Brain-targeted hypoxia-inducible factor stabilization reduces neonatal hypoxic-ischemic brain injury.

Hypoxia-inducible factor-1α (HIF1α) is a major regulator of cellular adaptation to hypoxia and oxidative stress, and recent advances of prolyl-4-hydroxylase (P4H) inhibitors have produced powerful tools to stabilize HIF1α for clinical applications. However, whether HIF1α provokes or resists neonatal hypoxic-ischemic (HI) brain injury has not been established in previous studies. We hypothesize that systemic and brain-targeted HIF1α stabilization may have divergent effects. To test this notion, herein we compared the effects of GSK360A, a potent P4H inhibitor, in in-vitro oxygen-glucose deprivation (OGD) and in in-vivo neonatal HI via intracerebroventricular (ICV), intraperitoneal (IP), and intranasal (IN) drug-application routes. We found that GSK360A increased the erythropoietin (EPO), heme oxygenase-1 (HO1) and glucose transporter 1 (Glut1) transcripts, all HIF1α target-genes, and promoted the survival of neurons and oligodendrocytes after OGD. Neonatal HI insult stabilized HIF1α in the ipsilateral hemisphere for up to 24 h, and either ICV or IN delivery of GSK360A after HI increased the HIF1α target-gene transcripts and decreased brain damage. In contrast, IP-injection of GSK360A failed to reduce HI brain damage, but elevated the risk of mortality at high doses, which may relate to an increase of the kidney and plasma EPO, leukocytosis, and abundant vascular endothelial growth factor (VEGF) mRNAs in the brain. These results suggest that brain-targeted HIF1α-stabilization is a potential treatment of neonatal HI brain injury, while systemic P4H-inhibition may provoke unwanted adverse effects.

Osteoarthritis-associated basic calcium phosphate crystals alter immune cell metabolism and promote M1 macrophage polarization.

OBJECTIVE: A number of studies have demonstrated that molecules called 'alarmins' or danger-associated molecular patterns (DAMPs), contribute to inflammatory processes in the OA joint. Metabolic reprogramming of immune cells, including macrophages, is emerging as a prominent player in determining immune cell phenotype and function. The aim of this study was to investigate if basic calcium phosphate (BCP) crystals which are OA-associated DAMPs, impact on macrophage phenotype and metabolism. METHODS: Human monocyte derived macrophages were treated with BCP crystals and expression of M1 (CXCL9, CXCL10) and M2 (MRC1, CCL13)-associated markers was assessed by real-time PCR while surface maturation marker (CD40, CD80 & CD86) expression was assessed by flow cytometry. BCP induced metabolic changes were assessed by Seahorse analysis and glycolytic marker expression (hexokinase 2(HK2), Glut1 and HIF1α) was examined using real-time PCR and immunoblotting. RESULTS: Treatment with BCP crystals upregulated mRNA levels of CXCL9 and CXCL10 while concomitantly downregulating expression of CCL13 and MRC1. Furthermore, BCP-treated macrophages enhanced surface expression of the maturation makers, CD40, CD80 and CD86. BCP-treated cells also exhibited a shift towards glycolysis as evidenced by an increased ECAR/OCR ratio and enhanced expression of the glycolytic markers, HK2, Glut1 and HIF1α. Finally, BCP-induced macrophage activation and alarmin expression was reduced in the presence of the glycolytic inhibitor, 2-DG. CONCLUSIONS: This study not only provides further insight into how OA-associated DAMPs impact on immune cell function, but also highlights metabolic reprogramming as a potential therapeutic target for calcium crystal-related arthropathies.

The expression of glucose transporters and mitochondrial division and fusion proteins in rats exposed to hypoxic preconditioning to attenuate propofol neurotoxicity.

