated Caco-2 cells as a model for the intestinal barrier. The results revealed that, in contrast to other polyphenols, the flavanon homoeriodictyol promotes glucose uptake by 29.0 ± 3.83% at a concentration of 100 μM. The glucose uptake stimulating effect was sensitive to phloridzin, but not to phloretin, indicating an involvement of the sodium-coupled glucose transporter SGLT-1, but not of sodium-independent glucose transporters (GLUT). In addition, in contrast to the increased extracellular serotonin levels by stimulation with 500 mM D-(+)-glucose, treatment with 100 μM homoeriodictyol decreased serotonin release by –48.8 ± 7.57% in Caco-2 cells via a phloridzin-sensitive signaling pathway. Extracellular serotonin levels were also reduced by –57.1 ± 5.43% after application of 0.01 μM homoeriodictyol to human neural SH-SY5Y cells. In conclusion, we demonstrate that homoeriodictyol affects both the glucose metabolism and the serotonin system in Caco-2 cells via a SGLT-1-meditated pathway. Furthermore, the results presented here support the usage of Caco-2 cells as a model for peripheral serotonin release. Further investigations may address the value of homoeriodictyol in the treatment of anorexia and malnutrition through the targeting of SGLT-1.

Evaluation of the hypoglycemic potential of a black bean hydrolyzed protein isolate and its pure peptides using in silico, in vitro and in vivo approaches

The objective was to evaluate the in silico, in vitro and in vivo effects of a black bean hydrolyzed protein isolate (HPI) and its pure peptides on glucose absorption. By interacting with their protein motif, pure peptides blocked glucose transporters GLUT2 and SGLT1. HPI (10mg/mL) reduced formation of intracellular oxygen reactive species by 71%. Glucose absorption decreased 21.5% after 24h treatment when using the Caco-2 cell model. Oral glucose tolerance test in rats showed a 24.5% decrease on postprandial glucose (p<0.05) (50mg HPI /kg BW). In the hyperglycemic rat model, HPI caused a reduction of blood glucose, in a dose-dependent manner. The lowest fasting glucose was found in rats receiving 150 and 200mg/kg BW/day HPI, (p<0.05). The black bean HPI is an inexpensive food source of bioactive compounds that could be used in the management of blood glucose.

Effect of egg storage duration and brooding temperatures on chick growth, intestine morphology and nutrient transporters

The effects of egg storage duration (ESD) and brooding temperature (BT) on BW, intestine development and nutrient transporters of broiler chicks were investigated. A total of 396 chicks obtained from eggs stored at 18°C for 3 days (ESD3-18°C) or at 14°C for 14 days (ESD14-14°C) before incubation were exposed to three BTs. Temperatures were initially set at 32°C, 34°C and 30°C for control (BT-Cont), high (BT-High) and low (BT-Low) BTs, respectively. Brooding temperatures were decreased by 2°C each at days 2, 7, 14 and 21. Body weight was measured at the day of hatch, 2, 7, 14, 21, 28 and 42. Cloacal temperatures of broilers were recorded from 1 to 14 days. Intestinal morphology and gene expression levels of H+-dependent peptide transporter (PepT1) and Na-dependent glucose (SGLT1) were evaluated on the day of hatch and 14. Cloacal temperatures of chicks were affected by BTs from days 1 to 8, being the lowest for BT-Low chicks. BT-High resulted in the heaviest BWs at 7 days, especially for ESD14-14°C chicks. This result was consistent with longer villus and larger villus area of ESD14-14°C chicks at BT-High conditions. From 14 days to slaughter age, BT had no effect on broiler weight. ESD3-18°C chicks were heavier than ESD14-14°C chicks up to 28 days. The PepT1 and SGLT1 expression levels were significantly higher in ESD3-18°C chicks than ESD14-14°C on the day of hatch. There was significant egg storage by BT interaction for PepT1 and SGLT1 transporters at day 14. ESD14-14°C chicks had significantly higher expression of PepT1 and SGLT1 at BT-Low than those at BT-Cont. ESD14-14°C chicks upregulated PepT1 gene expression 1.15 and 1.57-fold at BT-High and BT-Low, respectively, compared with BT-Cont, whereas PepT1 expression was downregulated 0.67 and 0.62-fold in ESD3-18°C chicks at BT-High and BT-Low. These results indicated that pre-incubation egg storage conditions and BTs affected intestine morphology and PepT1 and SGLT1 nutrient transporters expression in broiler chicks.

