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

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

Ivermectin accelerates autophagic death of glioma cells by inhibiting glycolysis through blocking GLUT4 mediated JAK/STAT signaling pathway activation.

OBJECTIVE: This study aimed to investigate the regulatory effect of ivermectin (IVM) on energy metabolism in glioma progression, and provide a reference for the treatment of glioma. METHODS: Glioma cells were treated with IVM to measure cell viability, autophagy marker protein expression, ATP content, glucose uptake, pyruvate content, and expression of key enzymes of glycolysis. Glucose transporter 4 (GLUT4) or siGLUT4 was transfected in IVM treated U87 cells to investigate the effect of GLUT4 on cellular glycolysis and autophagy. The JAK2 inhibitor AZD-1480 was introduced to explore the specific mechanism by which IVM regulates glycolysis and autophagy. Rat models of glioma xenograft were constructed and treated with 10 mg/kg IVM to observe tumor growth and examine the expression levels of GLUT4 and autophagy marker proteins in tumor tissues. RESULTS: IVM inhibited glioma cell survival and promoted cell death. IVM promoted LC3-II protein expression and inhibited p62/SQSTM1 protein expression in glioma cells. IVM decreased adenosine-triphosphate (ATP) and pyruvate content, promoted glucose uptake, and reduced HK2 and PFK1 protein expression in U87 cells. IVM inhibited GLUT4 protein expression, and overexpression of GLUT4 promoted glycolysis and inhibited autophagic cell death in U87 cells. IVM inhibited glycolysis by blocking GLUT4 mediated the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway activation. IVM inhibited tumor growth in vivo, decreased the protein expression of GLUT4, JAK2, HK2, and PFK1 in tumor tissues, decreased the phosphorylation levels of STAT3/STAT5, and promoted the expression of autophagy marker proteins. CONCLUSIONS: IVM accelerates autophagic death of glioma cells by inhibiting glycolysis through blocking GLUT4 mediated JAK/STAT signaling pathway activation.

Low-Dose Acrolein, an Endogenous and Exogenous Toxic Molecule, Inhibits Glucose Transport via an Inhibition of Akt-Regulated GLUT4 Signaling in Skeletal Muscle Cells.

Urinary acrolein adduct levels have been reported to be increased in both habitual smokers and type-2 diabetic patients. The impairment of glucose transport in skeletal muscles is a major factor responsible for glucose uptake reduction in type-2 diabetic patients. The effect of acrolein on glucose metabolism in skeletal muscle remains unclear. Here, we investigated whether acrolein affects muscular glucose metabolism in vitro and glucose tolerance in vivo. Exposure of mice to acrolein (2.5 and 5 mg/kg/day) for 4 weeks substantially increased fasting blood glucose and impaired glucose tolerance. The glucose transporter-4 (GLUT4) protein expression was significantly decreased in soleus muscles of acrolein-treated mice. The glucose uptake was significantly decreased in differentiated C2C12 myotubes treated with a non-cytotoxic dose of acrolein (1 µM) for 24 and 72 h. Acrolein (0.5-2 µM) also significantly decreased the GLUT4 expression in myotubes. Acrolein suppressed the phosphorylation of glucose metabolic signals IRS1, Akt, mTOR, p70S6K, and GSK3α/ß. Over-expression of constitutive activation of Akt reversed the inhibitory effects of acrolein on GLUT4 protein expression and glucose uptake in myotubes. These results suggest that acrolein at doses relevant to human exposure dysregulates glucose metabolism in skeletal muscle cells and impairs glucose tolerance in mice.

Mitofusion is required for MOTS-c induced GLUT4 translocation.

MOTS-c (mitochondrial ORF of the twelve S-c) is a 16-amino-acid mitochondrial peptide that has been shown to counter insulin resistance and alleviate obesity in vivo. However, the mechanisms involved in the pharmacological action of MOTS-c remain elusive. Based on the ability of MOTS-c to improve insulin resistance and promote cold adaptation, we hypothesized that MOTS-c might play a role in boosting the number of mitochondria in a cell. We found that treatment of mammalian cells with MOTS-c increased protein levels of TFAM, COX4, and NRF1, which are markers for mitochondrial biogenesis. However, flow cytometry analysis using MitoTracker Green revealed a sharp reduction in the mitochondrial count after MOTS-c treatment. We then anticipated possible synchronized activation of mitofusion/mitochondrial fusion by MOTS-c following the onset of mitochondrial biogenesis. This was confirmed after a significant increase in protein levels two GTPases, OPA1, and MFN2, both vital for the fusion of mammalian mitochondria. Finally, we found that inhibition of the two GTPases by TNFα abrogated the ability of MOTS-c to prompt GLUT4 translocation and glucose uptake. Similar results were obtained by siRNA KD of MFN2 as well. Our results reveal for the first time a pathway that links mitofusion to MOTS-c-induced GLUT4 translocation.