Purpose: Evidence has shown that propofol may cause widespread apoptotic neurodegeneration. Hypoxic preconditioning has been demonstrated to provide neuroprotection and brain recovery from both acute and chronic neurodegeneration in several cellular and animal models. However, the mechanism has not been well elucidated. Therefore, the present study was designed to investigate the expression of glucose transporters (GLUT1 and GLUT3) and mitochondrial division and fusion (Drp1 and Mfn2) proteins in rats exposed to hypoxic preconditioning to attenuate propofol neurotoxicity.Methods: Propofol (100 mg/kg) was given to 7-day-old Sprague-Dawley rats; in some rats, hypoxic preconditioning was administered before intraperitoneal propofol injection by subjecting rats to five cycles of 10 min of hypoxia (8% O2) and 10 min of normoxia (21% O2). Then, the rats were allowed to breathe room air for 2 h. Neuronal mitochondrial morphology was observed by transmission electron microscopy. ATP content was detected using an ATP assay kit. The expression levels of GLUT1, GLUT3, pDrp1, Drp1 and Mfn2 were detected by Western blot, and the expression levels of GLUT1 and GLUT3 were further examined by immunohistochemistry.Results: Propofol damaged mitochondria, and decreased ATP content and GLUT3 and pDrp1 protein expression. However, our results suggested that hypoxic preconditioning could attenuate propofol neurotoxicity by reducing mitochondrial damage and increasing ATP content and pDrp1, GLUT1 and GLUT3 protein expression.Conclusion: Hypoxic preconditioning reduced propofol-induced damage in the hippocampus of neonatal rats by attenuating the increase in mitochondrial division and decrease in GLUT3 expression.

Pioglitazone reversed the fructose-programmed astrocytic glycolysis and oxidative phosphorylation of female rat offspring.

Excessive maternal high-fructose diet (HFD) during pregnancy and lactation has been reported to cause metabolic disorders in the offspring. Whether the infant's brain metabolism is disturbed by maternal HFD is largely unknown. Brain energy metabolism is elevated dramatically during fetal and postnatal development, whereby maternal nutrition is a key factor that determines cellular metabolism. Astrocytes, a nonneuronal cell type in the brain, are considered to support the high-energy demands of neurons by supplying lactate. In this study, the effects of maternal HFD on astrocytic glucose metabolism were investigated using hippocampal primary cultures of female infants. We found that glycolytic capacity and mitochondrial respiration and electron transport chain were suppressed by maternal HFD. Mitochondrial DNA copy number and mitochondrial transcription factor A expression were suppressed by maternal HFD. Western blots and immunofluorescent images further indicated that the glucose transporter 1 was downregulated whereas the insulin receptor-α, phospho-insulin receptor substrate-1 (Y612) and the p85 subunit of phosphatidylinositide 3-kinase were upregulated in the HFD group. Pioglitazone, which is known to increase astrocytic glucose metabolism, effectively reversed the suppressed glycolysis, and lactate release was restored. Moreover, pioglitazone also normalized oxidative phosphorylation with an increase of cytosolic ATP. Together, these results suggest that maternal HFD impairs astrocytic energy metabolic pathways that were reversed by pioglitazone.

[High glucose dialysate enhances peritoneal fibrosis through upregulating glucose transporters GLUT1 and SGLT1].

Objective: To investigate the role of glucose transporter 1 (GLUT1) and sodium-glucose cotransporter 1 (SGLT1) in high glucose dialysate-induced peritoneal fibrosis. Methods: Thirty six male SD rats were randomly divided into 6 groups (6 in each):normal control group, sham operation group, peritoneal dialysis group (PD group), PD+phloretin group (PD+T group), PD+phlorizin group (PD+Z group), PD+phloretin+phlorizin group (PD+T+Z group). Rat model of uraemia was established using 5/6 nephrotomy, and 2.5% dextrose peritoneal dialysis solution was used in peritoneal dialysis. Peritoneal equilibration test was performed 24 h after dialysis to evaluate transport function of peritoneum in rats; HE staining was used to observe the morphology of peritoneal tissue; and immunohistochemistry was used to detect the expression of GLUT1, SGLT1, TGF-ß1 and connective tissue growth factor (CTGF) in peritoneum. Human peritoneal microvascular endothelial cells (HPECs) were divided into 5 groups:normal control group, peritoneal dialysis group (PD group), PD+phloretin group (PD+T group), PD+phlorezin group (PD+Z group), and PD+phloretin+phlorezin group (PD+T+Z group). Real time PCR and Western blotting were used to detect mRNA and protein expressions of GLUT1, SGLT1, TGF-ß1, CTGF in peritoneal membrane and HPECs. Results:In vivo, compared with sham operation group, rats in PD group had thickened peritoneum, higher ultrafiltration volume, and the mRNA and protein expressions of GLUT1, SGLT1, CTGF, TGF-ß1 were significantly increased (all P<0.05); compared with PD group, thickened peritoneum was attenuated, and the mRNA and protein expressions of GLUT1, SGLT1, CTGF, TGF-ß1 were significantly decreased in PD+T, PD+Z and PD+T+Z groups (all P<0.05). Pearson's correlation analysis showed that the expressions of GLUT1, SGLT1 in peritoneum were positively correlated with the expressions of TGF-ß1 and CTGF (all P<0.05). In vitro, the mRNA and protein expressions of GLUT1, SGLT1, TGF-ß1, CTGF were significantly increased in HPECs of peritoneal dialysis group (all P<0.05), and those in PD+T, PD+Z, and PD+T+Z groups were decreased (all P<0.05). Pearson's correlation analysis showed that the expressions of GLUT1, SGLT1 in HPECs were positively correlated with the expressions of TGF-ß1 and CTGF (all P<0.05). Conclusion: High glucose peritoneal dialysis fluid may promote peritoneal fibrosis by upregulating the expressions of GLUT1 and SGLT1.

