GLUT3 inhibitor discovery through in silico ligand screening and in vivo validation in eukaryotic expression systems.

The passive transport of glucose and related hexoses in human cells is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT3 is a high-affinity glucose transporter primarily responsible for glucose entry in neurons. Changes in its expression have been implicated in neurodegenerative diseases and cancer. GLUT3 inhibitors can provide new ways to probe the pathophysiological role of GLUT3 and tackle GLUT3-dependent cancers. Through in silico screening of an ~ 8 million compounds library against the inward- and outward-facing models of GLUT3, we selected ~ 200 ligand candidates. These were tested for in vivo inhibition of GLUT3 expressed in hexose transporter-deficient yeast cells, resulting in six new GLUT3 inhibitors. Examining their specificity for GLUT1-5 revealed that the most potent GLUT3 inhibitor (G3iA, IC50 ~ 7 µM) was most selective for GLUT3, inhibiting less strongly only GLUT2 (IC50 ~ 29 µM). None of the GLUT3 inhibitors affected GLUT5, three inhibited GLUT1 with equal or twofold lower potency, and four showed comparable or two- to fivefold better inhibition of GLUT4. G3iD was a pan-Class 1 GLUT inhibitor with the highest preference for GLUT4 (IC50 ~ 3.9 µM). Given the prevalence of GLUT1 and GLUT3 overexpression in many cancers and multiple myeloma's reliance on GLUT4, these GLUT3 inhibitors may discriminately hinder glucose entry into various cancer cells, promising novel therapeutic avenues in oncology.

Identification of new GLUT2-selective inhibitors through in silico ligand screening and validation in eukaryotic expression systems.

Glucose is an essential energy source for cells. In humans, its passive diffusion through the cell membrane is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT2 transports both glucose and fructose with low affinity and plays a critical role in glucose sensing mechanisms. Alterations in the function or expression of GLUT2 are involved in the Fanconi-Bickel syndrome, diabetes, and cancer. Distinguishing GLUT2 transport in tissues where other GLUTs coexist is challenging due to the low affinity of GLUT2 for glucose and fructose and the scarcity of GLUT-specific modulators. By combining in silico ligand screening of an inward-facing conformation model of GLUT2 and glucose uptake assays in a hexose transporter-deficient yeast strain, in which the GLUT1-5 can be expressed individually, we identified eleven new GLUT2 inhibitors (IC50 ranging from 0.61 to 19.3 µM). Among them, nine were GLUT2-selective, one inhibited GLUT1-4 (pan-Class I GLUT inhibitor), and another inhibited GLUT5 only. All these inhibitors dock to the substrate cavity periphery, close to the large cytosolic loop connecting the two transporter halves, outside the substrate-binding site. The GLUT2 inhibitors described here have various applications; GLUT2-specific inhibitors can serve as tools to examine the pathophysiological role of GLUT2 relative to other GLUTs, the pan-Class I GLUT inhibitor can block glucose entry in cancer cells, and the GLUT2/GLUT5 inhibitor can reduce the intestinal absorption of fructose to combat the harmful effects of a high-fructose diet.

Investigation of Hypoglycemic Peptides Derived from Conserved Regions of adMc1 to Reveal Their Antidiabetic Activities.

Diabetes mellitus is the most common chronic disorder and leading cause of renal, neurological, and gastrointestinal manifestations in developed and developing countries. Despite of many drugs and combinational therapies, the complications of diabetes are still listed due to severe consequences of those drugs. In past few years, plant-derived drugs draw special attention due to their higher efficacy and fewer side-effects. Momordica charantia also known as bitter melon is referred as an antidiabetic and hypoglycemic plant in native populations of Asia and East Africa. In current study, an in silico approach was used to evaluate the interactions and binding patterns of plant-derived peptides devised from a hypoglycemic protein adMc1 of M. charantia as potential inhibitor of DPP-IV, SGLT1, and GLUT2 receptor proteins. The study has described a novel approach to investigate hypoglycemic peptides to cure diabetes. A total of eighty tetra-, penta-, and hexapeptides were devised from conserved regions of adMc1 homologs. The molecular docking approach using MOE software was employed to reveal inhibiting potentials of devised peptides against three selected proteins. Out of 30 shortlisted ligands six peptides (i.e. SMCG, DECC, TTIT, RTTI, ARNL and TVEV) accomplished the criteria of being good drug candidates against selected receptor proteins following the drugability assessment test. The overall results are acceptable on the basis of ADMET profiling for being good drug candidates against selected proteins.

