Increase In GLUT4 Research Articles

Conditioning-induced expression of novel glucose transporters in canine skeletal muscle homogenate.

Athletic conditioning can increase the capacity for insulin-stimulated skeletal muscle glucose uptake through increased sarcolemmal expression of GLUT4 and potentially additional novel glucose transporters. We used a canine model that has previously demonstrated conditioning-induced increases in basal, insulin- and contraction-stimulated glucose uptake to identify whether expression of glucose transporters other than GLUT4 was upregulated by athletic conditioning. Skeletal muscle biopsies were obtained from 12 adult Alaskan Husky racing sled dogs before and after a full season of conditioning and racing, and homogenates from those biopsies were assayed for expression of GLUT1, GLUT3, GLUT4, GLUT6, GLUT8, and GLUT12 using western blots. Athletic conditioning resulted in a 1.31 ± 0.70 fold increase in GLUT1 (p <0.0001), 1.80 ± 1.99 fold increase in GLUT4 (p = 0.005), and 2.46 ± 2.39 fold increase in GLUT12 (p = 0.002). The increased expression of GLUT1 helps explain the previous findings of conditioning-induced increases in basal glucose clearance in this model, and the increase in GLUT12 provides an alternative mechanism for insulin- and contraction-mediated glucose uptake and likely contributes to the substantial conditioning-induced increases in insulin sensitivity in highly trained athletic dogs. Furthermore, these results suggest that athletic dogs can serve as a valuable resource for the study of alternative glucose transport mechanisms in higher mammals.

Metformin acts in the gut and induces gut-liver crosstalk

Metformin is the most prescribed drug for DM2, but its site and mechanism of action are still not well established. Here, we investigated the effects of metformin on basolateral intestinal glucose uptake (BIGU), and its consequences on hepatic glucose production (HGP). In diabetic patients and mice, the primary site of metformin action was the gut, increasing BIGU, evaluated through PET-CT. In mice and CaCo2 cells, this increase in BIGU resulted from an increase in GLUT1 and GLUT2, secondary to ATF4 and AMPK. In hyperglycemia, metformin increased the lactate (reducing pH and bicarbonate in portal vein) and acetate production in the gut, modulating liver pyruvate carboxylase, MPC1/2, and FBP1, establishing a gut-liver crosstalk that reduces HGP. In normoglycemia, metformin-induced increases in BIGU is accompanied by hypoglycemia in the portal vein, generating a counter-regulatory mechanism that avoids reductions or even increases HGP. In summary, metformin increases BIGU and through gut-liver crosstalk influences HGP.

Effects of branched-chain amino acids on glucose uptake and lactose synthesis rates in bovine mammary epithelial cells and lactating mammary tissue slices

Even though supplementations of essential AA (EAA) are often related to increased lactose yields in dairy cows, underlying mechanisms connecting EAA availability to the mammary glands and lactose synthesis are poorly understood. The objective of this study was to examine the effects of branched-chain AA (BCAA) including Leu, Ile, and Val on (1) glucose transporter (GLUT1) abundance and glucose uptake, (2) the abundance of proteins regulating lactose synthesis pathway, and (3) fractional synthesis rates of lactose (FSR) using bovine mammary epithelial cells (BMEC) and mammary tissues slices (MTS). The BMEC (n = 4) were allocated randomly to regular Dulbecco's Modified Eagle Medium with Ham's F12 (DMEM/F12) medium (+EAA) or +EAA deficient (by 90%) in all EAA (-EAA), all BCAA (-BCAA), only Leu (-Leu), only Ile (-Ile) or only Val (-Val). Western immunoblotting analyses, depletion of glucose in media, and a proteomic analysis were performed to determine the abundance of GLUT1 in the cell membrane, net glucose uptake, and the abundance of enzymes involved in lactose synthesis pathway in BMEC, respectively. The MTS (n = 6) were allocated randomly to DMEM/F12 medium having all EAA and 13C-glucose at concentrations similar to plasma concentrations of cows (+EAAp), and +EAAp deprived of all BCAA (-BCAAp) or only Leu (-Leup) for 3 h. The 13C enrichments of free glucose pool in MTS (EGlu-free) and the enrichments of glucose incorporated into lactose in MTS and media [ELactose-bound (T&M)] were determined and used in calculating FSR. In BMEC, -BCAA increased the fraction of total GLUT1 translocated to the cell membrane and the fraction that was potentially glycosylated compared with +EAA. Among individual BCAA, only -Leu was associated with a 63% increase in GLUT1 translocated to the cell membrane and a 40% increase in glucose uptake of BMEC. The -BCAA tended to be related to a 75% increase in the abundance of hexokinase in BMEC. Deprivation of Leu tended to increase glucose uptake of MTS but did not affect EGlu-free, ELactose-bound (T&M), or FSR relative to +EAAp. On the other hand, -BCAAp did not affect glucose uptake of MTS but was related to lower ELactose-bound (T&M), or FSR relative to +EAAp. Considering together, decreasing Leu supply to mammary tissues enhances GLUT1 and thus glucose uptake, which, however, does not affect lactose synthesis rates. Moreover, the deficiency of other BCAA, Ile, and Val alone or together with the deficiency of Leu seemed to decrease lactose synthesis rates without affecting glucose uptake. The data also emphasize the importance of addressing the effect of the supply of other nutrients to the mammary glands than the precursor supply in describing the synthesis of a milk component.