Therapeutic role of EF24 targeting glucose transporter 1-mediated metabolism and metastasis in ovarian cancer cells.

Cancer cells require glucose to support their rapid growth through a process known as aerobic glycolysis, or the Warburg effect. As in ovarian cancer cells, increased metabolic activity and glucose concentration has been linked to aggressiveness of cancer. However, it is unclear as to whether targeting the glycolytic pathway may kill the malignant cells and likely have broad therapeutic implications against ovarian cancer metastasis. In the present research, we found that EF24, a HIF-1α inhibitor, could significantly block glucose uptake, the rate of glycolysis, and lactate production compared with vehicle treatment in SKOV-3, A2780 and OVCAR-3 cells. These results might possibly contribute to the further observation that EF24 could inhibit ovarian cancer cell migration and invasion from wound healing and Transwell assays. Furthermore, as an important mediator of glucose metabolism, glucose transporter 1 (Glut1) was found to contribute to the function of EF24 in both energy metabolism and metastasis. To examine the effect of EF24 and the mediated role of Glut1 in vivo in a xenograph subcutaneous tumor model, intraperitoneal metastasis and lung metastasis model were introduced. Our results indicated that EF24 treatment could inhibit tumor growth, intraperitoneal metastasis and lung metastasis of SKOV-3 cells, and Glut1 is a possible mediator for the role of EF24. In conclusion, our results highlight that an anti-cancer reagent with an inhibiting effect on energy metabolism could inhibit metastasis, and EF24 is a possible candidate for anti-metastasis therapeutic applications for ovarian cancer.

Capsaicin inhibits HIF-1α accumulation through suppression of mitochondrial respiration in lung cancer cells.

Hypoxia inducible factor (HIF)-1α is an important transcription factor regulating cancer metabolism in hypoxic environment. Capsaicin is known to inhibit hypoxia-induced HIF activity in lung cancer. Hence, in this study we tried to elucidate its inhibitory mechanism of action. In lung cancer cells, including H1299, H23, A549, and H2009 cells, capsaicin inhibited cell growth and HIF activation. Under hypoxic conditions, capsaicin reduced the accumulation of HIF-1α protein and the expression of its target genes, including pyruvate dehydrogenase kinase 1 (PDK1) and glucose transporter 1 (GLUT1), with no effect on overall HIF-1α mRNA levels in the H1299 cells. In addition, capsaicin increased intracellular oxygen levels by suppressing mitochondrial respiration, resulting in a reduction of HIF-1α accumulation. Furthermore, mitochondrial ATP production was reduced by capsaicin through the inhibition of mitochondrial respiration in the H1299, H23, A549, and H2009 cells. These results indicate that capsaicin potentially exhibits anticancer therapeutic effects in lung cancer under hypoxic conditions.

Correction of glycaemia and GLUT1 level by mildronate in rat streptozotocin diabetes mellitus model.