Inhibition of the facilitative sugar transporters (GLUTs) by tea extracts and catechins.

Tea polyphenolics have been suggested to possess blood glucose lowering properties by inhibiting sugar transporters in the small intestine and improving insulin sensitivity. In this report, we studied the effects of teas and tea catechins on the small intestinal sugar transporters, SGLT1 and GLUTs (GLUT1, 2 and 5). Green tea extract (GT), oolong tea extract (OT), and black tea extract (BT) inhibited glucose uptake into the intestinal Caco-2 cells with GT being the most potent inhibitor (IC50 : 0.077 mg/mL), followed by OT (IC50 : 0.136 mg/mL) and BT (IC50 : 0.56 mg/mL). GT and OT inhibition of glucose uptake was partial non-competitive, with an inhibitor constant (Ki ) = 0.0317 and 0.0571 mg/mL, respectively, whereas BT was pure non-competitive, Ki  = 0.36 mg/mL. Oocytes injected to express small intestinal GLUTs were inhibited by teas, but SGLT1 was not. Furthermore, catechins present in teas were the predominant inhibitor of glucose uptake into Caco-2 cells, and gallated catechins the most potent: CG > ECG > EGCG ≥ GCG when compared to the non-gallated catechins (C, EC, GC, and EGC). In Caco-2 cells, individual tea catechins reduced the SGLT1 gene, but not protein expression levels. In contrast, GLUT2 gene and protein expression levels were reduced after 2 hours exposure to catechins but increased after 24 hours. These in vitro studies suggest teas containing catechins may be useful dietary supplements capable of blunting postprandial glycaemia in humans, including those with or at risk to Type 2 diabetes mellitus.

Small-Molecule Inhibition of Glucose Transporters GLUT-1-4.

Glucose addiction is observed in cancer and other diseases that are associated with hyperproliferation. The development of compounds that restrict glucose supply and decrease glycolysis has great potential for the development of new therapeutic approaches. Addressing facilitative glucose transporters (GLUTs), which are often upregulated in glucose-dependent cells, is therefore of particular interest. This article reviews a selection of potent, isoform-selective GLUT inhibitors and their biological characterization. Potential therapeutic applications of GLUT inhibitors in oncology and other diseases that are linked to glucose addiction are discussed.

Inhibition of GLUT2 transporter by geraniol from Cymbopogon martinii: a novel treatment for diabetes mellitus in streptozotocin-induced diabetic rats.

OBJECTIVE: To isolate and identify the bioactive component from Cymbopogon martinii having GLUT2 transporter inhibitory activity - towards development of a novel strategy for treatment of diabetes mellitus. METHOD: Isolation of bioactive component was carried out using differential solvent extraction, HPTLC and HPLC, and identification was done by GC-MS. In-vitro studies on intestine, liver, kidney and in-vivo assessment by OGTT and long-term treatment on diabetic rats were carried out. KEY FINDINGS: Geraniol was isolated and identified as bioactive component. Intestinal glucose absorption demonstrated 60.28% inhibition of transport at 648.34 µm of geraniol. It was found to inhibit glucose release from liver on adrenaline challenge by 89.82% at 324.17 µm/ml. Kidney glycogen content doubled using 648.34 µm of geraniol as compared to control. Geraniol demonstrated 2.14 times higher renal glucose output than diabetic control. OGTT demonstrated prevention of postprandial spikes. Prolonged treatment for 60 days with 29.37 mm/kg B.W. twice a day of geraniol improved the lipid profile, HbA1C levels and renal parameters. In mRNA studies for 10 days, over expression of GLUT2 was prevented by geraniol. CONCLUSIONS: Inhibition of GLUT2 by geraniol has the potential to reduce hyperglycaemia and prevent secondary complications in diabetes.