Effect of high salinity acclimation on glucose homeostasis in Mozambique tilapia (Oreochromis mossambicus).

During salinity stress, osmoregulatory processes in euryhaline fish need to modify for their survival, and glucose is the preferred mode of extra energy during such conditions. These organisms must have a proper mechanism to maintain glucose homeostasis during such modified osmoregulatory process across different body fluids. Hence, we studied high salinity effect on regulation of glucose homeostasis in Mozambique tilapia. The fish were induced to 15‰ salinity for 21 days. Glucose, glycogen, ion concentrations, Na+-K+-ATPase, pyruvate kinase, γ-amylase activities and GLUT mRNA expressions were investigated in liver, intestine, gill and white muscle tissues. At the end of experiment, Na+ ion concentrations, glucose content and activity of Na+-K+-ATPase especially in the gill and intestine were increased, while decrease in liver and gill glycogen content was seen. Lower concentration of glycogen decrease was observed in the intestine and white muscle of the treated group. High pyruvate kinase activity was noticed in liver and gill tissues that correlates with high Na+-K+-ATPase activity. Elevated γ-amylase activity was observed in the liver and intestine suggesting breakdown of glycogen; however, gill and white muscle did not show any increased activity. Increase in GLUT1 and GLUT4 mRNA expressions was observed especially in the gill and intestine, while increase in GLUT2 mRNA expressions was observed in the liver. Upregulations of GLUTs suggest higher influx of glucose into the cell for catabolism to provide energy and further to drive the enhanced osmoregulatory process. These findings suggest glucose homeostasis being regulated in Mozambique tilapia during salinity acclimation.

Expression of GLUT-1 and GLUT-3 on spermatogenesis and fertility in rat testis with diabetes mellitus: an experimental study

Introduction: Diabetes mellitus is a major public health problem worldwide that may cause organ failure and dysfunction such as reproduction dysfunction, including infertility. Glucose metabolism is an important process in spermatogenesis. Glucose Transporters (GLUTs) play an important role in transferring glucose into cells to carry out the sperm capacitation process and maintain sperm motility. Although glucose is needed in spermatogenesis, hyperglycaemic conditions may cause detrimental effects on male fertility. This study was conducted to analyze the impact of hyperglycaemic status in rats on testicular function of spermatogenesis, specifically regarding GLUT-1 and GLUT-3.Methods: This study was an experimental study done in the integrated research and testing laboratory of Universitas Gadjah Mada from June to August 2018. This study used male Wistar rats, randomly divided into three groups as two intervention groups given Streptozotizin (STZ) induction for 4 and 8 weeks and one control group. The minimum sample size in each group was 9. Furthermore, the mRNA expression of GLUT-1 and GLUT-3 genes were assayed by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR).Results: This study showed an increase in GLUT-1 and GLUT-3 expression in rats that experienced hyperglycemia for 4 weeks (p = 0.008 and p = 0.002, respectively). Increased expression of GLUT-1 and GLUT-3 also occurred in rats that experienced hyperglycemia for 8 weeks, but the increase was not statistically significant (p=0.36 and p=0.079).Conclusion: Hyperglycaemia may cause an increase in GLUT-1 and GLUT-3 expression. This expression indicates that GLUT-1 and GLUT-3 are very sensitive to glucose levels changes and may play a role in spermatogenesis changes in patients with diabetes mellitus.