Anti-ischaemic drug mildronate suppresses fatty acid metabolism and increases glucose utilization in myocardium. It was proposed that it could produce a favourable effect on metabolic parameters and glucose transport in diabetic animals. Rats with streptozotocin diabetes mellitus were treated with mildronate (100 mg/kg daily, per os, 6 weeks). Therapeutic effect of mildronate was monitored by measuring animal weight, concentrations of blood glucose, insulin, blood triglycerides, free fatty acids, blood ketone bodies and cholesterol, glycated haemoglobin per cent (HbA1c%) and glucose tolerance. GLUT1 mRNA and protein expression in kidneys, heart, liver and muscles were studied by means of real time RT-PCR and immunohistochemistry correspondingly. In the streptozotocin + mildronate group, mildronate treatment caused a significant decrease in mean blood glucose, cholesterol, free fatty acid and HbA1c concentrations and improved glucose tolerance. Induction of streptozotocin diabetes mellitus provoked increase of both GLUT1 gene and protein expression in kidneys, heart and muscle, mildronate treatment produced normalization of the GLUT1 expression levels. In the liver a similar effect was observed for GLUT1 protein expression, while GLUT1 gene expression was increased by mildronate. Mildronate produces therapeutic effect in streptozotocin diabetes model. Mildronate normalizes the GLUT1 expression up-regulated by streptozotocin diabetes mellitus in kidneys, heart, muscle and liver. Copyright © 2011 John Wiley & Sons, Ltd.

GLUT1 targeting and hypoxia-activating polymer-drug conjugate-based micelle for tumor chemo-thermal therapy.

PURPOSE: Mitochondria are closely correlated with the proliferation and metastasis of tumor for providing suitable micro-environment and energy supply. Herein, we construct a glucose transporter 1 (GLUT1) targeting and hypoxia activating polyprodrug-based micelle (Glu-PEG-Azo-IR808-S-S-PTX) for mitochondria-specific drug delivery and tumor chemo-thermal therapy. RESULTS: The micelle was characterized by hypoxia-sensitive PEG outer layer detachment, high photo-thermal conversion efficiency, and glutathione (GSH)-sensitive paclitaxel （PTX） release. It showed GLUT1 specifically cellular uptake and hypoxia-sensitive mitochondria targeting on A549 cell. In vivo fluorescence imaging confirmed the micelle also could selectively accumulate in tumor and its mitochondria on A549 tumor-bearing nude mice. Consequently, it not only exhibited higher cytotoxicity, apoptosis rate, and metastasis inhibition rate on A549 cells, but also better tumor growth and metastasis inhibition rate on tumor-bearing nude mice and lower whole-body toxicity. The mechanism might be caused by destroying mitochondria and down-regulating ATP production. CONCLUSION: This study provided a GLUT1 targeting, hypoxia, and reductive responsive nanomedicine that hold the potential to be exploited for tumor therapy.

Monoethanolamine-induced glucose deprivation promotes apoptosis through metabolic rewiring in prostate cancer.

Rationale: Cancer cells rely on glucose metabolism for fulfilling their high energy demands. We previously reported that monoethanolamine (Etn), an orally deliverable lipid formulation, reduced intracellular glucose and glutamine levels in prostate cancer (PCa). Glucose deprivation upon Etn treatment exacerbated metabolic stress in PCa, thereby enhancing cell death. Moreover, Etn was potent in inhibiting tumor growth in a PCa xenograft model. However, the precise mechanisms underlying Etn-induced metabolic stress in PCa remain elusive. The purpose of the present study was to elucidate the mechanisms contributing to Etn-mediated metabolic rewiring in PCa. Methods: Glucose transporters (GLUTs) facilitate glucose transport across the plasma membrane. Thus, we assessed the expression of GLUTs and the internalization of GLUT1 in PCa. We also evaluated the effects of Etn on membrane dynamics, mitochondrial structure and function, lipid droplet density, autophagy, and apoptosis in PCa cells. Results: Compared to other GLUTs, GLUT1 was highly upregulated in PCa. We observed enhanced GLUT1 internalization, altered membrane dynamics, and perturbed mitochondrial structure and function upon Etn treatment. Etn-induced bioenergetic stress enhanced lipolysis, decreased lipid droplet density, promoted accumulation of autophagosomes, and increased apoptosis. Conclusion: We provide the first evidence that Etn alters GLUT1 trafficking leading to metabolic stress in PCa. By upregulating phosphatidylethanolamine (PE), Etn modulates membrane fluidity and affects mitochondrial structure and function. Etn also induces autophagy in PCa cells, thereby promoting apoptosis. These data strongly suggest that Etn rewires cellular bioenergetics and could serve as a promising anticancer agent for PCa.

Berberine exerts its antineoplastic effects by reversing the Warburg effect via downregulation of the Akt/mTOR/GLUT1 signaling pathway.