Do Endocannabinoids Regulate Glucose Reabsorption in the Kidney?

Diabetic nephropathy (DN), a distinct manifestation of diabetic kidney disease, affects approximately 30% of patients with diabetes. While most attention has been focused on glomerular changes related to DN, there is growing evidence that tubulopathy is a key feature in the pathogenesis of this disease. The renal proximal tubule cells (RPTCs) are particularly sensitive to the deleterious effect of chronic hyperglycemia. However, the cellular changes that control the dysfunction of the RPTCs are not fully understood. Controlling glucose reabsorption in the proximal tubules via inhibition of glucose transporters (GLUT) has emerged as a promising therapeutic in ameliorating DN. Overactivation of the renal endocannabinoid (eCB) system via the cannabinoid-1 receptor (CB1R) contributes to the development of DN, and its blockade by globally acting or peripherally restricted CB1R antagonists has been shown to ameliorate renal dysfunction in different murine models for diabetes. Recently, we have utilized various pharmacological and genetic tools to show that the eCB/CB1R system contributes to the development of DN via regulating the expression, translocation, and activity of the facilitative GLUT2 located in the RPTCs. These findings have the potential to be translated into therapy, and support the rationale for the preclinical development of novel renal-specific CB1R and/or GLUT2 inhibitors for the treatment of DN.

Developmental exposure to DEHP alters hepatic glucose uptake and transcriptional regulation of GLUT2 in rat male offspring.

Type-2-diabetes (T2D) is a long term metabolic disorder characterized by high blood glucose and insulin resistance. It has become an alarming issue globally due to tremendous increase in number of new subjects every year. Apart from the classical factors, there are few non-classical factors such as environmental pollutants, endocrine disrupting chemicals (EDCs) which also play a major role in pathogenesis of T2D. Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer which is an endocrine disrupting chemical. It is used in the plastic industry to give flexibility and durability. Its widespread use resulted in constant presence in the environment and human are under high risk of exposure to this compound. There are literature available stating that DEHP has an impact on glucose homeostasis. Glucose transporter 2 (GLUT2) is a principal transporter of glucose in liver and it is a bi-directional transporter. We investigated whether DEHP exposure during gestation and lactation alters transcriptional regulation of GLUT2 and epigenetics changes in the rat F1 male offspring at adulthood. Pregnant rats were divided into three groups and administered with DEHP (10 and 100 mg /kg /day) or olive oil from gestational day (GD) 9- to postnatal day (PND) 21 through oral gavage. DEHP treated rats showed decreased glucose uptake and oxidation, decreased mRNA levels of insulin receptor (IR), GLUT2 and reduced GLUT2 protein in cytosol but unaltered level in plasma membrane. There are three main transcription factors (SREBP1c, HNF3ß and HNF1α) involved in the regulation of GLUT2 gene and all these proteins were reduced in DEHP exposed groups. A weak interaction of the transcription factors (SREBP1c & HNF1α) with GLUT2 gene promoter was observed in DEHP-treated groups. Hyper- methylation of IR and GLUT2 gene promoter was observed in both the DEHP-exposed groups compared to control. The present study reveals that DEHP exposure alters transcriptional regulation of GLUT2 and imposes epigenetic alteration in IR and GLUT2 gene promoters which plays a significant role in the development of metabolic abnormality in F1 male offspring at adulthood.

High Basolateral Glucose Increases Sodium-Glucose Cotransporter 2 and Reduces Sirtuin-1 in Renal Tubules through Glucose Transporter-2 Detection.