Insight Into The Molecular Basis Of The Anti‑Hyperglycemic Activity Of Ra‑3 In Type 2 Diabetic Rats And Its Cardioprotective Potential In Cultured Cardiomyoblasts

Insulin resistance, a hallmark of type 2 diabetes mellitus (T2DM), is the underlying cause of various metabolic changes observed in type 2 diabetic patients. This study investigated the molecular basis of the anti-hyperglycemic activity of RA-3, the lanosteryl triterpene from Protorhus longifolia, in type 2 diabetic rats and its cardioprotective potential in cultured cardiomyoblasts. To mimic a hyperglycemic state, H9c2 cells were exposed to high glucose concentration (33 mM) for 24 hours. Thereafter, the cells wells were treated with RA3 (1 μM). The high-fat diet fed (HFD) and STZ-induced T2DM in rat model was used to evaluate the anti-hyperglycemic activity of RA-3. The T2DM mimetic rats were orally administered with RA-3 (100 mg/kg) daily for 28 days. Blood and skeletal muscles were collected for biochemical and protein (IRS-1, AKT, GSK and GLUT 4) expression analysis. Biochemical analysis of the cell extracts revealed that RA3 improves glucose uptake via insulin- dependent (IRS-1/PI3K/Akt signaling) and independent (AMPK) pathways whilst ameliorating the activity of antioxidant enzymes defense in the H9c2 cells. The in vivo results showed significant decrease in p-GSK-3β and p-AKT expression, whereas the expression of IRS- 1ser307 were increased when compared to the model control group. This effect was ablated upon treatment of the animals with RA-3 and this was concomitant to an observed increase in GLUT 4 expression. It was concluded that the anti-hyperglycemic effect of RA-3 could partly be associated with its ability to improve cellular glucose uptake in muscle tissue and thus alleviating hyperglycemia- induced tissue damage.

Pancreatic β-cell-specific deletion of insulin-degrading enzyme leads to dysregulated insulin secretion and β-cell functional immaturity.

Inhibition of insulin-degrading enzyme (IDE) has been proposed as a possible therapeutic target for type 2 diabetes treatment. However, many aspects of IDE's role in glucose homeostasis need to be clarified. In light of this, new preclinical models are required to elucidate the specific role of this protease in the main tissues related to insulin handling. To address this, here we generated a novel line of mice with selective deletion of the Ide gene within pancreatic beta-cells, B-IDE-KO mice, which have been characterized in terms of multiple metabolic end points, including blood glucose, plasma C-peptide, and intraperitoneal glucose tolerance tests. In addition, glucose-stimulated insulin secretion was quantified in isolated pancreatic islets and beta-cell differentiation markers and insulin secretion machinery were characterized by RT-PCR. Additionally, IDE was genetically and pharmacologically inhibited in INS-1E cells and rodent and human islets, and insulin secretion was assessed. Our results show that, in vivo, life-long deletion of IDE from beta-cells results in increased plasma C-peptide levels. Corroborating these findings, isolated islets from B-IDE-KO mice showed constitutive insulin secretion, a hallmark of beta-cell functional immaturity. Unexpectedly, we found 60% increase in Glut1 (a high-affinity/low-Km glucose transporter), suggesting increased glucose transport into the beta-cell at low glucose levels, which may be related to constitutive insulin secretion. In parallel, IDE inhibition in INS-1E and islet cells resulted in impaired insulin secretion after glucose challenge. We conclude that IDE is required for glucose-stimulated insulin secretion. When IDE is inhibited, insulin secretion machinery is perturbed, causing either inhibition of insulin release at high glucose concentrations or constitutive secretion.

Annona stenophylla aqueous extract stimulate glucose uptake in established C2Cl2 muscle cell lines.

Annona stenophylla is a folk medicine popularly used in Zimbabwe for the treatment of many ailments. This study was carried out to determine some of the possible anti diabetic mechanisms of its action using in vitro cell culturing methods. A. stenophylla's effects on glucose uptake were tested using muscle cells (C2Cl2). Expression of glucose 4 transporters was determined by treating cell lines with plant extract. Total RNA was isolated and using RT-PCR, GLUT 4 expression levels were quantified. Translocation of GLUT 4 was assessed using FITC fluorescence measured by flow cytometry. Treatment of cells with plant extract significantly increased glucose uptake in a concentration dependent manner, with the highest concentration (250 µg/ml) giving 28% increased uptake compared to the negative control. The increase in glucose uptake (2.5 times more than control) was coupled to increase in GLUT 4 mRNA and subsequently GLUT 4 translocation. Wortmannin expunged the A. stenophylla induced increase in GLUT 4 mRNA and glucose uptake. The results suggest that A. stenophylla aqueous extract increases glucose uptake partly through increasing the GLUT 4 mRNA and translocation potentially acting via the PI-3-K pathway. This study confirms the ethnopharmacological uses of A. stenophylla indicating potential for anti-diabetic products formulation.

