Mangiferin promotes intestinal elimination of uric acid by modulating intestinal transporters.

Increasing evidence shows that the intestinal tract plays an important role in maintaining urate homeostasis and might be a potential therapeutic target for hyperuricaemia. However, uric acid-lowering drugs available in the clinic do not target intestinal excretion as a therapeutic strategy. We previously reported that mangiferin had potent hypouricaemic effects in hyperuricaemic animals. However, the underlying mechanisms are not completely clear. Here, we investigated the effects of mangiferin on the intestinal excretion of urate and its underlying mechanisms. The data revealed that mangiferin concentration-dependently promoted the intestinal secretion of endogenous urate in in situ intestinal closed loops in normal and hyperuricaemic mice, as well as inhibited the absorption of exogenous uric acid perfused into the intestinal loops in rats. Administration of mangiferin not only decreased the serum urate levels in the hyperuricaemic mice but also increased the protein expression of ATP-binding cassette transporter, subfamily G, member 2 (ABCG2) and inhibited the protein expression of glucose transporter 9 (GLUT 9) in the intestine. These findings suggested that intestinal ABCG2 and GLUT9 might be pivotal and possible action sites for the observed hypouricaemic effects. Moreover, no significant changes in intestinal xanthine oxidoreductase activities were observed, suggesting that mangiferin did not affect intestinal uric acid generation in the hyperuricaemic mice. Overall, promoting intestinal elimination of urate by upregulating ABCG2 expression and downregulating GLUT9 expression might be an important mechanism underlying mangiferin lowering serum uric acid levels. Mangiferin supplementation might be beneficial for the prevention and treatment of hyperuricaemia.

Tea catechin suppresses adipocyte differentiation accompanied by down-regulation of PPARgamma2 and C/EBPalpha in 3T3-L1 cells.

Obesity is a serious health problem, and its prevention is promoted through life style including diet and exercise. In this study, we investigated the suppressive effects of tea catechin on the differentiation of 3T3-L1 preadipocytes to adipocytes. (-)-Catechin 3-gallate (CG), (-)-epigallocatechin (EGC), (-)-epicatechin 3-gallate, and (-)-epigallocatechin 3-gallate at 5 muM suppressed intracellular lipid accumulation. The suppressive effects of CG and EGC were stronger than the others, and CG and EGC also suppressed the activity of glycerol-3-phosphate dehydrogenase as a differentiation marker. These catechins inhibited the expression of peroxisome proliferator-activated receptor (PPAR) gamma2 and CCAAT/enhancer-binding protein (C/EBP) alpha, both of which act as key transcription factors at an early stage of differentiation, followed by the expression of glucose transporter (GLUT) 4 at a later stage. In addition, the catechins did not affect the phosphorylation status of the insulin signal pathway. Thus, catechin suppressed adipocyte differentiation accompanied by the down-regulation of PPARgamma2, C/EBPalpha, and GLUT4. These results suggest that tea catechin prevents obesity through the suppression of adipocyte differentiation.

Wild ginseng prevents the onset of high-fat diet induced hyperglycemia and obesity in ICR mice.

Ginseng is a shade-loving perennial herb that is cultivated mainly in Korea, Japan, and China. The ginseng root has been used as a tonic remedy, and its antidiabetic activity has been demonstrated as early as 1920s. Although wild ginseng was anecdotally thought to be superior to cultivated ginseng as far as pharmacological properties were concerned, there have been no prior reports on the antidiabetic effect of wild ginseng. In this study, we investigated the preventative anti-diabetic and anti-obese effects of wild ginseng ethanol extract (WGEE). In the preventive experiment, WGEE co-administered with a high fat diet significantly inhibited body weight gain, fasting blood glucose, triglyceride, and free fatty acid levels in a dose dependent manner. WGEE-treated mice at doses of 250 and 500 mg/kg improved the insulin resistance index by 55% and 61% compared to the high fat diet (HFD) control, respectively. Diameters of white and brown adipocytes were also decreased by 62% and 46% in the WG500-treated group compared to those in HFD fed control mice. Taken together, WGEE has potential as a preventive agent for type 2 diabetes mellitus (and possibly obesity) and deserves clinical trial in the near future.