Abordaje de la diabetes mellitus tipo 2 a través del cotransportador sodio-glucosa tipo 2: ¿tiene sentido?

The kidney is involved in glucose homeostasis through three main mechanisms: renal gluconeogenesis, renal glucose consumption and glucose reabsorption in the proximal tubule. Glucose reabsorption is one of the most relevant physiological functions of the kidney, through which filtered glucose is fully recovered, urine is free of glucose, and calorie loss is prevented. Approximately 90% of the glucose is reabsorbed in the S1 segment of the proximal tubule, where GLUT2 and SGLT2 transporters are located, while the remaining 10% is reabsorbed in the S3 segment by SGLT1 and GLUT1 transporters. In patients with hyperglycaemia, the kidney continues reabsorbing glucose, and hyperglycaemia is maintained. Most renal glucose reabsorption is mediated by the SGLT2 transporter. Several experimental and clinical studies suggest that pharmacological blockade of this transporter might be beneficial in the management of hyperglycemia in patients with type 2 diabetes.

NO EFFECT OF PRE-EXERCISE SUCRALOSE INGESTION ON GASTRIC EMPTYING RATE OF A CARBOHYDRATE-ELECTROLYTE SOLUTION

Ingestion of carbohydrate during exercise is common in order to provide an exogenous substrate for oxidation and to improve performance. The rate at which a solution is emptied from the...

Implicaciones hemodinámicas y renales de los inhibidores del cotransportador sodio-glucosa tipo 2 en el contexto de la diabetes mellitus tipo 2

In DM2, there is increased expression of the proximal glucose transporter SGLT2. The increased glucose reabsorption from the urine to the proximal tubule and subsequently to the bloodstream, has three direct effects on the prognosis of patients with DM2: a) it increases the daily glucose load by raising the renal threshold for glucose, thus augmenting requirements for oral antidiabetics and insulin. This progressive increase occurs throughout the course of the disease and in parallel with the increase in renal mass (renal hypertrophy); b) because of the greater glucose reabsorption, glycosuria is lower than the level corresponding to glycaemia, decreasing the stimulus on the tubuloglomerular feedback system of the distal nephron. As a result, the glomerular vasodilation caused by hyperglycaemia is not arrested, maintaining glomerular hyperfiltration, and c) the excess glucose transported to the proximal tubular cells modifies their redox status, increasing local production of glycosylating products and activating local production of proinflammatory and profibrotic proliferative mediators. These mediators are responsible for the direct free radical damage to proximal tubular cells, for increased SGLT2 expression, increased production of collagen IV and extracellular matrix, and activation of monocyte/macrophages able to cause endothelial injury. The use of SGLT2 inhibitors not only reduces the reabsorption of glucose from the glomerular filtrate back into the circulationthus improving metabolic control in diabetesbut also restores tubuloglomerular feedback by increasing glycosuria and distal urinary flow. However, the most notable effect is due to inhibition of glucose entry to the proximal tubular cells. Glycosuria is toxic to the kidney: it harms glucosetransporting cells, that is, the proximal cells, which contain SGLT2. In animal models, SGLT2 inhibition reduces local production of oxygen-free radicals, the formation of mesangial matrix and collagen IV, glomerular infiltration by inflammatory cells and monocyte/macrophage-dependent arteriosclerosis. In humans, SGLT2 have a demonstrated ability to reduce renal injury and cardiovascular risk in patients with type 2 diabetes mellitus.