Molecular basis of the anti-hyperglycemic activity of RA-3 in hyperlipidemic and streptozotocin-induced type 2 diabetes in rats

BackgroundInsulin resistance is a hallmark of type 2 diabetes mellitus (T2DM) and the underlying cause of various metabolic changes observed in type 2 diabetic patients. This study investigated the molecular basis of the anti-hyperglycemic activity of the lanosteryl triterpene (RA-3), from Protorhus longifolia stem bark, in hyperlipidemic and streptozotocin (STZ)-induced T2DM in rats.MethodsThe high-fat diet fed (HFD) and STZ-induced T2DM in rat model was used to evaluate the anti-hyperglycemic activity of RA-3. The hyperlipidemic rats received a single intraperitoneal injection of STZ (35 mg/kg body weight) to induce T2DM. The experimental animals received a daily oral single dose of RA-3 (100 mg/kg body) for a period of 28 days, whiles the control group received distilled water only. The animals were euthanized, and skeletal muscle was collected for protein (IRS-1, AKT, GSK and GLUT 4) expression analysis. Western blot confirmed expression of the proteins.ResultsTreatment of the diabetic animals with the RA-3 showed marked reduction in fasting plasma glucose levels in comparison to the untreated diabetic group animals. A significant decrease in p-GSK-3β and p-AKT expression was observed, whereas the expression of IRS-1ser307 were increased when compared to the diabetic control group. This effect was ablated upon treatment with RA-3 and this was concomitant to an observed increase in GLUT 4 expression.ConclusionsThe results obtained in the present study strongly suggested that the anti-hyperglycemic effect of RA-3 could partly be associated with its ability to improve cellular glucose uptake in muscle tissue from T2DM.

Contribution of SGLT1 in cardiac glucose transport

Sodium-glucose transporter 1 (SGLT1) is the major SGLT isoform expressed in the heart. Under baseline condition, its absence does not affect heart function and structure. Using the pharmacological SGLT1 inhibitor, phlorizin, several data suggested that SGLT1 could contribute to overall glucose transport under physiological as well as under pathological conditions, i.e. in the ischemic heart. However, SGL1 role in glucose transport has never been formerly demonstrated. This study aimed to evaluate, in physiological and pathological conditions, the consequence of SGLT1 transporter invalidation on cardiomyocyte glucose uptake. Glucose uptake was measured in isolated cardiomyocytes using 2,3[H]-glucose and 3[H]-2-deoxyglucose. SGLT1 contribution was evaluated by pharmacologic (phlorizin) and genetic means (KO mice). GLUT transporters expression was examined by RT-qPCR and Western Blot. Under basal and hyperglycemic conditions as well as under insulin or oligomycin stimulation, glucose uptake was similar in SGLT1 + / + and SGLT1 − / − cardiomyocytes. We verified that SGLT1 absence was not compensated by an increase in GLUT1 or GLUT4 expression. Phlorizin inhibited insulin-stimulated glucose transport in a dose dependent manner, leading to its complete inhibition at high concentration (1 mM). However, this effect was independent of SGLT1 as phlorizin impact on glucose transport was similar in SGLT1 + / + and SGLT1 − / − cardiomyocytes. Furthermore, the phlorizin-induced glucose uptake inhibition was not explained by any change on insulin signaling or GLUT4 translocation, suggesting that phlorizin directly affected the GLUTs facilitated glucose transporters. Phlorizin effect on glucose transport does not involve SGLT1 but rather a direct effect on GLUTs. We also demonstrated that SGLT1 does not significantly contribute to glucose uptake in the cardiomyocyte in physiological and pathological conditions.

Melatonin regulates tumor aggressiveness under acidosis condition in breast cancer cell lines.

Breast cancer progression is composed of multiple steps that are influenced by tumor cell adaptations to survive under acidic conditions in the tumor microenvironment. Regulation of this cell survival behavior is a promising strategy to avoid cancer development. Melatonin is a natural hormone produced and secreted by the pineal gland capable of modulating different biological pathways in cancer. Although the anti-cancer effects of melatonin are currently widespread, its role in the acid tumor microenvironment remains poorly understood. The aim of the present study was to investigate the effect of low pH (6.7) on human breast cancer cell lines MCF-7 and MDA-MB-231, and the effectiveness of melatonin in acute acidosis survival mechanisms. Cell viability was measured by a MTT assay and the protein expression of glucose transporter (GLUT)-1, Ki-67 and caspase-3 was evaluated by immunocytochemical (ICC) analysis following low pH media and melatonin treatment. In both cell lines the viability was decreased after melatonin treatment (1 mM) under acidosis conditions for 24 h. ICC analysis showed a significant increase in GLUT-1 and Ki-67 expression at pH 6.7, and a decrease after treatment with melatonin for 12 and 24 h. The low pH media decreased the expression of caspase-3, which was increased after melatonin treatment for 12 and 24 h. Overall, the results of the present study revealed melatonin treatment increases apoptosis, as indicated by changes in caspase-3, and decreases proliferation, indicated by changes to Ki-67, and GLUT-1 protein expression under acute acidosis conditions in breast cancer cell lines.