Effect of ascorbate on the activity of hypoxia-inducible factor in cancer cells.

Hypoxia-inducible factor (HIF) plays an important role in determining patterns of gene expression in cancer. HIF is down-regulated in oxygenated cells by a series of Fe (II) and 2-oxoglutarate dependent dioxygenases that hydroxylate specific residues in the regulatory HIF-alpha subunits. Because these enzymes require ascorbate for activity in vitro we analyzed the effects of ascorbate on HIF in human cancer cell lines. Ascorbate at physiological concentrations (25 micro M) strikingly suppressed HIF-1alpha protein levels and HIF transcriptional targets, particularly when the system was oncogenically activated in normoxic cells. Similar results were obtained with iron supplementation. These results indicate that both ascorbate and iron availability have major effects on HIF, and imply that the system is commonly regulated by limiting hydroxylase activity under normoxic tissue culture conditions.

Regulation of glucose transport and transporter 4 (GLUT-4) in muscle and adipocytes of sucrose-fed rats: effects of N-3 poly- and monounsaturated fatty acids.

The goal of this study was to compare the short-term effects of dietary n-3 polyunsaturated (fish oil) and monounsaturated (olive oil) fatty acids on glucose transport, plasma glucose and lipid controls in a dietary insulin resistance model using sucrose-fed rats. The underlying cellular and molecular mechanisms were also determined in the muscle and adipose tissue. Male Sprague-Dawley rats (5 weeks old) were randomized for diets containing 57.5 % (w/w) sucrose and 14 % lipids as either fish oil (SF), olive oil (SO) or a mixture of standard oils (SC) for 3 weeks. A fourth control group (C) was fed a diet containing 57.5 % starch and 14 % standard oils. After three weeks on the diet, body weight was comparable in the four groups. The sucrose-fed rats were hyperglycemic and hyperinsulinemic in response to glucose load. The presence of fish oil in the sucrose diet prevented sucrose-induced hyperinsulinemia and hypertriglyceridemia, but had no effect on plasma glucose levels. Insulin-stimulated glucose transport in adipocytes increased after feeding with fish oil (p < 0.005). These modifications were associated with increased Glut-4 protein (p < 0.05) and mRNA levels in adipocytes. In the muscle, no effect was found on Glut-4 protein levels. Olive oil, however, could not bring about any improvement in plasma insulin, plasma lipids or Glut-4 protein levels. We therefore conclude that the presence of fish oil, in contrast to olive oil, prevents insulin resistance and hypertriglyceridemia in rats on a sucrose diet, and restores Glut-4 protein quantity in adipocytes but not in muscle at basal levels. Dietary regulation of Glut-4 proteins appears to be tissue specific and might depend on insulin stimulation and/or duration of dietary interventions.

Identification of a mammalian H(+)-myo-inositol symporter expressed predominantly in the brain.

Inositol and its phosphorylated derivatives play a major role in brain function, either as osmolytes, second messengers or regulators of vesicle endo- and exocytosis. Here we describe the identification and functional characterization of a novel H(+)-myo- inositol co-transporter, HMIT, expressed predominantly in the brain. HMIT cDNA encodes a 618 amino acid polypeptide with 12 predicted transmembrane domains. Functional expression of HMIT in Xenopus oocytes showed that transport activity was specific for myo-inositol and related stereoisomers with a Michaelis-Menten constant of approximately 100 microM, and that transport activity was strongly stimulated by decreasing pH. Electrophysiological measurements revealed that transport was electrogenic with a maximal transport activity reached at pH 5.0. In rat brain membrane preparations, HMIT appeared as a 75-90 kDa protein that could be converted to a 67 kDa band upon enzymatic deglycosylation. Immunofluorescence microscopy analysis showed HMIT expression in glial cells and some neurons. These data provide the first characterization of a mammalian H(+)-coupled myo- inositol transporter. Predominant central expression of HMIT suggests that it has a key role in the control of myo-inositol brain metabolism.