Under diabetic conditions, sodium-glucose cotransporter 2 (SGLT2) for glucose uptake in proximal tubules (PTs) increases, whereas NAD+-dependent protein deacetylase silent mating type information regulation 2 homolog 1 (Sirtuin-1; SIRT1) for PT survival decreases. Therefore, we hypothesized that increased glucose influx by SGLT2 reduces SIRT1 expression. To test this hypothesis, db/db mice with diabetes and high-glucose (HG)-cultured porcine PT LLC-PK1 cells in a two-chamber system were treated with the SGLT2 inhibitor canagliflozin. We also examined SIRT1 and SGLT2 expression in human kidney biopsies. In db/db mice, SGLT2 expression increased with concomitant decreases in SIRT1, but was inhibited by canagliflozin. For determination of the polarity of SGLT2 and SIRT1 expression, LLC-PK1 cells were seeded into Transwell chambers (pore size, 0.4 µm; Becton Dickinson, Oxford, UK). HG medium was added to either or to both of the upper and lower chambers, which corresponded to the apical and basolateral sides of the cells, respectively. In this system, the lower chamber with HG showed increased SGLT2 and decreased SIRT1 expression. Canagliflozin reversed HG-induced SIRT1 downregulation. Gene silencing and inhibitors for glucose transporter 2 (GLUT2) blocked HG-induced SGLT2 expression upregulation. Gene silencing for the hepatic nuclear factor-1α (HNF-1α), whose nuclear translocation was enhanced by HG, blocked HG-induced SGLT2 expression upregulation. Similarly, gene silencing for importin-α1, a chaperone protein bound to GLUT2, blocked HG-induced HNF-1α nuclear translocation and SGLT2 expression upregulation. In human kidney, SIRT1 immunostaining was negatively correlated with SGLT2 immunostaining. Thus, under diabetic conditions, SIRT1 expression in PTs was downregulated by an increase in SGLT2 expression, which was stimulated by basolateral HG through activation of the GLUT2/importin-α1/HNF-1α pathway.

Differential patterns of inhibition of the sugar transporters GLUT2, GLUT5 and GLUT7 by flavonoids.

Only limited data are available on the inhibition of the sugar transporter GLUT5 by flavonoids or other classes of bioactives. Intestinal GLUT7 is poorly characterised and no information exists concerning its inhibition. We aimed to study the expression of GLUT7 in Caco-2/TC7 intestinal cells, and evaluate inhibition of glucose transport by GLUT2 and GLUT7, and of fructose transport by GLUT2, GLUT5 and GLUT7, by flavonoids. Differentiated Caco-2/TC7 cell monolayers were used to investigate GLUT7 expression, as well as biotinylation and immunofluorescence to assess GLUT7 location. For mechanistic sugar transport studies, X. laevis oocytes were injected with individual mRNA, and GLUT protein expression on oocyte membranes was confirmed. Oocytes were incubated with D-[14C(U)]-glucose or D-[14C(U)]-fructose in the presence of flavonoids, and uptake was estimated by liquid scintilation counting. In differentiated Caco-2/TC7 cell monolayers, GLUT7 was mostly expressed apically. When applied apically, or to both compartments, sorbitol, galactose, L-glucose or sucrose did not affect GLUT7 mRNA expression. Fructose applied to both sides increased GLUT7 mRNA (13%, p ≤ 0.001) and total GLUT7 protein (2.7-fold, p ≤ 0.05), while the ratio between apical, basolateral and total GLUT7 protein was unchanged. In the X. laevis oocyte model, GLUT2-mediated glucose and fructose transport were inhibited by quercetin, (-)-epigallocatechin gallate (EGCG) and apigenin, GLUT5-mediated fructose transport was inhibited by apigenin and EGCG, but not by quercetin, and GLUT7-mediated uptake of both glucose and fructose was inhibited by apigenin, but not by quercetin nor EGCG. Expression of GLUT7 was increased by fructose, but only when applied to Caco-2/TC7 cells both apically and basolaterally. Since GLUT2, GLUT5 and GLUT7 show different patterns of inhibition by the tested flavonoids, we suggest that they have the potential to be used as investigational tools to distinguish sugar transporter activity in different biological settings.