Participation of HIFs in the regulation of Sertoli cell lactate production

Hypoxia Inducible Factors (HIFs) are master regulators of glycolytic metabolism. HIFs consist of a constitutive HIFbeta (HIFβ) subunit and a HIFalpha (HIFα) subunit, whose half-life depends on prolyl-hydroxylases activity. Inhibition of prolyl-hydroxylases by hypoxia or transition metals, or augmentation of HIFα subunit levels by hormonal stimuli lead to a higher HIF transcriptional activity. On the other hand, it is well known that lactate produced by Sertoli cells is delivered to and used by germ cells as an energy substrate. The aim of this work was to investigate whether HIFs participate in the regulation of lactate production in rat Sertoli cells and whether they are involved in the FSH mechanism of action. In order to reach a higher HIF transcriptional activity, Sertoli cells were treated with CoCl2. We observed that a higher HIF transcriptional activity leads to an augmentation of: lactate production, glucose uptake and LDH activity. Besides, an increase in Glut1, Pkm2 and Ldha mRNA levels was observed. These findings suggested that HIFs may participate in the modulation of Sertoli cell nutritional function. As FSH regulates lactate production, we evaluated whether HIFs were involved in FSH action. Sertoli cells were stimulated with FSH in the absence or presence of LW6, a drug which promotes HIFα subunit degradation. On the one hand, we observed that FSH increases HIF1α protein, Hif1α and Hif2α mRNA levels and, on the other hand, that LW6 inhibits FSH-stimulated lactate production, glucose uptake, Glut1, Pkm2 and Ldha expression. It is proposed that HIFs are key components of the intricate pathways utilized by FSH to regulate the provision of lactate for germ cells. Considering that FSH is the master endocrine regulator of Sertoli cells, it is not surprising that this hormone may employ several regulatory mechanisms to fulfill the nourishing functions of this cell type.

Changes in glucose transporter expression and nitric oxide production are associated with liver injury in diabetes.

In diabetes mellitus (DM), both hyperglycaemia and hyperlipidaemia can initiate accumulation of fat in the liver, which might be further mediated by inducible nitric oxide synthase. We have studied changes in GLUT1, nitric oxide (NO(·)) concentration and liver damage in two rat DM models. STZ model was induced by strepozotocin 50 mg/kg. HS model was induced by high-fat diet and 30 mg/kg streptozotocin. GLUT1 expression was studied by means of real-time RT-PCR and immunohistochemistry. Production of NO(·) was monitored by means of erythrocyte sedimentation rate spectroscopy of Fe-DETC-NO complex. Liver damage was assessed using histological activity index (HAI). NO(·) concentration was increased in the liver of STZ rats, but it did not change in HS rats (control 36.8 ± 10.3; STZ 142.1 ± 31.1; HS 35.4 ± 9.8 ng/g). Liver HAI was higher in STZ group, 8.6 ± 0.17 versus HS 4.7 ± 0.31, p < 0.05. GLUT1 protein expression was elevated only in STZ group, 16 ± 3 cells/mm(2) versus Control 5 ± 2 cells/mm(2), p = 0.007. Hyperglycaemia sooner causes severe liver damage in rat models of DM, compared with hyperlipidaemia, and is associated with increased NO(·) production. GLUT1 transporter expression might be involved in toxic effects of glucose in the liver. We have obtained novel data about association of GLUT1 expression and NO(·) metabolism in the pathogenesis of liver injury in DM. Increased GLUT1 expression was observed together with overproduction of NO(·) and pronounced liver injury in severely hyperglycaemic rats. On the contrary, moderately hyperglycaemic hyperlipidaemic rats developed only moderate liver steatosis and no increase in GLUT1 and NO(·). GLUT1 overexpression might be implicated in the toxic effects of glucose in the liver. Glycotoxicity is associated with oxidative stress and NO(·) hyperproduction. GLUT1 and NO(·) metabolism might become novel therapeutic targets in liver steatosis.