Glucose transporter 1 (GLUT1) plays a primary role in the glucose metabolism of cancer cells. However, to the best of our knowledge, there are currently no anticancer drugs that inhibit GLUT1 function. The present study aimed to investigate the antineoplastic activity of berberine (BBR), the main active ingredient in numerous Traditional Chinese medicinal herbs, on HepG2 and MCF7 cells. The results of Cell Counting Kit­8 assay, colony formation assay and flow cytometry revealed that BBR effectively inhibited the proliferation of tumor cells, and induced G2/M cell cycle arrest and apoptosis. Notably, the results of luminescence ATP detection assay and glucose uptake assay showed that BBR also significantly inhibited ATP synthesis and markedly decreased the glucose uptake ability, which suggested that the antitumor effect of BBR may occur via reversal of the Warburg effect. In addition, the results of reverse transcription­quantitative PCR, western blotting and immunofluorescence staining indicated that BBR downregulated the protein expression levels of GLUT1, maintained the cytoplasmic internalization of GLUT1 and suppressed the Akt/mTOR signaling pathway in both HepG2 and MCF7 cell lines. Augmentation of Akt phosphorylation levels by the Akt activator, SC79, abolished the BBR­induced decrease in ATP synthesis, glucose uptake, GLUT1 expression and cell proliferation, and reversed the proapoptotic effect of BBR. These findings indicated that the antineoplastic effect of BBR may involve the reversal of the Warburg effect by downregulating the Akt/mTOR/GLUT1 signaling pathway. Furthermore, the results of the co­immunoprecipitation assay demonstrated that BBR increased the interaction between ubiquitin conjugating enzyme E2 I (Ubc9) and GLUT1, which suggested that Ubc9 may mediate the proteasomal degradation of GLUT1. On the other hand, BBR decreased the interaction between Gα­interacting protein­interacting protein at the C­terminus (GIPC) and GLUT1, which suggested that the retention of GLUT1 in the cytoplasm may be achieved by inhibiting the interaction between GLUT1 and GIPC, thereby suppressing the glucose transporter function of GLUT1. The results of the present study provided a theoretical basis for the application of the Traditional Chinese medicine component, BBR, for cancer treatment.

An inhibitory and beneficial effect of chlorpromazine and promethazine (C + P) on hyperglycolysis through HIF-1α regulation in ischemic stroke.

BACKGROUND: After ischemic stroke, the increased catabolism of glucose (hyperglycolysis) results in the production of reactive oxygen species (ROS) via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). A depressive or hibernation-like effect of C + P on brain activity was reported to induce neuroprotection. The current study assesses the effect of C + P on hyperglycolysis and NOX activation. METHODS: Adult male Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) followed by 6 or 24 h of reperfusion. At the onset of reperfusion, rats received C + P with or without temperature control, or phloretin [glucose transporter (GLUT)-1 inhibitor], or cytochalasin B (GLUT-3 inhibitor). We detected brain ROS, apoptotic cell death, and ATP levels along with HIF-1α expression. Cerebral hyperglycolysis was measured by glucose, protein expression of GLUT-1/3, and phosphofructokinase-1 (PFK-1), as well as lactate and lactate dehydrogenase (LDH) at 6 and 24 h of reperfusion. The enzymatic activity of NOX and protein expression of its subunits (gp91phox) were detected. Neural SHSY5Y cells were placed under 2 h of oxygen-glucose deprivation (OGD) followed by reoxygenation for 6 and 24 h with C + P treatment. Cell viability and protein levels of HIF-1α, GLUT-1/3, PFK-1, LDH, and gp91phox were measured. A HIF-1α overexpression vector was transfected into the cells, and then protein levels of HIF-1α, GLUT-1/3, PFK-1, and LDH were quantitated. In sham-operated rats and control cells, the protein levels of HIF-1α, GLUT-1/3, PFK-1, LDH, and gp91phox were measured at 6 and 24 h after C + P administration. RESULTS: C + P reduced the protein elevations after stroke in HIF-1α, glycolytic enzymes, as well as in ROS, cell death, glucose and lactate, but raised ATP levels in the brain. In ischemic rats exposed to GLUT-1/3 inhibitors, ROS, cell death, glucose, and lactate were all decreased, as well as GLUT-1, GLUT-3, LDH, and PFK-1 protein levels. C + P decreased ischemia-induced NOX activation by reducing the enzymatic activity and protein expression of the NOX subunit gp91phox, as was observed in the presence of GLUT-1/3 inhibitors. These markers were significantly decreased following C + P administration with the induced hypothermia, while C + P administration with temperature control at 37 °C induced lesser protection after ischemia stroke. In the OGD/reoxygenation model, C + P treatment increased cell viability and diminished protein levels of HIF-1α, GLUT-1, GLUT-3, PFK-1, LDH, and gp91phox. However, in OGD with HIF-1α overexpression, C + P was unable to effectively reduce the upregulated GLUT-1, GLUT-3, and LDH. In normal conditions, C + P reduced HIF-1α and the levels of key glycolytic enzymes depending on its pharmacological effect. CONCLUSION: C + P, partially depending on hypothermia, attenuates hyperglycolysis and NOX activation through HIF-1α regulation.

