Effects of supplementing different chromium histidinate complexes on glucose and lipid metabolism and related protein expressions in rats fed a high-fat diet.

BACKGROUND: The objective of this study was to investigate the effects of different chromium histidinate (CrHis) complexes added to the diet of rats fed a high-fat diet (HFD) on body weight changes, glucose and lipid metabolism parameters, and changes in biomarkers such as PPAR-γ, IRS-1, GLUTs, and NF-κB proteins. METHODS: Forty-two Sprague-Dawley rats were divided equally into six groups and fed either a control, an HFD, or an HFD supplemented with either CrHis1, CrHis2, CrHis3, or a combination of the CrHis complexes as CrHisM. RESULTS: Feeding an HFD to rats increased body weights, HOMA-IR values, fasting serum glucose, insulin, leptin, free fatty acid, total cholesterol, low-density lipoprotein cholesterol, and MDA concentrations as well as AST activities, and decreased serum and brain serotonin concentrations compared with rats fed a control diet (P < 0.0001). The levels of the PPAR-γ, IRS-1, and GLUTs in the liver and brain decreased, while NF-κB level increased, with feeding an HFD (P < 0.05). Although all the CrHis supplements reversed the negative effects of feeding an HFD (P < 0.05), the CrHis1 complex was most effective in changing the protein levels, while CrHisM was most effective in influencing certain parameters such as body weight and serum metabolites. CONCLUSION: The results of the present work suggest that the CrHis1 complex is most potent for alleviating the negative effects of feeding an HFD. The efficacy of CrHisM is likely due to the presence of the CrHis1 complex.

Discovery of novel N-substituted thiazolidinediones (TZDs) as HDAC8 inhibitors: in-silico studies, synthesis, and biological evaluation.

Epigenetics plays a fundamental role in cancer progression, and developing agents that regulate epigenetics is crucial for cancer management. Among Class I and Class II HDACs, HDAC8 is one of the essential epigenetic players in cancer progression. Therefore, we designed, synthesized, purified, and structurally characterized novel compounds containing N-substituted TZD (P1-P25). Cell viability assay of all compounds on leukemic cell lines (CEM, K562, and KCL22) showed the cytotoxic potential of P8, P9, P10, P12, P19, and P25. In-vitro screening of different HDACs isoforms revealed that P19 was the most potent and selective inhibitor for HDAC8 (IC50 - 9.3 µM). Thermal shift analysis (TSA) confirmed the binding of P19 to HDAC8. In-vitro screening of all compounds on the transport activity of GLUT1, GLUT4, and GLUT5 indicated that P19 inhibited GLUT1 (IC50 - 28.2 µM). P10 and P19 induced apoptotic cell death in CEM cells (55.19% and 60.97% respectively) and P19 was less cytotoxic on normal WBCs (CC50 - 104.2 µM) and human fibroblasts (HS27) (CC50 - 105.0 µM). Thus, among this novel series of TZD derivatives, compound P19 was most promising HDAC8 inhibitor and cytotoxic on leukemic cells. Thus, P19 could serve as a lead for further development of optimized molecules with enhanced selectivity and potency.

DT-13 inhibited the proliferation of colorectal cancer via glycolytic metabolism and AMPK/mTOR signaling pathway.

BACKGROUND: Emerging hallmark of cancer is reprogrammed cellular metabolism, increased glycolytic metabolism is physiological characteristic of human malignant neoplasms. Saponin monomer 13 of the dwarf lilyturf tuber (DT-13) is the main steroidal saponin from Liriopes Radix, which has been reported to exert anti-inflammation and anti-tumor activities but low toxicity to normal tissue. However, the effect of DT-13 on metabolism process is still unclear. PURPOSE: This study aims to characterize the role of DT-13 in glucose metabolism in colorectal cancer cells, and investigate whether the metabolism process is involved in the anti-cancer response of DT-13. METHODS: Colony formation assay was employed to determine anti-proliferative effect induced by DT-13 at 2.5, 5, 10 µM. Apoptosis and cell cycle arrest were detected by Annexin V/PI staining and PI staining, respectively. Genetic inhibition of glycolytic metabolism was carried out by knockdown of GLUT1. Orthotopic implantation mouse model of colorectal cancer was used to assess in vivo antitumor effect of DT-13 (0.625, 1.25, 2.5 mg/kg). The chemoprevention effect of DT-13 (10mg/kg) was evaluated by using C57BL/6J APCmin mice model. Glycolytic-related key enzymes and AMPK pathway were detected by using quantitative real-time PCR, western blotting, and immunohistochemical staining. RESULTS: Our results showed that cell proliferation was significantly inhibited by DT-13 in a dose-dependent manner. DT-13 inhibited glucose uptake, ATP generation, and reduced lactate production. Furthermore, DT-13 remarkably inhibited GLUT1 expression in both mRNA and protein levels. Knocking down of GLUT1 led to reduced inhibition of glucose uptake after DT-13 treatment. Moreover, deletion of GLUT1 decreased inhibitory ratio of DT-13 on cancer growth. Orthotopic implantation mouse model of colorectal cancer further confirmed that DT-13 inhibited colorectal cancer growth via blocking GLUT1 in vivo. In addition, C57BL/6J APCmin mice model revealed that DT-13 dramatically reduced the total number of spontaneous adenomas in intestinal, which further confirmed the anti-tumor activity of DT-13 in colorectal cancer. Furthermore, the mechanistically investigation showed DT-13 activated AMPK and inhibited m-TOR to block cancer growth in vitro. CONCLUSION: DT-13 is a potent anticancer agent for colorectal cancer.