Abstract 2451: Insulin-like growth factor-1 receptor (IGF-1R) inhibitors downregulate p53 expression and upregulate the Warburg effect in paediatric glioblastoma cells

Abstract Introduction: Previously we showed that Picropodophyllin (PPP) - an inhibitor of Insulin-Like Growth Factor-1 Receptor (IGF-1R) promotes the Warburg effect and this effect is associated with the downregulation of p53 after 24h of PPP-treatment. Activation of IKK-NF-κB, by loss of p53, has been reported to increase glycolysis in cancer (1). Aims: To understand the molecular mechanism that links p53 downregulation with increased glycolysis after PPP treatment, we studied the changes in glucose metabolism in 3 different IGF-1R inhibitors - PPP, OSI906 and NVP-AEW541 (NVP) and related these changes to IKK-NF-κB expression and other molecular markers. Methods: KNS42 cells were treated for 24 hours with 5xIC50 of either PPP (2.4µM), OSI906 (220µM) or NVP (40µM). Culture media from each treated group and controls were analyzed by 1H-MRS. Lactate dehydrogenase-A (LDH-A), p53, pAKT, pS6, MCT-1, MCT-4, Glut-1, pIKK and β-actin (loading control) were examined by western blot (WB). LDH enzyme activity was also measured in PPP- and NVP-treated cells. Results: After 24 hours of OSI906, NVP and PPP treatment, decreases of 30% (p&amp;lt;0.01), 30% (p&amp;lt;0.01) and 50% (p&amp;lt;0.001) in cell number were found respectively, when compared with their respective controls. 1H-MRS analysis showed increases in glucose uptake (p&amp;lt;0.01) and lactate excretion (p&amp;lt;0.0001) in cells treated with the 3 IGF-1R inhibitors when compared with their corresponding controls. Larger increases were observed in the PPP-treated group when compared to the other 2 groups. In all 3 treatments, WB showed decreases in p53 and pAkt, an increase in Glut-1 and no change in MCT-1 expression. PPP-treated cells showed an increase in pS6 but a decreased pS6 expression was shown in OSI906- and NVP-treated cells. MCT-4 and pIKK expressions were higher in OSI906- and NVP-treated cells, but unchanged in the PPP-treated cells. LDH enzymatic activities were found to be significantly increased in PPP (p&amp;lt;0.01) and NVP (p&amp;lt;0.0001) treated groups, when compared with controls. Discussions and Conclusion: Our data indicated that the observed increased Warburg effect after IGF-1R inhibition is associated with increased Glut-1 expression, LDH activity and downregulation of p53 but the molecular mechanism is different in PPP when compared to OSI906 and NVP-treated cells. The increased Warburg effect after OSI906 and NVP treatment may be driven by the activation of the IKK-NF-κB pathway following the downregulation of p53, whereas upregulation of pS6 was seen in the PPP-treated cells. The activation of the mTOR-S6K pathway has been linked with the reduction of p53 expression (2). Further investigation is needed to examine why KNS42 cells have a different molecular response to PPP compared with OSI906 and NVP treatment. 1) Kawauchi et al., Nature Cell Biology 30(9): 1792-5, 2007. 2) Lai et al., EMBO 29: 2994-3006, 2010. Citation Format: Anne-Christine Wong Te Fong, Gabriela Andrejeva, Aleksandra Bielen, Chris Jones, John Griffiths, Martin O. Leach, Yuen-Li Chung. Insulin-like growth factor-1 receptor (IGF-1R) inhibitors downregulate p53 expression and upregulate the Warburg effect in paediatric glioblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2451. doi:10.1158/1538-7445.AM2014-2451

Regulation of human trophoblast GLUT1 glucose transporter by insulin-like growth factor I (IGF-I).

Glucose transport to the fetus across the placenta takes place via glucose transporters in the opposing faces of the barrier layer, the microvillous and basal membranes of the syncytiotrophoblast. While basal membrane content of the GLUT1 glucose transporter appears to be the rate-limiting step in transplacental transport, the factors regulating transporter expression and activity are largely unknown. In view of the many studies showing an association between IGF-I and fetal growth, we investigated the effects of IGF-I on placental glucose transport and GLUT1 transporter expression. Treatment of BeWo choriocarcinoma cells with IGF-I increased cellular GLUT1 protein. There was increased basolateral (but not microvillous) uptake of glucose and increased transepithelial transport of glucose across the BeWo monolayer. Primary syncytial cells treated with IGF-I also demonstrated an increase in GLUT1 protein. Term placental explants treated with IGF-I showed an increase in syncytial basal membrane GLUT1 but microvillous membrane GLUT1 was not affected. The placental dual perfusion model was used to assess the effects of fetally perfused IGF-I on transplacental glucose transport and syncytial GLUT1 content. In control perfusions there was a decrease in transplacental glucose transport over the course of the perfusion, whereas in tissues perfused with IGF-I through the fetal circulation there was no change. Syncytial basal membranes from IGF-I perfused tissues showed an increase in GLUT1 content. These results demonstrate that IGF-I, whether acting via microvillous or basal membrane receptors, increases the basal membrane content of GLUT1 and up-regulates basal membrane transport of glucose, leading to increased transepithelial glucose transport. These observations provide a partial explanation for the mechanism by which IGF-I controls nutrient supply in the regulation of fetal growth.