The alternative medicine pawpaw and its acetogenin constituents suppress tumor angiogenesis via the HIF-1/VEGF pathway.

Products that contain twig extracts of pawpaw (Asimina triloba) are widely consumed anticancer alternative medicines. Pawpaw crude extract (CE) and purified acetogenins inhibited hypoxia-inducible factor-1 (HIF-1)-mediated hypoxic signaling pathways in tumor cells. In T47D cells, pawpaw CE and the acetogenins 10-hydroxyglaucanetin (1), annonacin (2), and annonacin A (3) inhibited hypoxia-induced HIF-1 activation with IC(50) values of 0.02 microg/mL, 12 nM, 13 nM, and 31 nM, respectively. This inhibition correlates with the suppression of the hypoxic induction of HIF-1 target genes VEGF and GLUT-1. The induction of secreted VEGF protein represents a key event in hypoxia-induced tumor angiogenesis. Both the extract and the purified acetogenins blocked the angiogenesis-stimulating activity of hypoxic T47D cells in vitro. Pawpaw extract and acetogenins inhibited HIF-1 activation by blocking the hypoxic induction of nuclear HIF-1alpha protein. The inhibition of HIF-1 activation was associated with the suppression of mitochondrial respiration at complex I. Thus, the inhibition of HIF-1 activation and hypoxic tumor angiogenesis constitutes a novel mechanism of action for these anticancer alternative medicines.

Cytokine stimulation promotes glucose uptake via phosphatidylinositol-3 kinase/Akt regulation of Glut1 activity and trafficking.

Cells require growth factors to support glucose metabolism for survival and growth. It is unclear, however, how noninsulin growth factors may regulate glucose uptake and glucose transporters. We show that the hematopoietic growth factor interleukin (IL)3, maintained the glucose transporter Glut1 on the cell surface and promoted Rab11a-dependent recycling of intracellular Glut1. IL3 required phosphatidylinositol-3 kinase activity to regulate Glut1 trafficking, and activated Akt was sufficient to maintain glucose uptake and surface Glut1 in the absence of IL3. To determine how Akt may regulate Glut1, we analyzed the role of Akt activation of mammalian target of rapamycin (mTOR)/regulatory associated protein of mTOR (RAPTOR) and inhibition of glycogen synthase kinase (GSK)3. Although Akt did not require mTOR/RAPTOR to maintain surface Glut1 levels, inhibition of mTOR/RAPTOR by rapamycin greatly diminished glucose uptake, suggesting Akt-stimulated mTOR/RAPTOR may promote Glut1 transporter activity. In contrast, inhibition of GSK3 did not affect Glut1 internalization but nevertheless maintained surface Glut1 levels in IL3-deprived cells, possibly via enhanced recycling of internalized Glut1. In addition, Akt attenuated Glut1 internalization through a GSK3-independent mechanism. These data demonstrate that intracellular trafficking of Glut1 is a regulated component of growth factor-stimulated glucose uptake and that Akt can promote Glut1 activity and recycling as well as prevent Glut1 internalization.

Vitexin, an inhibitor of hypoxia-inducible factor-1α, enhances the radiotherapy sensitization of hyperbaric oxygen on glioma.