Structural and functional significance of water permeation through cotransporters

Membrane transporters, in addition to their major role as specific carriers for ions and small molecules, can also behave as water channels. However, neither the location of the water pathway in the protein nor their functional importance is known. Here, we map the pathway for water and urea through the intestinal sodium/glucose cotransporter SGLT1. Molecular dynamics simulations using the atomic structure of the bacterial transporter vSGLT suggest that water permeates the same path as Na+ and sugar. On a structural model of SGLT1, based on the homology structure of vSGLT, we identified and mutated residues lining the sugar transport pathway to cysteine. The mutants were expressed in Xenopus oocytes, and the unitary water and urea permeabilities were determined before and after modifying the cysteine side chain with reversible methanethiosulfonate reagents. The results demonstrate that water and urea follow the sugar transport pathway through SGLT1. The changes in permeability, increases or decreases, with side-chain modifications depend on the location of the mutation in the region of external or internal gates, or the sugar binding site. These changes in permeability are hypothesized to be due to alterations in steric hindrance to water and urea, and/or changes in protein folding caused by mismatching of side chains in the water pathway. Water permeation through SGLT1 and other transporters bears directly on the structural mechanism for the transport of polar solutes through these proteins. Finally, in vitro experiments on mouse small intestine show that SGLT1 accounts for two-thirds of the passive water flow across the gut.

T1 High glucose and myocardial ischaemia/reperfusion injury: the role of the sodium/glucose transporter, SGLT1

Background Hyperglycaemia, frequently found at the time of acute myocardial infarction, is associated with excess cardiovascular mortality. The sodium-glucose transporter SGLT1 is present in mammalian myocardium, but its role in acute ischaemia/reperfusion injury is currently unknown. We hypothesise high glucose at reperfusion increases infarct size following injurious ischaemia and this excess injury can be ameliorated by inhibition of SGLT1. Methods and results Langendorff-perfused Sprague-Dawley rat (SDR) heart were subjected to 35 min regional ischaemia and 60min variable glucose (3.5–22 mmol/L) reperfusion, with osmolality controlled by reciprocal dose mannitol. Reperfusion High Glucose (RHG) significantly dose-dependently increased infarct size (45 to 65%, p Discussion and conclusion We demonstrate for the first time that high glucose at reperfusion exacerbates myocardial injury following lethal myocardial ischaemia that can be ameliorated by inhibition of SGLT, which appears not directly targeted at cardiomyocytes, but rather non-myocyte myocardial cell populations. These data therefore provide the first data for a novel repurposing of SGLT inhibitors in non-diabetic patients presenting with acute coronary syndromes complicated by hyperglycaemia, and provides a tantalising insight into the mechanisms of hyperglycaemic-driven excess cardiovascular mortality.

Protein RS1 (RSC1A1) Downregulates the Exocytotic Pathway of Glucose Transporter SGLT1 at Low Intracellular Glucose via Inhibition of Ornithine Decarboxylase.