Red wine and green tea flavonoids are cis-allosteric activators and competitive inhibitors of glucose transporter 1 (GLUT1)-mediated sugar uptake.

The antioxidant- and flavonoid-rich contents of red wine and green tea are reported to offer protection against cancer, cardiovascular disease, and diabetes. Some studies, however, show that flavonoids inhibit GLUT1-mediated, facilitative glucose transport, raising the possibility that their interaction with GLUT1 and subsequent downstream effects on carbohydrate metabolism may also impact health. The present study explores the structure-function relationships of flavonoid-GLUT1 interactions. We find that low concentrations of flavonoids act as cis-allosteric activators of sugar uptake, whereas higher concentrations competitively inhibit sugar uptake and noncompetitively inhibit sugar exit. Studies with heterologously expressed human GLUT1, -3, or -4 reveal that quercetin-GLUT1 and -GLUT4 interactions are stronger than quercetin-GLUT3 interactions, that epicatechin gallate (ECG) is more selective for GLUT1, and that epigallocatechin gallate (EGCG) is less GLUT isoform-selective. Docking studies suggest that only one flavonoid can bind to GLUT1 at any instant, but sugar transport and ligand-binding studies indicate that human erythrocyte GLUT1 can bind at least two flavonoid molecules simultaneously. Quercetin and EGCG are each characterized by positive, cooperative binding, whereas ECG shows negative cooperative binding. These findings support recent studies suggesting that GLUT1 forms an oligomeric complex of interacting, allosteric, alternating access transporters. We discuss how modulation of facilitative glucose transporters could contribute to the protective actions of the flavonoids against diabetes and Alzheimer's disease.

Silybin counteracts doxorubicin resistance by inhibiting GLUT1 expression.

Despite significant advances in the diagnosis and treatment of cancer, the development of drug resistance still remains one of the principal causes that hampers the effectiveness of the therapy. Emerging evidences support the idea that the dysregulated metabolism could be related to drug resistance. The major goal of this study was to target cancer metabolic pathways using new pharmacological approaches coming from natural sources in order to possibly prevent or overcome this phenomenon. Firstly, the metabolic profile of human colorectal adenocarcinoma cells sensitive (LoVo WT) and resistant to doxorubicin (LoVo DOX) was delineated demonstrating that resistant cells remodel their metabolism toward a glycolytic phenotype. In particular it was observed that doxorubicin-resistant cancer cells exhibit an increased dependency from glucose for their survival, associated with overexpression of the glycolytic pathway. Moreover, both GLUT1 mRNA and protein expression significantly increased in LoVo DOX cells. Given the results about the metabolic profile, silybin, modulator of GLUTs, was selected as potential candidate to overcome doxorubicin resistance and, intriguingly, data revealed not only that silybin is more active in resistant cells than in wild type cells, but also that the combined treatment with doxorubicin and silybin presents a synergistic effect in LoVo DOX cells. Although many unanswered questions still remain about the molecular mechanism of silybin, these data suggest that targeting GLUTs may be a good strategy to restore doxorubicin sensitivity and elude drug resistance.

Insights into novel anticancer applications for apigenin.

Flavonoids, naturally occuring derivatives of 2-phenyl-benzo-γ-pyrone, are widespread in plants as coloring substances. Apigenin (4',5,7,-trihydroxyflavone (5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one), molecular formula C15H10O5, is a flavonoid present in many fruits and vegetables, primarily in citrus fruits, apples, parsley and celery leaves. It is also found in some medicinal plants, including chamomile flowers, thyme, oregano, peppermint, lemon balm and yarrow, as a 7-O-glycoside with anti-inflammatory and antioxidant activity. In recent years it has attracted a great deal of interest as a bioactive substance reported to have anticancer properties. According to recent literature data, apigenin is able to reduce cancer cell glucose uptake, inhibit remodeling of the extracellular matrix, inhibit cell adhesion molecules that participate in cancer progression and hinder the development of blood vessels needed by growing tumors. It is reported to protect against a wide variety of cancers. The mechanism of anticancer activity is still under investigation and further research is needed.