Elevated expression of glucose transporter-1 in hypothalamic ependymal cells not involved in the formation of the brain-cerebrospinal fluid barrier.

Glucose transporters play an essential role in the acquisition of glucose by the brain. Elevated expression of glucose transporter-1 has been detected in endothelial cells of the blood-brain barrier and in choroid plexus cells of the blood-cerebrospinal fluid barrier. On the other hand, there is a paucity of information on the expression of glucose transporters in the ependymal cells that line the walls of the cerebral ventricles. The tanycytes are specialized ependymal cells localized in circumventricular organs such as the median eminence that can be segregated into at least three types, alpha, beta1 and beta2. The beta2 tanycytes form tight junctions and participate in the formation of the cerebrospinal fluid-median eminence barrier. Using immunocytochemistry and in situ hybridization, we analyzed the expression of hexose transporters in rat and mouse hypothalamic tanycytes. In both species, immunocytochemical analysis revealed elevated expression of glucose transporter-1 in alpha and beta1 tanycytes. Intense anti-glucose transporter-1 staining was observed in cell processes located throughout the arcuate nucleus, in the end-feet reaching the lateral sulcus of the infundibular region, and in cell processes contacting the hypothalamic capillaries. On the other hand, there was very low expression of glucose transporter-1 in beta2 tanycytes involved in barrier function. In contrast with the results of the cytochemical analysis, in situ hybridization revealed that tanycytes alpha, beta1, and beta2 express similar levels of glucose transporter-1 mRNA. Further analysis using anti-glial fibrillary acidic protein antibodies to identify areas rich in astrocytes revealed that astrocytes were absent from areas containing alpha and beta1 tanycytes, but were abundant in regions containing the barrier-forming beta2 tanycytes. Overall, our data reveal a lack of correlation between participation in barrier function and expression of glucose transporter-1 in hypothalamic tanycytes. Given the virtual absence of astrocytes in areas rich in alpha and beta1 tanycytes, we speculate whether the tanycytes might have astrocyte-like functions and participate in the metabolic coupling between glia and neurons in the hypothalamic area.

[Effect of alcohol extract of Cornus officinalis Sieb. et Zucc on GLUT4 expression in skeletal muscle in type 2 (non-insulin-dependent) diabetic mellitus rats].

OBJECTIVE: Based on its effects of decreasing postprandial plasma glucose and increasing insulin level in non-insulin-dependent diabetic mellitus (NIDDM) rats, we studied the effects of Alcohol extract of Cornus officinalis Sieb. et Zucc on the GLUT4 expression in NIDDM model rats. METHOD: The rat model of NIDDM was made. The animals were divided into three groups(six for each group): group I: control; group II: NIDDM model; group III: NIDDM model + Cornus officinalis Sieb. et Zucc. The drug was given orally to animals one time a day, uninterrupted for a month. The GLUT4 mRNA and its protein expression in skeletal muscle were observed with Northern blot and Western blot method, respectively. RESULT: The GLUT4 mRNA expression in skeletal muscle was decreased remarkably in NIDDM rats (P < 0.01, compared to control group). Alcohol extract of Cornus officinalis Sieb. et Zucc increased GLUT4 mRNA expression in NIDDM rats(P < 0.01), compared to NIDDM model group). The result of GLUT4 protein expression was similar to GLUT4 mRNA expression. CONCLUSION: This experiment demonstrated that alcohol extract of Cornus officinalis Sieb. et Zucc can increase GLUT4 mRNA and its protein expression in NIDDM rats through promoting proliferation of islet and increasing postprandial secretion of insulin and therefore accelerate glucose transport.

Exercise-induced overexpression of key regulatory proteins involved in glucose uptake and metabolism in tetraplegic persons: molecular mechanism for improved glucose homeostasis.