Chronic valproic acid administration enhances oxidative stress, upregulates IL6 and downregulates Nrf2, Glut1 and Glut4 in rat's liver and brain.

Valproic acid (VPA) is a powerful antiepileptic drug that was associated with several neurological and hepatic problems especially with increasing its dose and duration. These problems may be metabolic in origin and related to glucose homeostasis. So, the present study investigated the effect of different doses and durations of VPA on the expression of glucose transporters (Glut1 and Glut4), oxidative stress and inflammatory cytokine (IL-6) in the liver and specific brain regions. Seventy-two male Sprague-Dawley rats were randomly allocated into three equal groups: (1) saline group, (2) 200 mg VPA group and (3) 400 mg VPA group. By the end of experiments, the expressions of Glut1, Glut4 nuclear factor erythroid-like 2 related factor (Nrf2), IL-6 and oxidative stress markers [malondialdehyde (MDA) and glutathione (GSH)] in the liver, corpus striatum, prefrontal cortex (PFC) and cerebellum were assessed. We found that administration of VPA (200 mg and 400 mg) caused a significant decrease in the Glut1 and Glut4 expression in different tissues in a dose- and time-dependent manner (P < 0.01). Also, VPA (200 and 400 mg) caused a significant increase in MDA with a decrease in GSH in tissues at different times. Moreover, VPA (200 and 400 mg) caused significant upregulation in IL-6 expression and downregulation in Nrf2 expression (P < 0.01). The results suggest that increasing the dose and time of VPA therapy downregulates Glut1 and Glut4 in the liver and brain which may impair glucose uptake in these tissues. This effect was associated with enhanced oxidative stress, downregulation of nrf2 and upregulation of IL-6 in liver and brain tissues.

miR-335-5p aggravates type 2 diabetes by inhibiting SLC2A4 expression.

Globally, type 2 diabetes (T2D) is the most common chronic disease. It affects approximately 500 million people worldwide. Dysregulation of the solute carrier family 2 member 4 (SLC2A4) gene and miR-335-5p has been associated with T2D progression. However, the mechanisms underlying this dysregulation are unclear. The levels of miR-335-5p and SLC2A4 in blood samples collected from patients with T2D (T2D blood samples) and pancreatic cell lines were measured by Real Time quantitative PCR (RT-qPCR). The relationship between miR-335-5p and SLC2A4 was investigated using a luciferase assay. The role of the miR-335-5p-SLC2A4 axis was detected by CCK8, BrdU, and caspase-3 assays in pancreatic cells treated with 25 mM glucose. Increased miR-335-5p and decreased SLC2A4 expression was observed in both T2D blood samples and pancreatic cell lines. The miR-335-5p mimic markedly suppressed proliferation and elevated apoptosis in glucose-treated pancreatic cells. SLC2A4 overexpression significantly enhanced proliferation but inhibited apoptosis in glucose-treated pancreatic cells. Moreover, miR-335-5p inhibited the expression of SLC2A4 in the pancreatic cells and suppressed the growth of these cells. The data indicated that miR-335-5p targeting of SLC2A4 could hamper the growth of T2D cell model by inhibiting their proliferation and elevating apoptosis. Collectively, our findings implicate miR-335-5p and SLC2A4 as potentially effective therapeutic targets for patients with T2D.

Upregulation of glucocorticoid receptor-mediated glucose transporter 4 in enzalutamide-resistant prostate cancer.

Enzalutamide (Enz) is a second-generation androgen receptor (AR) antagonist for castration-resistant prostate cancer (CRPC) therapy, and it prolongs survival time in these patients. However, during Enz treatment, CRPC patients usually acquire resistance to Enz and often show cross-resistance to other AR signaling inhibitors. Although glucocorticoid receptor (GR) is involved in this resistance, the role of GR has not yet been clarified. Here, we report that chronic Enz treatment induced GR-mediated glucose transporter 4 (GLUT4) upregulation, and that upregulation was associated with resistance to Enz and other AR signaling inhibitors. Additionally, inhibition of GLUT4 suppressed cell proliferation in Enz-resistant prostate cancer cells, which recovered from Enz resistance and cross-resistance without changes in GR expression. Thus, a combination of Enz and a GLUT4 inhibitor could be useful in Enz-resistant CRPC patients.