PURPOSE: Vitexin, an inhibitor of hypoxia-inducible factor (HIF)-1α, has anti-tumor effect. However, whether it can enhance the radiotherapy sensitization of hyperbaric oxygen (HBO) on glioma is unclear. This study aimed to investigate the effect of vitexin. METHODS: The nude mice with paw-transplanted glioma were divided into four groups: control group, HBO + radiation group, HBO + vitexin group, and HBO + vitexin + radiation group. The mice of last two groups were daily given vitexin 75 mg/kg by intraperitoneal injection. 30 min after administration of vitexin, the HBO-treated mice were daily placed in HBO chamber for 60 min. The radiation-treated mice were given local tumor irradiation once every week during the HBO treatment, and the dose of irradiation was 10 Gy/time. The experimental treatment lasted for 21 days. RESULTS: Compared with the HBO + radiation group, the tumor volume, tumor weight, and tumor weight coefficient in the HBO + vitexin + radiation group were lower (p < 0.05). Importantly, the contents of reduced glutathione and glutathione peroxidase as well as expressions of HIF-1α, vascular endothelial growth factor, glucose transporter (GLUT)-1, and GLUT-3 proteins in tumor tissues were also lower in the HBO + vitexin + radiation group than in the HBO + radiation group (p < 0.01). CONCLUSIONS: Vitexin can cooperate with HBO to sensitize the glioma radiotherapy, and its mechanisms may be correlated to the inhibition of HIF-1α protein expression and subsequent decrements of its downstream protein expressions, which finally cause the reduction of antioxidant capacity.

Rosiglitazone enhances glucose uptake in glomerular podocytes using the glucose transporter GLUT1.

AIMS/HYPOTHESIS: Peroxisome proliferator-activated receptor (PPAR) gamma agonists are used increasingly in the treatment of type 2 diabetes. In the context of renal disease, PPARgamma agonists reduce microalbuminuria in diabetic nephropathy; however, the mechanisms underlying this effect are unknown. Glomerular podocytes are newly characterised insulin-sensitive cells and there is good evidence that they are targeted in diabetic nephropathy. In this study we investigated the functional and molecular effects of the PPARgamma agonist rosiglitazone on human podocytes. METHODS: Conditionally immortalised human podocytes were cultured with rosiglitazone and functional effects were measured with glucose-uptake assays. The effect of rosiglitazone on glucose uptake was also measured in 3T3-L1 adipocytes, nephrin-deficient podocytes, human glomerular endothelial cells, proximal tubular cells and podocytes treated with the NEFA palmitate. The role of the glucose transporter GLUT1 was investigated with immunofluorescence and small interfering RNA knockdown and the plasma membrane expression of GLUT1 was determined with bis-mannose photolabelling. RESULTS: Rosiglitazone significantly increased glucose uptake in wild-type podocytes and this was associated with translocation of GLUT1 to the plasma membrane. This effect was blocked with GLUT1 small interfering RNA. Nephrin-deficient podocytes, glomerular endothelial cells and proximal tubular cells did not increase glucose uptake in response to either insulin or rosiglitazone. Furthermore, rosiglitazone significantly increased basal and insulin-stimulated glucose uptake when podocytes were treated with the NEFA palmitate. CONCLUSIONS/INTERPRETATION: In conclusion, rosiglitazone has a direct and protective effect on glucose uptake in wild-type human podocytes. This represents a novel mechanism by which PPARgamma agonists may improve podocyte function in diabetic nephropathy.

Is transketolase like 1 a target for the treatment of differentiated thyroid carcinoma? A study on thyroid cancer cell lines.

Radioactive iodine-refractory [(18)F] fluorodeoxy-glucose-positron emission tomography-positive thyroid carcinomas represent especially aggressive tumors. Targeting glucose metabolism by the transketolase isoenzyme transketolase like 1 (TKTL-1) which is over-expressed in various neoplasms, may be effective. The correlation of TKTL-1 expression and the response to oxythiamine as the currently best-characterized inhibitor of transketolases was studied in differentiated thyroid cancer cell lines. We determined TKTL-1 expression, proliferation, glucose uptake and GLUT-1 expression in non-treated thyroid cells and recorded the effect of oxythiamine on iodide uptake and on thymidine uptake. TKTL 1 was highest expressed in cell lines derived from more invasive tumors but the expression level was not strongly correlated to proliferation rate, to GLUT-1 expression or to the response to oxythiamine. Oxythiamine showed only a weak effect in the TKTL-1 expressing cell lines. Over-expression of TKTL-1 is not an indicator for responsiveness to oxythiamine. More specific inhibitors should be tested.

Normoxic induction of cerebral HIF-1alpha by acetazolamide in rats: role of acidosis.