Complete spinal cord lesion leads to profound metabolic abnormalities and striking changes in muscle morphology. Here we assess the effects of electrically stimulated leg cycling (ESLC) on whole body insulin sensitivity, skeletal muscle glucose metabolism, and muscle fiber morphology in five tetraplegic subjects with complete C5-C7 lesions. Physical training (seven ESLC sessions/wk for 8 wk) increased whole body insulin-stimulated glucose uptake by 33+/-13%, concomitant with a 2.1-fold increase in insulin-stimulated (100 microU/ml) 3-O-methylglucose transport in isolated vastus lateralis muscle. Physical training led to a marked increase in protein expression of GLUT4 (378+/-85%), glycogen synthase (526+/-146%), and hexokinase II (204+/-47%) in vastus lateralis muscle, whereas phosphofructokinase expression (282+/-97%) was not significantly changed. Hexokinase II activity was significantly increased, whereas activity of phosphofructokinase, glycogen synthase, and citrate synthase was not changed after training. Muscle fiber type distribution and fiber area were markedly altered compared to able-bodied subjects before ESLC training, with no change noted in either parameter after ECSL training. In conclusion, muscle contraction improves insulin action on whole body and cellular glucose uptake in cervical cord-injured persons through a major increase in protein expression of key genes involved in the regulation of glucose metabolism. Furthermore, improvements in insulin action on glucose metabolism are independent of changes in muscle fiber type distribution.

Na/D-glucose cotransport and SGLT1 expression in hen colon correlates with dietary Na+.

We have tested whether separately varying the content of either Na or Cl in diets causes earlier observed increase in Na-coupled sugar and amino acid transport induced by high NaCl diets in hen colon. A comparison was also made between the dependence of the Na-coupled transport on a pure wheat/barley/soya diet against a diet with supplements of essential amino acids, fatty acids, vitamins, and trace elements, as a test for possible elimination of the cotransporters due to a deficient diet. Na/nutrient-coupled transport was measured as changes in short circuit current. The level of expressed Na/glucose cotransporters, SGLT1, due to dietary alterations was followed by quantitative Western blot and immunodetection of SGLT1 in colon, and the dietary effects on plasma aldosterone were assessed as well. An observed switch in transport from amiloride-sensitive electrodiffusive Na transport to phlorizin-sensitive Na/D-glucose cotransport and Na/amino acid-coupled transport is caused solely by increasing Na+ in the diet. Thus, neither dietary Cl- nor the dietary supplements altered the expression of Na(+)-coupled nutrient transport processes. Corroborating these findings, only Na+ in the diet increased the expression of SGLT1 in colon epithelium and suppressed aldosterone level in plasma.

Colocalization of glucagon-like peptide-1 (GLP-1) receptors, glucose transporter GLUT-2, and glucokinase mRNAs in rat hypothalamic cells: evidence for a role of GLP-1 receptor agonists as an inhibitory signal for food and water intake.