Na+-d-glucose cotransporter 1 (SGLT1) is rate-limiting for glucose absorption in the small intestine. Shortly after intake of glucose-rich food, SGLT1 abundance in the luminal membrane of the small intestine is increased. This upregulation occurs via glucose-induced acceleration of the release of SGLT1-containing vesicles from the trans-Golgi network (TGN), which is regulated by a domain of protein RS1 (RSC1A1) named RS1-Reg. Dependent on phosphorylation, RS1-Reg blocks release of vesicles containing SGLT1 or concentrative nucleoside transporter 1. The hypothesis has been raised that RS1-Reg binds to different receptor proteins at the TGN, which trigger release of vesicles with different transporters. To identify the presumed receptor proteins, two-hybrid screening was performed. Interaction with ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme of polyamine synthesis, was observed and verified by immunoprecipitation. Binding of RS1-Reg mutants to ODC1 was characterized using surface plasmon resonance. Inhibition of ODC1 activity by RS1-Reg mutants and the ODC1 inhibitor difluoromethylornithine (DFMO) was measured in the absence and presence of glucose. In addition, short-term effects of DFMO, RS1-Reg mutants, the ODC1 product putrescine, and/or glucose on SGLT1 expressed in oocytes of Xenopus laevis were investigated. High-affinity binding of RS1-Reg to ODC1 was demonstrated, and evidence for a glucose binding site in ODC1 was provided. Binding of RS1-Reg to ODC1 inhibits the enzymatic activity at low intracellular glucose, which is blunted at high intracellular glucose. The data suggest that generation of putrescine by ODC1 at the TGN stimulates release of SGLT1-containing vesicles. This indicates a biomedically important role of ODC1 in regulation of glucose homeostasis.

Real-time monitoring of glucose and phenols intestinal absorption through an integrated Caco-2TC7cells/biosensors telemetric device: Hypoglycemic effect of fruit phytochemicals

An integrated device for real-time monitoring of glucose and phenols absorption, that consists of a sensors/biosensors system (SB) and a Caco-2TC7 human intestinal cell culture, is described in this study. The SB is composed of a glucose oxidase-based biosensor, a sentinel platinum sensor, a laccase/tyrosinase-based biosensor and a sentinel carbon sensor, all located in the basolateral compartment (BC) of a cell culture plate. Caco-2TC7 cells, differentiated on culture inserts, separated the apical compartment that simulates the intestinal lumen, from the BC which represented the bloodstream. The system recorded currents relative to glucose (1mM) absorption, obtaining bioavailability values (5.1%) comparable to HPLC analysis (4.8%). Phloridzin and phloretin, specific phenolic inhibitors of SGLT1 and GLUT2 glucose transporters, reduced the glucose transport of almost 10 times. They were minimally absorbed in the BC with a bioavailability of 0.13% and 0.49% respectively. The hypoglycemic potential of blueberry and pomegranate juices was also studied. In particular, the amount of glucose absorbed through the Caco-2TC7 monolayer was 8‰ for pomegranate and 1.7‰ for blueberry, demonstrating the potential hypoglycemic effect of the juices. Polyphenols absorption was also monitored by the SB and an increase was recorded during the first 50min in presence of both blueberry and pomegranate juices, then a constant decrease occurred. The proposed device has been developed as innovative tool for the dynamic monitoring of natural compounds effects on glucose absorption, in order to manage postprandial hyperglycemia.

Antidiabetic effect of chitosan oligosaccharide (GO2KA1) is mediated via inhibition of intestinal alpha-glucosidase and glucose transporters and PPARγ expression.

We have previously reported that administration of low molecular weight chitosan oligosaccharide (GO2KA1) significantly suppressed postprandial blood glucose rise with increased plasma adiponectin and HbA1c levels in animals and humans. However, the cellular mechanisms whereby GO2KA1 exerts antihyperglycemic effects still remain to be determined. Using intestinal Caco-2 cells and 3T3-L1 cells, here we show that GO2KA1 has dual modes of antidiabetic action by (1) inhibiting intestinal α-glucosidase as well as glucose transporters SGLT1 and GLUT2 that were distinct from the acarbose effect; (2) enhancing adipocyte differentiation, PPARγ expression and its target genes, such as FABP4, adiponectin, and GLUT4, whereas the effects were abolished by co-treatment with BADGE, a PPARγ antagonist. Moreover, GO2KA1 significantly increased glucose uptake, which was reduced in the presence of BADGE. Our data show that GO2KA1 may prevent hyperglycemia by inhibiting intestinal glucose digestion and transport and also enhance glucose uptake, at least in part, by upregulating adiponectin expression through PPARγ in adipocytes. These findings may provide potential molecular modes of action for the antidiabetic effects of chitosan oligosaccharide observed in clinical and animal studies. © 2016 BioFactors, 43(1):90-99, 2017.