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.

Glucose is a key driver for GLUT1-mediated nanoparticles internalization in breast cancer cells.

The mesenchymal state in cancer is usually associated with poor prognosis due to the metastatic predisposition and the hyper-activated metabolism. Exploiting cell glucose metabolism we propose a new method to detect mesenchymal-like cancer cells. We demonstrate that the uptake of glucose-coated magnetic nanoparticles (MNPs) by mesenchymal-like cells remains constant when the glucose in the medium is increased from low (5.5 mM) to high (25 mM) concentration, while the MNPs uptake by epithelial-like cells is significantly reduced. These findings reveal that the glucose-shell of MNPs plays a major role in recognition of cells with high-metabolic activity. By selectively blocking the glucose transporter 1 channels we showed its involvement in the internalization process of glucose-coated MNPs. Our results suggest that glucose-coated MNPs can be used for metabolic-based assays aimed at detecting cancer cells and that can be used to selectively target cancer cells taking advantage, for instance, of the magnetic-thermotherapy.

Inhibition of glutamine utilization sensitizes lung cancer cells to apigenin-induced apoptosis resulting from metabolic and oxidative stress.

Recent studies have shown anticancer activity of apigenin by suppressing glucose transporter 1 (GLUT1) expression in cultured cancer cells; however, it is not clear whether apigenin can suppress glucose metabolism in lung cancer cells or sensitize them to inhibition of glutamine utilization-mediated apoptosis through metabolic and oxidative stress. We show that apigenin significantly decreases GLUT1 expression in mice. Furthermore, we demonstrate that apigenin induces growth retardation and apoptosis through metabolic and oxidative stress caused by suppression of glucose utilization in lung cancer cells. The underlying mechanisms were defined that the anticancer effects of apigenin were reversed by ectopic GLUT1 overexpression and galactose supplementation, through activation of pentose phosphate pathway-mediated NADPH generation. Importantly, we showed that severe metabolic stress using a glutaminase inhibitor, compound 968, was involved in the mechanism of sensitization by apigenin. Taken together, the combination of apigenin with inhibitors of glutamine metabolism may provide a promising therapeutic strategy for cancer treatment.

Inhibition of human GLUT1 and GLUT5 by plant carbohydrate products; insights into transport specificity.

Glucose transporters GLUT1 (transports glucose) and GLUT5 (transports fructose), in addition to their functions in normal metabolism, have been implicated in several diseases including cancer and diabetes. While GLUT1 has several inhibitors, none have been described for GLUT5. By transport activity assays we found two plant products, rubusoside (from Rubus suavissimus) and astragalin-6-glucoside (a glycosylated derivative of astragalin, from Phytolacca americana) that inhibited human GLUT5. These plants are utilized in traditional medicine: R. suavissimus for weight loss and P. americana for cancer treatment, but the molecular interactions of these products are unknown. Rubusoside also inhibited human GLUT1, but astragalin-6-glucoside did not. In silico analysis of rubusoside:protein interactions pinpointed a major difference in substrate cavity between these transporters, a residue that is a tryptophan in GLUT1 but an alanine in GLUT5. Investigation of mutant proteins supported the importance of this position in ligand specificity. GLUT1W388A became susceptible to inhibition by astragalin-6-glucoside and resistant to rubusoside. GLUT5A396W transported fructose and also glucose, and maintained inhibition by rubusoside and astragalin-6-glucoside. Astragalin-6-glucoside can serve as a starting point in the design of specific inhibitors for GLUT5. The application of these studies to understanding glucose transporters and their interaction with substrates and ligands is discussed.

Glucose restriction induces cell death in parental but not in homeodomain-interacting protein kinase 2-depleted RKO colon cancer cells: molecular mechanisms and implications for tumor therapy.

Tumor cell tolerance to nutrient deprivation can be an important factor for tumor progression, and may depend on deregulation of both oncogenes and oncosuppressor proteins. Homeodomain-interacting protein kinase 2 (HIPK2) is an oncosuppressor that, following its activation by several cellular stress, induces cancer cell death via p53-dependent or -independent pathways. Here, we used genetically matched human RKO colon cancer cells harboring wt-HIPK2 (HIPK2(+/+)) or stable HIPK2 siRNA interference (siHIPK2) to investigate in vitro whether HIPK2 influenced cell death in glucose restriction. We found that glucose starvation induced cell death, mainly due to c-Jun NH2-terminal kinase activation, in HIPK2(+/+)cells compared with siHIPK2 cells that did not die. (1)H-nuclear magnetic resonance quantitative metabolic analyses showed a marked glycolytic activation in siHIPK2 cells. However, treatment with glycolysis inhibitor 2-deoxy-D-glucose induced cell death only in HIPK2(+/+) cells but not in siHIPK2 cells. Similarly, siGlut-1 interference did not re-establish siHIPK2 cell death under glucose restriction, whereas marked cell death was reached only after zinc supplementation, a condition known to reactivate misfolded p53 and inhibit the pseudohypoxic phenotype in this setting. Further siHIPK2 cell death was reached with zinc in combination with autophagy inhibitor. We propose that the metabolic changes acquired by cells after HIPK2 silencing may contribute to induce resistance to cell death in glucose restriction condition, and therefore be directly relevant for tumor progression. Moreover, elimination of such a tolerance might serve as a new strategy for cancer therapy.