Homoisoflavonoids Are Potent Glucose Transporter 2 (GLUT2) Inhibitors: A Potential Mechanism for the Glucose-Lowering Properties of Polygonatum odoratum.

Foods of high carbohydrate content such as sucrose or starch increase postprandial blood glucose concentrations. The glucose absorption system in the intestine comprises two components: sodium-dependent glucose transporter-1 (SGLT1) and glucose transporter 2 (GLUT2). Here five sappanin-type (SAP) homoisoflavonoids were identified as novel potent GLUT2 inhibitors, with three of them isolated from the fibrous roots of Polygonatum odoratum (Mill.) Druce. SAP homoisolflavonoids had a stronger inhibitory effect on 25 mM glucose transport (41.6 ± 2.5, 50.5 ± 7.6, 47.5 ± 1.9, 42.6 ± 2.4, and 45.7 ± 4.1% for EA-1, EA-2, EA-3, MOA, and MOB) than flavonoids (19.3 ± 2.2, 11.5 ± 3.7, 16.4 ± 2.4, 5.3 ± 1.0, 3.7 ± 2.2, and 18.1 ± 2.4% for apigenin, luteolin, quercetin, naringenin, hesperetin, and genistein) and phloretin (28.1 ± 1.6%) at 15 µM. SAP homoisoflavonoids and SGLT1 inhibitors were found to synergistically inhibit the uptake of glucose using an in vitro model comprising Caco-2 cells. This observed new mechanism of the glucose-lowering action of P. odoratum suggests that SAP homoisoflavonoids and their combination with flavonoid monoglucosides show promise as naturally functional ingredients for inclusion in foods and drinks designed to control postprandial glucose levels.

Mice Fed a High-Cholesterol Diet Supplemented with Quercetin-3-Glucoside Show Attenuated Hyperlipidemia and Hyperinsulinemia Associated with Differential Regulation of PCSK9 and LDLR in their Liver and Pancreas.

SCOPE: Hepatic LDL receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) regulate the clearance of plasma LDL-cholesterol (LDL-C): LDLR promotes it, and PCSK9 opposes it. These proteins also express in pancreatic ß cells. Using cultured hepatocytes, we previously showed that the plant flavonoid quercetin-3-glucoside (Q3G) inhibits PCSK9 secretion, stimulated LDLR expression, and enhanced LDL-C uptake. Here, we examine whether Q3G supplementation could reverse the hyperlipidemia and hyperinsulinemia of mice fed a high-cholesterol diet, and how it affects hepatic and pancreatic LDLR and PCSK9 expression. METHODS AND RESULTS: For 12 weeks, mice are fed a low- (0%) or high- (1%) cholesterol diet (LCD or HCD), supplemented or not with Q3G at 0.05 or 0.1% (w/w). Tissue LDLR and PCSK9 is analyzed by immunoblotting, plasma PCSK9 and insulin by ELISA, and plasma cholesterol and glucose by colorimetry. In LCD-fed mice, Q3G has no effect. In HCD-fed mice, it attenuates the increase in plasma cholesterol and insulin, accentuates the decrease in plasma PCSK9, and increases hepatic and pancreatic LDLR and PCSK9. In cultured pancreatic ß cells, however, it stimulates PCSK9 secretion. CONCLUSION: In mice, dietary Q3G could counter HCD-induced hyperlipidemia and hyperinsulinemia, in part by oppositely modulating hepatic and pancreatic PCSK9 secretion.

Modulation of Renal GLUT2 by the Cannabinoid-1 Receptor: Implications for the Treatment of Diabetic Nephropathy.