Resveratrol improves glycemic control in insulin-treated diabetic rats: participation of the hepatic territory.

BackgroundResveratrol is a natural polyphenol that has been proposed to improve glycemic control in diabetes, by mechanisms that involve improvement in insulin secretion and activity. In type 1 diabetes (T1D), in which insulin therapy is obligatory, resveratrol treatment has never been investigated. The present study aimed to evaluate resveratrol as an adjunctive agent to insulin therapy in a T1D-like experimental model.MethodsRats were rendered diabetic by streptozotocin (STZ) treatment. Twenty days later, four groups of animals were studied: non-diabetic (ND); diabetic treated with placebo (DP); diabetic treated with insulin (DI) and diabetic treated with insulin plus resveratrol (DIR). After 30 days of treatment, 24-hour urine was collected; then, blood, soleus muscle, proximal small intestine, renal cortex and liver were sampled. Specific glucose transporter proteins were analyzed (Western blotting) in each territory of interest. Solute carrier family 2 member 2 (Slc2a2), phosphoenolpyruvate carboxykinase (Pck1) and glucose-6-phosphatase catalytic subunit (G6pc) mRNAs (qPCR), glycogen storage and sirtuin 1 (SIRT1) activity were analyzed in liver.ResultsDiabetes induction increased blood glucose, plasma fructosamine concentrations, and glycosuria. Insulin therapy partially recovered the glycemic control; however, resveratrol as adjunctive therapy additionally improved glycemic control and restored plasma fructosamine concentration to values of non-diabetic rats. Resveratrol did not alter the expression of the glucose transporters GLUT2 and SGLT1 in the intestine, GLUT2 and SGLT2 in kidney and GLUT4 in soleus, suggesting that fluxes of glucose in these territories were unaltered. Differently, in liver, resveratrol promoted a reduction in Slc2a2, Pck1, and G6pc mRNAs, as well as in GLUT2 protein (P < 0.05, DIR vs. DI); besides, it increased (P < 0.01, DIR vs. DI) the hepatic glycogen content, and SIRT1 protein.ConclusionsResveratrol is able to improve glycemic control in insulin-treated T1D-like rats. This effect seems not to involve changes in glucose fluxes in the small intestine, renal proximal tubule, and soleus skeletal muscle; but to be related to several changes in the liver, where downregulation of Slc2a2/GLUT2, Pck1, and G6pc expression was observed, favoring reduction of glucose production and efflux. Besides, resveratrol increased SIRT1 nuclear protein content in liver, which may be related to the observed gene expression regulations.

MAP17 Is a Necessary Activator of Renal Na+/Glucose Cotransporter SGLT2.

The renal proximal tubule reabsorbs 90% of the filtered glucose load through the Na+-coupled glucose transporter SGLT2, and specific inhibitors of SGLT2 are now available to patients with diabetes to increase urinary glucose excretion. Using expression cloning, we identified an accessory protein, 17 kDa membrane-associated protein (MAP17), that increased SGLT2 activity in RNA-injected Xenopus oocytes by two orders of magnitude. Significant stimulation of SGLT2 activity also occurred in opossum kidney cells cotransfected with SGLT2 and MAP17. Notably, transfection with MAP17 did not change the quantity of SGLT2 protein at the cell surface in either cell type. To confirm the physiologic relevance of the MAP17-SGLT2 interaction, we studied a cohort of 60 individuals with familial renal glucosuria. One patient without any identifiable mutation in the SGLT2 coding gene (SLC5A2) displayed homozygosity for a splicing mutation (c.176+1G>A) in the MAP17 coding gene (PDZK1IP1). In the proximal tubule and in other tissues, MAP17 is known to interact with PDZK1, a scaffolding protein linked to other transporters, including Na+/H+ exchanger 3, and to signaling pathways, such as the A-kinase anchor protein 2/protein kinase A pathway. Thus, these results provide the basis for a more thorough characterization of SGLT2 which would include the possible effects of its inhibition on colocalized renal transporters.