Lyophilized tea extracts of Brickellia cavanillesii (Asteraceae): in vitro characterization of biological activity.

Lyophilized Brickellia cavanillesii (LBC) tea extracts and identified chemical compounds of LBC were examined using in vitro human carcinoma liver (HepG2) cells with and without fetal bovine serum (FBS). Cells were incubated for 24 h with varying concentrations of FBS and LBC, respectively; cytotoxicity was determined spectrophotometrically using MTT (Formazan 3-(4,5-dimethyl-thiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide) assay. Furthermore, the potential hypoglycemic activity of LBC tea extracts was investigated using glucose transport and metabolism proteins biomarkers. FBS (0% to 10%) increased the viability of HepG2 cells steadily with increasing concentration. Possible therapeutic effects of LBC were concentration dependent with and without FBS. The cytotoxicity of 12 identified compounds from the LBC extract suggests that the individual compounds inhibited the proliferation of HepG2 cells differentially and do not reflect the inhibition of the whole aqueous LBC. Western blot analysis of glucose facilitated transporter protein 2 (GLUT 2) expression of HepG2 cells exposed to 0 mg/mL (Control) and 0.2 mg/mL LBC for 2, 4, 6, and 24 h suggests that GLUT 2 expression was increased. Increase in GLUT 2 expression in the absence of FBS was statistically significant with time of exposure. Significant difference was observed for GLUT 2 expression between 6 and 24 h and also between 4 and 24 h at 0.2 mg/mL LBC. Results obtained indicate that LBC may exhibit antidiabetic activity. However, further studies will be necessary to clearly delineate LBC potential therapeutic benefit and biological activities in animal studies as well as other in vitro models. Brickellia cavanillesii (Asteraceae) is a herbal plant widely used (Central America, Mexico and South-Western U.S.A.) in Type 2 diabetes mellitus therapy. Unfortunately, there is insufficient scientific data to validate its presumed pharmacological properties. This study examines the cytotoxic properties of whole, and certain identified chemical compounds in B. cavanillesii. It also investigates the potential hypoglycemic activity of lyophilized B. cavanillesii using glucose transport and metabolism proteins as biomarkers.

Differential regulation of GLUT1 and GLUT8 expression by hypoxia in mammary epithelial cells.

Glucose is a major substrate for milk synthesis and is taken up from the blood by mammary epithelial cells (MECs) through facilitative glucose transporters (GLUTs). The expression levels of GLUT1 and GLUT8 are upregulated dramatically in the mammary gland from late pregnancy through early lactation stages. This study aimed to test the hypothesis that this increase in GLUT1 and GLUT8 expression involves hypoxia signaling through hypoxia inducible factor-1α (HIF-1α) in MECs. Mouse mammary glands showed significantly more hypoxia in midpregnancy through early lactation stages compared with in the virgin stage, as stained by the hypoxia marker pimonidazole HCl. Treatment with hypoxia (2% O2) significantly stimulated glucose uptake and GLUT1 mRNA and protein expression, but decreased GLUT8 mRNA expression in bovine MECs. In MECs, hypoxia also increased the levels of HIF-1α protein in the nuclei, and siRNA against HIF-1α completely abolished the hypoxia-induced upregulation of GLUT1, while having no effect on GLUT8 expression. A 5'-RCGTG-3' core HIF-1α binding sequence was identified 3.7 kb upstream of the bovine GLUT1 gene, and HIF-1α binding to this site was increased during hypoxia. In conclusion, the mammary glands in pregnant and lactating animals are hypoxic, and MECs respond to this hypoxia by increasing GLUT1 expression and glucose uptake through a HIF-1α-dependent mechanism. GLUT8 expression, however, is negatively regulated by hypoxia through a HIF-1α-independent pathway. The regulation of glucose transporters through hypoxia-mediated gene transcription in the mammary gland may provide an important physiological mechanism for MECs to meet the metabolic demands of mammary development and lactation.