Altered glucose reabsorption via the facilitative glucose transporter 2 (GLUT2) during diabetes may lead to renal proximal tubule cell (RPTC) injury, inflammation, and interstitial fibrosis. These pathologies are also triggered by activating the cannabinoid-1 receptor (CB1R), which contributes to the development of diabetic nephropathy (DN). However, the link between CB1R and GLUT2 remains to be determined. Here, we show that chronic peripheral CB1R blockade or genetically inactivating CB1Rs in the RPTCs ameliorated diabetes-induced renal structural and functional changes, kidney inflammation, and tubulointerstitial fibrosis in mice. Inhibition of CB1R also downregulated GLUT2 expression, affected the dynamic translocation of GLUT2 to the brush border membrane of RPTCs, and reduced glucose reabsorption. Thus, targeting peripheral CB1R or inhibiting GLUT2 dynamics in RPTCs has the potential to treat and ameliorate DN. These findings may support the rationale for the clinical testing of peripherally restricted CB1R antagonists or the development of novel renal-specific GLUT2 inhibitors against DN.

Dynamic Profiling of Insulin Secretion and ATP Generation in Isolated Human and Mouse Islets Reveals Differential Glucose Sensitivity.

BACKGROUND/AIMS: Rodent islets are often used for basic science research but they do not always recapitulate signalling events in human islets. This study evaluated the glucose-dependent responses of human and mouse islets in terms of dynamic insulin secretion, metabolic coupling and the role of glucose transporters. METHODS: Glucose-induced insulin secretion from isolated mouse and human islets was profiled by perifusion and islet ATP levels were measured by chemoluminescence assay. Glucose transporter expression was determined by qPCR and western blotting. RESULTS: Human islets show a left-shifted glucose concentration-insulin secretion profile compared to mouse islets. These data are consistent with glucose transporter expression, with human islets expressing mainly GLUT1 and GLUT3, and GLUT2 being the predominant transporter in mouse islets. Using the GLUT1 inhibitor STF-31 we unveiled an important role for GLUT1 for differences in glucose-induced insulin secretion profiles observed between the two species. CONCLUSION: The high affinity of GLUT1/3 for glucose reflects the left-shifted glucose-induced insulin secretory response of human islets and the impairment of insulin secretion from human islets after STF-31 treatment indicates an important role for GLUT1 in human islet stimulus-secretion coupling. Our data provide further insight into key differences between insulin secretion regulation in mouse and human islets.

Green and Chamomile Teas, but not Acarbose, Attenuate Glucose and Fructose Transport via Inhibition of GLUT2 and GLUT5.

SCOPE: High glycaemic sugars result in blood-glucose spikes, while large doses of post-prandial fructose inundate the liver, causing an imbalance in energy metabolism, both leading to increased risk of metabolic malfunction and type 2 diabetes. Acarbose, used for diabetes management, reduces post-prandial hyperglycaemia by delaying carbohydrate digestion. METHODS AND RESULTS: Chamomile and green teas both inhibited digestive enzymes (α-amylase and maltase) related to intestinal sugar release, as already established for acarbose. However, acarbose had no effect on uptake of sugars using both differentiated human Caco-2 cell monolayers and Xenopus oocytes expressing human glucose transporter-2 (GLUT2) and GLUT5. Both teas effectively inhibited transport of fructose and glucose through GLUT2 inhibition, while chamomile tea also inhibited GLUT5. Long term incubation of Caco-2/TC7 cells with chamomile tea for 16 h or 4 days did not enhance the observed effects, indicating that inhibition is acute. Sucrase activity was directly inhibited by green tea and acarbose, but not chamomile. CONCLUSION: These findings show that chamomile and green teas are potential tools to manage absorption and metabolism of sugars with efficacy against high sugar bolus stress inflicted, for example, by high fructose syrups, where the drug acarbose would be ineffective.

Natural Products as Lead Compounds for Sodium Glucose Cotransporter (SGLT) Inhibitors.