Acetazolamide has been recognized as an effective treatment for acute mountain sickness. The efficacy of acetazolamide is related to metabolic acidosis, which promotes chemoreceptors to respond to hypoxic stimuli at altitude. In this study, adult male Sprague-Dawley rats were treated with acetazolamide (100mg/kg or 50mg/kg, I.P.) for 3 days. Primary cultured cortical neurons and PC12 cell lines were exposed to acidosis-permissive (pH 6.5) or standard (pH 7.2) media for 20h. HIF-1alpha and its target genes were assayed by Western blot, real-time PCR, HIF-1 DNA-binding assay and chloramphenicol acetyltransferase reporter gene assay. HIF-1alpha protein level and HIF-1 DNA-binding activities were increased in cerebral cortices of rats treated with acetazolamide. Moreover, the mRNA levels of erythropoietin, vascular endothelial growth factor, and glucose transporter-1 also increased. The HIF-1alpha protein level and activity of HIF-driven chloramphenicol acetyltransferase reporters of cortical neurons and PC12 cells treated with acidosis media were significantly enhanced. We conclude that the normoxic induction of HIF-1alpha and HIF-1 mediated genes by acetazolamide may mediate the effect of acetazolamide in the reduction of symptoms of acute mountain sickness.

Effects of celecoxib on the expression of osteoprotegerin, energy metabolism and cell viability in cultured human osteoblastic cells.

BACKGROUND AND OBJECTIVE: The selective COX-2 inhibitor celecoxib is widely used to treat pain and inflammation in rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis. The drug has well-known important effects on immune cells but its direct and/or indirect influence on osteoblasts has not yet been explored in detail. This study aimed to investigate the dose-dependent effects of celecoxib on cell viability, energy metabolism and bone remodeling processes in cultured human osteoblastic cells. METHODS: Primary human osteoblasts and MG-63 cells were incubated with celecoxib (2, 10, 50microM). Cell viability and apoptosis were determined by trypan blue, 7AAD and Annexin-V staining. Effects on cellular oxygen consumption were measured amperometrically using a Clark electrode. mRNA expression of GLUT-1 and OPG was determined by RT-PCR; OPG protein secretion by ELISA and HIF-1alpha protein expression by immunoblotting. RESULTS: While celecoxib at a concentration of 2 and 10microM showed only marginal effects, a suprapharmacological concentration of 50microM influenced viability and energy metabolism, as well as OPG expression and secretion of osteoblastic cells. Cell viability was significantly reduced by celecoxib treatment. Celecoxib at 50microM stimulated oxygen consumption significantly. Corresponding experiments with the protonophore FCCP suggest that this effect is due to mitochondrial uncoupling. After 24h, GLUT-1 mRNA expression was significantly increased. HIF-1alpha protein was not expressed under any of our experimental conditions. We also showed that celecoxib at 50microM significantly inhibits OPG protein secretion leading to a compensative increase of mRNA expression. CONCLUSION: Pronounced effects of celecoxib on cell viability (reduction), oxygen consumption (stimulation), GLUT-1 mRNA expression (stimulation) and OPG protein secretion (inhibition) in osteoblastic cells were observed only at 50microM-a concentration not reached by therapeutic doses giving plasma concentrations less than 10microM. On the contrary, celecoxib at 2 and 10microM showed only marginal effects, suggesting that celecoxib administration is probably safe with respect to bone metabolism in cases requiring potent treatment of pain and inflammation. However, higher intracellular concentrations, which might occur through accumulation, necessitate investigations with high concentrations.

Increased transglutaminase 2 and GLUT-1 expression in breast tumors not susceptible to chemoprevention with antioxidants.

GOALS: Expression of GLUT-1 and transglutaminase 2 is increased in aggressive breast cancer, whereas claudin-1, which is expressed in normal tissues, is absent in such tumors. This experimental study was undertaken to establish the aggressiveness and prognosis of DMBA-induced mammary tumors in female Wistar rats based on the assessment of these markers. MATERIALS AND METHODS: The rats were divided into two groups, a control group (n = 70) and a chemoprevention group (n = 70). Breast tumors were induced in both groups by administration of 7,12-dimethylbenz[a] anthracene (DMBA). The chemoprevention group also received alpha-tocopherol and a solution of micronutrients containing ascorbic acid and selenium. Neoplastic lesions of both groups were randomly selected for immunohistochemical assessment of the expression of GLUT-1, transglutaminase 2 and claudin-1. RESULTS: A higher proportion of mammary tumors expressed GLUT-1 and transglutaminase 2 in the chemoprevention group. Claudin-1 expression was absent in all tumors of both groups. CONCLUSIONS: These results are suggestive of increased aggressiveness of tumors not susceptible to chemoprevention by the agents used in this study.