Homeostasis of the astrocytic glutamate transporter GLT-1 is altered in mouse models of Lafora disease

Lafora disease (LD, OMIM 254780) is a fatal rare disorder characterized by epilepsy and neurodegeneration. Although in recent years a lot of information has been gained on the molecular basis of the neurodegeneration that accompanies LD, the molecular basis of epilepsy is poorly understood. Here, we present evidence indicating that the homeostasis of glutamate transporter GLT-1 (EAAT2) is compromised in mouse models of LD. Our results indicate that primary astrocytes from LD mice have reduced capacity of glutamate transport, probably because they present a reduction in the levels of the glutamate transporter at the plasma membrane. On the other hand, the overexpression in cellular models of laforin and malin, the two proteins related to LD, results in an accumulation of GLT-1 (EAAT2) at the plasma membrane and in a severe reduction of the ubiquitination of the transporter. All these results suggest that the laforin/malin complex slows down the endocytic recycling of the GLT-1 (EAAT2) transporter. Since, defects in the function of this transporter lead to excitotoxicity and epilepsy, we suggest that the epilepsy that accompanies LD could be due, at least in part, to deficiencies in the function of the GLT-1 (EAAT2) transporter.

Chlorogenic Acid Maintains Glucose Homeostasis through Modulating the Expression of SGLT-1, GLUT-2, and PLG in Different Intestinal Segments of Sprague-Dawley Rats Fed a High-Fat Diet

ObjectiveTo reveal the effects and related mechanisms of chlorogenic acid (CGA) on intestinal glucose homeostasis. MethodsForty male Sprague-Dawley rats were randomly and equally divided into four groups: normal chow (NC), high-fat diet (HFD), HFD with low-dose CGA (20 mg/kg, HFD-LC), and HFD with high-dose CGA (90 mg/kg, HFD-HC). The oral glucose tolerance test was performed, and fast serum insulin (FSI) was detected using an enzyme-linked immunosorbent assay. The mRNA expression levels of glucose transporters (Sglt-1 and Glut-2) and proglucagon (Plg) in different intestinal segments (the duodenum, jejunum, ileum, and colon) were analyzed using quantitative real-time polymerase chain reaction. SGLT-1 protein and the morphology of epithelial cells in the duodenum and jejunum was localized by using immunofluorescence. ResultsAt both doses, CGA ameliorated the HFD-induced body weight gain, maintained FSI, and increased postprandial 30-min glucagon-like peptide 1 secretion. High-dose CGA inhibited the HFD-induced elevation in Sglt-1 expression. Both CGA doses normalized the HFD-induced downregulation of Glut-2 and elevated the expression of Plg in all four intestinal segments. ConclusionAn HFD can cause a glucose metabolism disorder in the rat intestine and affect body glucose homeostasis. CGA can modify intestinal glucose metabolism by regulating the expression of intestinal glucose transporters and Plg, thereby controlling the levels of blood glucose and insulin to maintain glucose homeostasis.

Alzheimer’s disease-like APP processing in wild-type mice identifies synaptic defects as initial steps of disease progression