Glucose homeostasis is maintained by antagonistic hormones such as insulin and glucagon as well as by regulation of glucose absorption, gluconeogenesis, biosynthesis and mobilization of glycogen, glucose consumption in all tissues and glomerular filtration, and reabsorption of glucose in the kidneys. Glucose enters or leaves cells mainly with the help of two membrane integrated transporters belonging either to the family of facilitative glucose transporters (GLUTs) or to the family of sodium glucose cotransporters (SGLTs). The intestinal glucose absorption by endothelial cells is managed by SGLT1, the transfer from them to the blood by GLUT2. In the kidney SGLT2 and SGLT1 are responsible for reabsorption of filtered glucose from the primary urine, and GLUT2 and GLUT1 enable the transport of glucose from epithelial cells back into the blood stream.The flavonoid phlorizin was isolated from the bark of apple trees and shown to cause glucosuria. Phlorizin is an inhibitor of SGLT1 and SGLT2. With phlorizin as lead compound, specific inhibitors of SGLT2 were developed in the last decade and some of them have been approved for treatment mainly of type 2 diabetes. Inhibition of SGLT2 eliminates excess glucose via the urine. In recent times, the dual SGLT1/SGLT2 inhibitory activity of phlorizin has served as a model for the development and testing of new drugs exhibiting both activities.Besides phlorizin, also some other flavonoids and especially flavonoid enriched plant extracts have been investigated for their potency to reduce postprandial blood glucose levels which can be helpful in the prevention and supplementary treatment especially of type 2 diabetes.

Grape seed proanthocyanidins protects against cadmium induced oxidative pancreatitis in rats by attenuating oxidative stress, inflammation and apoptosis via Nrf-2/HO-1 signaling.

The present study has been designed and carried out to explore the role of grape seed proanthocyanidins (GSP) in the pancreas of cadmium (Cd)-induced cellular oxidative stress-mediated toxicity in rats. Four groups of healthy rats were given oral doses of Cd (5-mg/kg BW) and to identify the possible mechanism of action of GSP 100-mg/kg BW was selected and was given 90 min before Cd intoxication. The causative molecular and cellular mechanism of Cd was determined using various biochemical assays, histology, western blotting and ELISA. Cd intoxication revealed increased levels of proinflammatory cytokines (TNF-α, IL1ß and IFN-γ), reduced levels of cellular defense proteins (Nrf-2 and HO-1) and glucose transporter (GLUT-2 and GLUT-4) along with the enhanced levels of signaling molecules of apoptosis (cleaved Caspase-12/9/8/3) in the pancreas of Cd-intoxicated rats. Results suggested that the treatment with GSP reduced blood glucose level, increased plasma insulin and mitigated oxidative stress-related markers. GSP protects pancreatic tissue by attenuated inflammatory responses and inhibited apoptosis. This uniqueness and absence of any detectable adverse effect of GSP proposes the possibility of using it as an effective protector in the oxidative stress-mediated pancreatic dysfunction in rats.

The ergogenic supplement ß-hydroxy-ß-methylbutyrate (HMB) attenuates insulin resistance through suppressing GLUT-2 in rat liver.

This study investigates the effect of the ergogenic supplement ß-hydroxy-ß-methylbutyrate (HMB) on insulin resistance induced by high-fructose diet (HFD) in rats. Male Sprague Dawley rats were fed 60% HFD for 12 weeks and HMB (320 mg·kg(-1)·day(-1), orally) for 4 weeks. HFD significantly increased fasting insulin, fasting glucose, glycosylated hemoglobin (HBA1C), liver glycogen content, and homeostasis model assessment of insulin resistance (HOMA-IR) index, while it decreased glucose and insulin tolerance. Furthermore, HFD significantly increased serum triglycerides (TG), low density lipoprotein cholesterol (LDL-C), and very low density lipoprotein cholesterol (VLDL-C) levels, while it significantly decreased high density lipoprotein cholesterol (HDL-C). Moreover, HFD significantly increased mRNA expression of glucose transporter type-2 (GLUT-2), the mammalian target of rapamycin (mTOR), and sterol regulatory element-binding protein-1c (SREBP-1c) but decreased peroxisome proliferator-activated receptor-alpha (PPAR-α) in liver. Aortic relaxation to acetylcholine (ACh) was impaired and histopathology showed severe hepatic steatosis. HMB significantly increased insulin tolerance and decreased fasting insulin, HOMA-IR, HBA1C, hepatic glycogen content, serum TG, LDL-C, and VLDL-C. Additionally, HMB enhanced ACh-induced relaxation, ameliorated hepatic steatosis, and decreased mRNA expression of GLUT-2. In conclusion, HMB may attenuate insulin resistance and hepatic steatosis through inhibiting GLUT-2 in liver.