This study was designed to determine the possible role of brain glucagon-like peptide-1 (GLP-1) receptors in feeding behavior. In situ hybridization showed colocalization of the mRNAs for GLP-1 receptors, glucokinase, and GLUT-2 in the third ventricle wall and adjacent arcuate nucleus, median eminence, and supraoptic nucleus. These brain areas are considered to contain glucose-sensitive neurons mediating feeding behavior. Because GLP-1 receptors, GLUT-2, and glucokinase are proteins involved in the multistep process of glucose sensing in pancreatic beta cells, the colocalization of specific GLP-1 receptors and glucose sensing-related proteins in hypothalamic neurons supports a role of this peptide in the hypothalamic regulation of macronutrient and water intake. This hypothesis was confirmed by analyzing the effects of both systemic and central administration of GLP-1 receptor ligands. Acute or subchronic intraperitoneal administration of GLP-1 (7-36) amide did not modify food and water intake, although a dose-dependent loss of body weight gain was observed 24 h after acute administration of the higher dose of the peptide. By contrast, the intracerebroventricular (i.c.v.) administration of GLP-1 (7-36) amide produced a biphasic effect on food intake characterized by an increase in the amount of food intake after acute i.c.v. delivery of 100 ng of the peptide. There was a marked reduction of food ingestion with the 1,000 and 2,000 ng doses of the peptide, which also produced a significant decrease of water intake. These effects seemed to be specific because i.c.v. administration of GLP-1 (1-37), a peptide with lower biological activity than GLP-1 (7-36) amide, did not change feeding behavior in food-deprived animals. Exendin-4, when given by i.c.v. administration in a broad range of doses (0.2, 1, 5, 25, 100, and 500 ng), proved to be a potent agonist of GLP-1 (7-36) amide. It decreased, in a dose-dependent manner, both food and water intake, starting at the dose of 25 ng per injection. Pretreatment with an i.c.v. dose of a GLP-1 receptor antagonist [exendin (9-39); 2,500 ng] reversed the inhibitory effects of GLP-1 (7-36) amide (1,000 ng dose) and exendin-4 (25 ng dose) on food and water ingestion. These findings suggest that GLP-1 (7-36) amide may modulate both food and drink intake in the rat through a central mechanism.

Sodium-glucose cotransporters display sodium- and phlorizin-dependent water permeability.

Expression of Na(+)-glucose cotransporters of the SGLT-1 type by Xenopus laevis oocytes increased the osmotic water permeability (Pf) of oocytes by a factor of 1.9-2.8, in the presence and in the absence of 5 mM extracellular glucose. The Pf increase was correlated with the amount of SGLT-1 cRNA injected. In oocytes expressing SGLT-1, either addition of phlorizin to the medium or the replacement of Na+ by choline inhibited the uptake of methyl-alpha-D-glucopyranoside, a specific substrate for SGLT-1, and returned oocyte Pf to its level in uninjected oocytes. Phlorizin inhibited the SGLT-1-attributable increase in Pf with an inhibition constant (Ki) of 6.1 microM, a value analogous to the Ki for phlorizin inhibition of sugar uptake. However, neither the presence of phlorizin nor the absence of extracellular Na+ significantly affected the increase in Pf elicited in oocytes expressing GLUT-1, a facilitative glucose transporter. These findings suggest that SGLT-1 forms a pore that allows the transmembrane passage of water and that water and glucose traverse the protein through this pore. The finding that removal of extracellular Na+ abolishes the increase in Pf attributable to SGLT-1 suggests that extracellular Na+ is required to maintain patency of this transporter's water-permeable transmembrane pore.

The glucose sensor protein glucokinase is expressed in glucagon-producing alpha-cells.

Expression of glucokinase in hepatocytes and pancreatic 6-cells is of major physiologic importance to mammalian glucose homeostasis. Liver glucokinase catalyzes the first committed step in the disposal of glucose, and beta-cell glucokinase catalyzes a rate-limiting step required for glucose-regulated insulin release. The present study reports the expression of glucokinase in rat glucagon-producing alpha-cells, which are negatively regulated by glucose. Purified rat alpha-cells express glucokinase mRNA and protein with the same transcript length, nucleotide sequence, and immunoreactivity as the beta-cell isoform. Glucokinase activity accounts for more than 50% of glucose phosphorylation in extracts of alpha-cells and for more than 90% of glucose utilization in intact cells. The glucagon-producing tumor MSL-G-AN also contained glucokinase mRNA, protein, and enzymatic activity. These data indicate that glucokinase may serve as a metabolic glucose sensor in pancreatic alpha-cells and, hence, mediate a mechanism for direct regulation of glucagon release by extracellular glucose. Since these cells do not express Glut2, we suggest that glucose sensing does not necessarily require the coexpression of Glut2 and glucokinase.

Effect of polygonati rhizoma on blood glucose and facilitative glucose transporter isoform 2 (GLUT2) mRNA expression in Wistar fatty rats.