Roles for Peroxisome Proliferator-activated Receptor γ (PPARγ) and PPARγ Coactivators 1α and 1β in Regulating Response of White and Brown Adipocytes to Hypoxia

Obese white adipose tissue is hypoxic but is incapable of inducing compensatory angiogenesis. Brown adipose tissue is highly vascularized, facilitating delivery of nutrients to brown adipocytes for heat production. In this study, we investigated the mechanisms by which white and brown adipocytes respond to hypoxia. Brown adipocytes produced lower amounts of hypoxia-inducible factor 1α (HIF-1α) than white adipocytes in response to low O(2) but induced higher levels of hypoxia-associated genes. The response of white adipocytes to hypoxia required HIF-1α, but its presence alone was incapable of inducing target gene expression under normoxic conditions. In addition to the HIF-1α targets, hypoxia also induced many inflammatory genes. Exposure of white adipocytes to a peroxisome proliferator-activated receptor γ (PPARγ) ligand (troglitazone) attenuated induction of these genes but enhanced expression of the HIF-1α targets. Knockdown of PPARγ in mature white adipocytes prevented the usual robust induction of HIF-1α targets in response to hypoxia. Similarly, knockdown of PPARγ coactivator (PGC) 1β in PGC-1α-deficient brown adipocytes eliminated their response to hypoxia. These data demonstrate that the response of white adipocytes requires HIF-1α but also depends on PPARγ in white cells and the PPARγ cofactors PGC-1α and PGC-1β in brown cells.

Differential Regulation of Glucose Transporters Mediated by CRH Receptor Type 1 and Type 2 in Human Placental Trophoblasts

Glucose transport across the placenta is mediated by glucose transporters (GLUT), which is critical for normal development and survival of the fetus. Regulatory mechanisms of GLUT in placenta have not been elucidated. Placental CRH has been implicated to play a key role in the control of fetal growth and development. We hypothesized that CRH, produced locally in placenta, could act to modulate GLUT in placenta. To investigate this, we obtained human placentas from uncomplicated term pregnancies and isolated and cultured trophoblast cells. GLUT1 and GLUT3 expressions in placenta were determined, and effects of CRH on GLUT1 and GLUT3 were examined. GLUT1 and GLUT3 were identified in placental villous syncytiotrophoblasts and the endothelium of vessels. Treatment of cultured placental trophoblasts with CRH resulted in an increase in GLUT1 expression while a decrease in GLUT3 expression in a dose-dependent manner. Cells treated with either CRH antibody or nonselective CRH receptor (CRH-R) antagonist astressin showed a decrease in GLUT1 and an increase in GLUT3 expression. CRH-R1 antagonist antalarmin decreased GLUT1 expression while increased GLUT3 expression. CRH-R2 antagonist astressin2b increased the expression of both GLUT1 and GLUT3. Knockdown of CRH-R1 decreased GLUT1 expression while increased GLUT3 expression. CRH-R2 knockdown caused an increase in both GLUT1 and GLUT3 expression. Our data suggest that, in placenta, CRH produced locally regulates GLUT1 and GLUT3 expression, CRHR1 and CRHR2-mediated differential regulation of GLUT1 and GLUT3 expression. Placental CRH may regulate the growth of fetus and placenta by modulating the expression of GLUT in placenta during pregnancy.

Prolonged fasting suppresses cellular insulin‐dependent activity in adipose tissue of the northern elephant seal

Throughout its life, the northern elephant seal (Mirounga angustirostris) experiences periods of prolonged fasting during which plasma glucose reaches hyperglycemic levels. As the fast progresses, the levels of circulating glucose decrease, despite relatively low or decreasing plasma insulin levels. Cellular glucose uptake is dependent on an insulin‐activated signaling cascade resulting in the translocation of the glucose transport protein, Glut4, into the plasma membrane. In order to understand how this pathway is affected by prolonged fasting PI3K, AKT and VAMP2 content, and plasma insulin were examined. Blubber biopsies as well as plasma samples were obtained from two groups of northern elephant seal pups (n = 10/group) sampled during their post weaning fast: early (2 weeks) and late (6 to 8 weeks). Tissue content of PI3K, AKT, and VAMP2 proteins was quantified by western blot. Plasma insulin did not change (1.5 ± 0.4 vs. 1.2 ± 0.2 uU/mL) between early and late fasting. While PI3K and VAMP2 also did not change, AKT decreased 29% with the fast. We have previously shown that fasting is associated with a decrease in insulin receptor (IR), but an increase in Glut 4. Because insulin stimulates PI3K and AKT, the decreases in insulin and IR may explain the lack of change in PI3K and the decrease in AKT suggesting that fasting suppresses cellular insulin‐dependent activity. Funded by NIH HL91767 and UC Startup funds.