Attenuation of glucose transport across Caco-2 cell monolayers by a polyphenol-rich herbal extract: interactions with SGLT1 and GLUT2 transporters.

Previous studies have indicated that secondary plant metabolites may modulate glucose absorption in the small intestine. We have characterized a polyphenol-rich herbal extract and its potential intestinal metabolites by LC-MS(2) and investigated the inhibition of glucose transporters SGLT1 and GLUT2 using the well-characterized Caco-2 intestinal model. Differentiated Caco-2 monolayers were incubated with an extract of a mixture of herbs and spices. Glucose transport under sodium-dependent and sodium-free conditions was determined by radiochemical detection of D-[U-(14) C]-glucose. A 54% decrease in transport was observed compared to control. Using sodium-dependent and sodium-free conditions, we demonstrate that the inhibition of GLUT2 was greater than SGLT1. Glycosidase and esterase enzymatic hydrolysis was used to assess the impact of metabolism on the efficacy of inhibition. Glucose transport across the membrane was reduced by 70% compared to the control and was associated with significant increases in flavonoid aglycones, caffeic acid, and p-coumaric acid. These results suggest that intact and hydrolyzed polyphenols, likely to be found in the lumen after ingestion of the supplement, play an important role in the attenuation of glucose absorption and may have potentially beneficial antiglycemic effects in the body.

The regulation of K- and L-cell activity by GLUT2 and the calcium-sensing receptor CasR in rat small intestine.

Intestinal enteroendocrine cells (IECs) secrete gut peptides in response to both nutrients and non-nutrients. Glucose and amino acids both stimulate gut peptide secretion. Our hypothesis was that the facilitative glucose transporter, GLUT2, could act as a glucose sensor and the calcium-sensing receptor, CasR, could detect amino acids in the intestine to modify gut peptide secretion. We used isolated loops of rat small intestine to study the secretion of gluco-insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY) secretion stimulated by luminal perfusion of nutrients or bile acid. Inhibition of the sodium-dependent glucose cotransporter 1 (SGLT1) with phloridzin partially inhibited GIP, GLP-1 and PYY secretion by 45%, suggesting another glucose sensor might be involved in modulating peptide secretion. The response was completely abolished in the presence of the GLUT2 inhibitors phloretin or cytochalasin B. Given that GLUT2 modified gut peptide secretion stimulated by glucose, we investigated whether it was involved in the secretion of gut peptide by other gut peptide secretagogues. Phloretin completely abolished gut peptide secretion stimulated by artificial sweetener (sucralose), dipeptide (glycylsarcosine), lipid (oleoylethanolamine), short chain fatty acid (propionate) and major rat bile acid (taurocholate) indicating a fundamental position for GLUT2 in the gut peptide secretory mechanism. We investigated how GLUT2 was able to influence gut peptide secretion mediated by a diverse range of stimulators and discovered that GLUT2 affected membrane depolarisation through the closure of K+(ATP)-sensitive channels. In the absence of SGLT1 activity (or presence of phloridzin), the secretion of GIP, GLP-1 and PYY was sensitive to K+(ATP)-sensitive channel modulators tolbutamide and diazoxide. L-amino acids phenylalanine (Phe), tryptophan (Trp), asparagine (Asn), arginine (Arg) and glutamine (Gln) also stimulated GIP, GLP-1 and PYY secretion, which was completely abolished when extracellular Ca2+ was absent. The gut peptide response stimulated by the amino acids was also blocked by the CasR inhibitor Calhex 231 and augmented by the CasR agonist NPS-R568. GLUT2 and CasR regulate K- and L-cell activity in response to nutrient and non-nutrient stimuli.