The intraperitoneal administration of the methanol extract of Polygonati Rhizoma (OM) to Wistar fatty rats caused a significant decrease in the blood glucose level 4h after its administration at 800 mg/kg (p < 0.01). The hepatic content of facilitative glucose transporter isoform 2, liver type glucose transporter (GLUT2) mRNA content from rat liver significantly decreased in the intraperitoneally OM-treated rats compared to that in the controls (p < 0.01). These results suggest that the hypoglycemic effect of OM is presumably due, at least in part, to the reduction of GLUT2 mRNA expression. Because of this unique therapeutic mechanism, OM could be a new category of therapeutic agent for non-insulin-dependent diabetes mellitus.

Central nervous system and peripheral abnormalities: clues to the understanding of obesity and NIDDM.

To study the impact on glucose handling of the observed hyperinsulinaemia and hypercorticism of the genetically obese fa/fa rats, simplified animal models were used. In the first model, normal rats were exposed to hyperinsulinaemia for 4 days and compared to saline-infused controls. At the end of this experimental period, the acute effect of insulin was assessed during euglycaemic-hyperinsulinaemic clamps. White adipose tissue lipogenic activity was much more insulin responsive in the "insulinized" than in the control groups. Conversely muscles from "insulinized" rats became insulin resistant. Such divergent consequences of prior "insulinization" on white adipose tissue and muscle were corroborated by similar divergent changes in glucose transporter (GLUT 4) mRNA and protein levels in these respective tissues. In the second model, normal rats were exposed to stress levels of corticosterone for 2 days. This resulted in an insulin resistance of all muscle types that was due to an increased glucose-fatty acid cycle, without measurable alteration of the GLUT 4 system. In genetically obese (fa/fa) rats, local cerebral glucose utilization was decreased compared to lean controls. This could be the reason for adaptive changes leading to increased levels in their hypothalamic neuropeptide Y levels and median eminence corticotropin-releasing-factor. Thus, in a third model, neuropeptide Y was administered intracerebroventricularly to normal rats for 7 days. This produced hyperinsulinaemia, hypercorticosteronaemia, as well as most of the metabolic changes observed in the genetically obese fa/fa rats, including muscle insulin resistance. These data together suggest that the aetiology of obesity-insulin resistance of genetically obese rodents has to be searched within the brain, not peripherally.

The amino terminus of GLUT4 functions as an internalization motif but not an intracellular retention signal when substituted for the transferrin receptor cytoplasmic domain.

Previous studies have demonstrated that the amino-terminal cytoplasmic domain of GLUT4 contains a phenylalanine-based targeting motif that determines its steady state distribution between the surface and the interior of cells (Piper, R. C., C. Tai, P. Kuleza, S. Pang, D. Warnock, J. Baenziger, J. W. Slot, H. J. Geuze, C. Puri, and D. E. James. 1993. J. Cell Biol. 121:1221). To directly measure the effect that the GLUT4 amino terminus has on internalization and subsequent recycling back to the cell surface, we constructed chimeras in which this sequence was substituted for the amino-terminal cytoplasmic domain of the human transferrin receptor. The chimeras were stably transfected into Chinese hamster ovary cells and their endocytic behavior characterized. The GLUT4-transferrin receptor chimera was recycled back to the cell surface with a rate similar to the transferrin receptor, indicating that the GLUT4 sequence was not promoting intracellular retention of the chimera. The GLUT4-transferrin receptor chimera was internalized at half the rate of the transferrin receptor. Substitution of an alanine for phenylalanine at position 5 slowed internalization of the chimera by twofold, to a level characteristic of bulk membrane internalization. However, substitution of a tyrosine increased the rate of internalization to the level of the transferrin receptor. Neither of these substitutions significantly altered the rate at which the chimeras were recycled back to the cell surface. These results demonstrate that the major function of the GLUT4 amino-terminal domain is to promote the effective internalization of the protein from the cell surface, via a functional phenylalanine-based internalization motif, rather than retention of the transporter within intracellular structures.