Complex behavioral and synaptic effects of dietary branched chain amino acids in a mouse model of amyotrophic lateral sclerosis.

SCOPE: We hypothesized that chronic supplementation with branched chain amino acids (BCAAs) affects neurobehavioral development in vulnerable gene backgrounds. METHODS AND RESULTS: A murine model of amyotrophic lateral sclerosis (ALS), G93A mice bearing the mutated human superoxide dismutase 1 (SOD1) gene, and control mice received from 4 to 16 wk of age dietary supplementation with BCAAs at doses comparable to human usage. Motor coordination, exploratory behaviors, pain threshold, synaptic activity and response to glutamatergic stimulation in primary motor cortex slices were evaluated between the 8th and 16th week. The glial glutamate transporter 1 (GLT-1) and metabotropic glutamate 5 receptor (mGlu5R) were analyzed by immunoblotting in cortex, hippocampus and striatum. BCAAs induced hyperactivity, decreased pain threshold in wild-type mice and exacerbated the motor deficits of G93A mice while counteracting their abnormal pain response. Electrophysiology on G93A brain slices showed impaired synaptic function, reduced toxicity of GLT-1 blocking and increased glutamate toxicity prevented by BCAAs. Immunoblotting indicated down-regulation of GLT-1 and mGlu5R in G93A, both effects counteracted by BCAAs. CONCLUSION: These results, though not fully confirming a role of BCAAs in ALS-like etiology in the genetic model, clearly indicate that BCAAs' complex effects on central nervous system depend on gene background and raise alert over their spread use.

Verapamil inhibits the glucose transport activity of GLUT1.

Calcium channel blocker toxicity has been associated with marked hyperglycemia responsive only to high-dose insulin therapy. The exact mechanism(s) of this induced hyperglycemia has not been clearly delineated. The glucose transporter GLUT1 is expressed in a wide variety of cell types and is largely responsible for a basal level of glucose transport. GLUT1 also is activated by cell stress. The specific purpose of this study was to investigate the effects of the calcium channel blocker verapamil on the glucose uptake activity of GLUT1 in L929 fibroblasts cells. Dose-dependent effects of verapamil on glucose uptake were studied using L929 fibroblast cells with 2-deoxyglucose. Verapamil had a dose-dependent inhibitory effect on both basal and stress-activated transport activity of GLUT1. Basal activity was inhibited 50% by 300 µM verapamil, while 150 µM verapamil completely inhibited the activation induced by the stress of glucose deprivation. These effects were reversible and required verapamil to be present during the stress. Alteration of calcium concentrations by addition of 5 mM CaCl2 or 4 mM EDTA had no effect on verapamil action. This study reveals the unique finding that verapamil has inhibitory effects on the transport activity of GLUT1 independent of its effects on calcium concentrations. The inhibition of GLUT1 may be one of the contributing factors to the hyperglycemia observed in CCB poisoning.

Comparison of effects of green tea catechins on apicomplexan hexose transporters and mammalian orthologues.

Here we have investigated the inhibitory properties of green tea catechins on the Plasmodium falciparum hexose transporter (PfHT), the Babesia bovis hexose transporter 1 (BboHT1) and the mammalian facilitative glucose transporters, GLUT1 and GLUT5, expressed in Xenopus laevis oocytes. (-)-Epicatechin-gallate (ECG) and (-)-epigallocatechin-gallate (EGCG) inhibited D-glucose transport by GLUT1 and PfHT, and D-fructose transport by GLUT5, with apparent K(i) values between 45 and 117 microM. BboHT1 was more potently inhibited by the ungallated catechins (-)-epicatechin (EC) and (-)-epigallocatechin (EGC), with apparent K(i) values of 108 and 168 microM, respectively. Site-directed mutagenesis experiments provided little further support for previously reported models of catechin binding to hexose transporters. Furthermore, P. falciparum growth inhibition by catechins was not affected by the external D-glucose concentration. Our results provide new data on the inhibitory action of catechins against sugar transporters but were unable to elucidate the antimalarial mechanism of action of these agents.