BackgroundAlzheimer’s disease (AD) is the most frequent form of dementia in the elderly and no effective treatment is currently available. The mechanisms triggering AD onset and progression are still imperfectly dissected. We aimed at deciphering the modifications occurring in vivo during the very early stages of AD, before the development of amyloid deposits, neurofibrillary tangles, neuronal death and inflammation. Most current AD models based on Amyloid Precursor Protein (APP) overproduction beginning from in utero, to rapidly reproduce the histological and behavioral features of the disease within a few months, are not appropriate to study the early steps of AD development. As a means to mimic in vivo amyloid APP processing closer to the human situation in AD, we used an adeno-associated virus (AAV)-based transfer of human mutant APP and Presenilin 1 (PS1) genes to the hippocampi of two-month-old C57Bl/6 J mice to express human APP, without significant overexpression and to specifically induce its amyloid processing.ResultsThe human APP, βCTF and Aβ42/40 ratio were similar to those in hippocampal tissues from AD patients. Three months after injection the murine Tau protein was hyperphosphorylated and rapid synaptic failure occurred characterized by decreased levels of both PSD-95 and metabolites related to neuromodulation, on proton magnetic resonance spectroscopy (1H-MRS). Astrocytic GLT-1 transporter levels were lower and the tonic glutamatergic current was stronger on electrophysiological recordings of CA1 hippocampal region, revealing the overstimulation of extrasynaptic N-methyl D-aspartate receptor (NMDAR) which precedes the loss of long-term potentiation (LTP). These modifications were associated with early behavioral impairments in the Open-field, Y-maze and Morris Mater Maze tasks.ConclusionsAltogether, this demonstrates that an AD-like APP processing, yielding to levels of APP, βCTF and Aβ42/Aβ40 ratio similar to those observed in AD patients, are sufficient to rapidly trigger early steps of the amyloidogenic and Tau pathways in vivo. With this strategy, we identified a sequence of early events likely to account for disease onset and described a model that may facilitate efforts to decipher the factors triggering AD and to evaluate early neuroprotective strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0070-y) contains supplementary material, which is available to authorized users.

Influence of Familial Renal Glycosuria Due to Mutations in the SLC5A2 Gene on Changes in Glucose Tolerance over Time

Familial renal glycosuria is an inherited disorder resulting in glucose excretion in the urine despite normal blood glucose concentrations. It is most commonly due to mutations in the SLC5A2 gene coding for the glucose transporter SGLT2 in the proximal tubule. Several drugs have been introduced as means to lower glucose in patients with type 2 diabetes targeting SGLT2 resulting in renal glycosuria, but no studies have addressed the potential effects of decreased renal glucose reabsorption and chronic glycosuria on the prevention of glucose intolerance. Here we present data on a large pedigree with renal glycosuria due to two mutations (c.300-303+2del and p.A343V) in the SLC5A2 gene. The mutations, which in vitro affected glucose transport in a cell line model, and the ensuing glycosuria were not associated with better glycemic control during a follow-up period of more than 10 years. One individual, who was compound heterozygous for mutations in the SLC5A2 gene suffered from severe urogenital candida infections and postprandial hypoglycemia. In conclusion, in this family with familial glycosuria we did not find any evidence that chronic loss of glucose in the urine would protect from deterioration of the glucose tolerance over time.

Expression of small intestinal nutrient transporters in embryonic and posthatch turkeys

Nutrients are absorbed in the small intestine through a variety of transporter proteins, which have not been as well characterized in turkeys as in chickens. The objective of this study was to profile the mRNA expression of amino acid and monosaccharide transporters in the small intestine of male and female turkeys. Jejunum was collected during embryonic development (embryonic d 21 and 24, and d of hatch (DOH)) and duodenum, jejunum, and ileum were collected in a separate experiment during posthatch development (DOH, d 7, 14, 21, and 28). Real-time PCR was used to determine expression of aminopeptidase N (APN), one peptide (PepT1), 6 amino acid (ASCT1, bo,+ AT, CAT1, EAAT3, LAT1, y+ LAT2) and 3 monosaccharide (GLUT2, GLUT5, SGLT1) transporters. Data were analyzed by ANOVA using JMP Pro 11.0. APN, bo,+ AT, PepT1, y+ LAT2, GLUT5, and SGLT1 showed increased expression from embryonic d 21 and 24 to DOH. During posthatch, all genes except GLUT2 and SGLT1 were expressed greater in females than males. GLUT2 was expressed the same in males as females and SGLT1 was expressed greater in males than females. All basolateral membrane transporters were expressed greater during early development then decreased with age, while the brush border membrane transporters EAAT3, GLUT5, and SGLT1 showed increased expression later in development. Because turkeys showed high-level expression of the anionic amino acid transporter EAAT3, a direct comparison of tissue-specific expression of EAAT3 between chicken and turkey was conducted. The anionic amino acid transporter EAAT3 showed 6-fold greater expression in the ileum of turkeys at d 14 compared to chickens. This new knowledge can be used not only to better formulate turkey diets to accommodate increased glutamate transport, but also to optimize nutrition for both sexes.

The Hydroxyl Side Chain of a Highly Conserved Serine Residue Is Required for Cation Selectivity and Substrate Transport in the Glial Glutamate Transporter GLT-1/SLC1A2

Glutamate transporters maintain synaptic concentration of the excitatory neurotransmitter below neurotoxic levels. Their transport cycle consists of cotransport of glutamate with three sodium ions and one proton, followed by countertransport of potassium. Structural studies proposed that a highly conserved serine located in the binding pocket of the homologous GltPh coordinates L-aspartate as well as the sodium ion Na1. To experimentally validate these findings, we generated and characterized several mutants of the corresponding serine residue, Ser-364, of human glutamate transporter SLC1A2 (solute carrier family 1 member 2), also known as glutamate transporter GLT-1 and excitatory amino acid transporter EAAT2. S364T, S364A, S364C, S364N, and S364D were expressed in HEK cells and Xenopus laevis oocytes to measure radioactive substrate transport and transport currents, respectively. All mutants exhibited similar plasma membrane expression when compared with WT SLC1A2, but substitutions of serine by aspartate or asparagine completely abolished substrate transport. On the other hand, the threonine mutant, which is a more conservative mutation, exhibited similar substrate selectivity, substrate and sodium affinities as WT but a lower selectivity for Na(+) over Li(+). S364A and S364C exhibited drastically reduced affinities for each substrate and enhanced selectivity for L-aspartate over D-aspartate and L-glutamate, and lost their selectivity for Na(+) over Li(+). Furthermore, we extended the analysis of our experimental observations using molecular dynamics simulations. Altogether, our findings confirm a pivotal role of the serine 364, and more precisely its hydroxyl group, in coupling sodium and substrate fluxes.

Experimental evidence that overexpression of NR2B glutamate receptor subunit is associated with brain vacuolation in adult glutaryl-CoA dehydrogenase deficient mice: A potential role for glutamatergic-induced excitotoxicity in GA I neuropathology

Glutaric aciduria type I (GA I) is biochemically characterized by accumulation of glutaric and 3-hydroxyglutaric acids in body fluids and tissues, particularly in the brain. Affected patients show progressive cortical leukoencephalopathy and chronic degeneration of the basal ganglia whose pathogenesis is still unclear. In the present work we investigated parameters of bioenergetics and redox homeostasis in various cerebral structures (cerebral cortex, striatum and hippocampus) and heart of adult wild type (Gcdh+/+) and glutaryl-CoA dehydrogenase deficient knockout (Gcdh−/−) mice fed a baseline chow. Oxidative stress parameters were also measured after acute lysine overload. Finally, mRNA expression of NMDA subunits and GLT1 transporter was determined in cerebral cortex and striatum of these animals fed a baseline or high lysine (4.7%) chow. No significant alterations of bioenergetics or redox status were observed in these mice. In contrast, mRNA expression of the NR2B glutamate receptor subunit and of the GLT1 glutamate transporter was higher in cerebral cortex of Gcdh−/− mice. Furthermore, NR2B expression was markedly elevated in striatum of Gcdh−/− animals receiving chronic Lys overload. These data indicate higher susceptibility of Gcdh−/− mice to excitotoxic damage, implying that this pathomechanism may contribute to the cortical and striatum alterations observed in GA I patients.