New insight in understanding the contribution of SGLT1 in cardiac glucose uptake: evidence for a truncated form in mice and humans.

Although sodium glucose cotransporter 1 (SGLT1) has been identified as one of the major SGLT isoforms expressed in the heart, its exact role remains elusive. Evidence using phlorizin, the most common inhibitor of SGLTs, has suggested its role in glucose transport. However, phlorizin could also affect classical facilitated diffusion via glucose transporters (GLUTs), bringing into question the relevance of SGLT1 in overall cardiac glucose uptake. Accordingly, we assessed the contribution of SGLT1 in cardiac glucose uptake using the SGLT1 knockout mouse model, which lacks exon 1. Glucose uptake was similar in cardiomyocytes isolated from SGLT1-knockout (Δex1KO) and control littermate (WT) mice either under basal state, insulin, or hyperglycemia. Similarly, in vivo basal and insulin-stimulated cardiac glucose transport measured by micro-PET scan technology did not differ between WT and Δex1KO mice. Micromolar concentrations of phlorizin had no impact on glucose uptake in either isolated WT or Δex1KO-derived cardiomyocytes. However, higher concentrations (1 mM) completely inhibited insulin-stimulated glucose transport without affecting insulin signaling nor GLUT4 translocation independently from cardiomyocyte genotype. Interestingly, we discovered that mouse and human hearts expressed a shorter slc5a1 transcript, leading to SGLT1 protein lacking transmembrane domains and residues involved in glucose and sodium bindings. In conclusion, cardiac SGLT1 does not contribute to overall glucose uptake, probably due to the expression of slc5a1 transcript variant. The inhibitory effect of phlorizin on cardiac glucose uptake is SGLT1-independent and can be explained by GLUT transporter inhibition. These data open new perspectives in understanding the role of SGLT1 in the heart.NEW & NOTEWORTHY Ever since the discovery of its expression in the heart, SGLT1 has been considered as similar as the intestine and a potential contributor to cardiac glucose transport. For the first time, we have demonstrated that a slc5a1 transcript variant is present in the heart that has no significant impact on cardiac glucose handling.

Structure guided design and synthesis of furyl thiazolidinedione derivatives as inhibitors of GLUT 1 and GLUT 4, and evaluation of their anti-leukemic potential.

Cancer cells increase their glucose uptake and glycolytic activity to meet the high energy requirements of proliferation. Glucose transporters (GLUTs), which facilitate the transport of glucose and related hexoses across the cell membrane, play a vital role in tumor cell survival and are overexpressed in various cancers. GLUT1, the most overexpressed GLUT in many cancers, is emerging as a promising anti-cancer target. To develop GLUT1 inhibitors, we rationally designed, synthesized, structurally characterized, and biologically evaluated in-vitro and in-vivo a novel series of furyl-2-methylene thiazolidinediones (TZDs). Among 25 TZDs tested, F18 and F19 inhibited GLUT1 most potently (IC50 11.4 and 14.7 µM, respectively). F18 was equally selective for GLUT4 (IC50 6.8 µM), while F19 was specific for GLUT1 (IC50 152 µM in GLUT4). In-silico ligand docking studies showed that F18 interacted with conserved residues in GLUT1 and GLUT4, while F19 had slightly different interactions with the transporters. In in-vitro antiproliferative screening of leukemic/lymphoid cells, F18 was most lethal to CEM cells (CC50 of 1.7 µM). Flow cytometry analysis indicated that F18 arrested cell cycle growth in the subG0-G1 phase and lead to cell death due to necrosis and apoptosis. Western blot analysis exhibited alterations in cell signaling proteins, consistent with cell growth arrest and death. In-vivo xenograft study in a CEM model showed that F18 impaired tumor growth significantly.

Glucose availability regulates nicotinamide N-methyltransferase expression in adipocytes.

BACKGROUND/OBJECTIVES: Nicotinamide N-methyltransferase (NNMT) is a novel regulator of energy homeostasis in adipocytes. NNMT expression in adipose tissue is increased in obesity and diabetes. Knockdown of NNMT prevents mice from developing diet-induced obesity, which is closely linked to insulin resistance. An early sign of systemic insulin resistance is reduced expression of glucose transporter 4 (GLUT4) selectively in adipose tissue. Adipose tissue-specific knockout and overexpression of GLUT4 cause reciprocal changes in NNMT expression. The aim of the current study was to elucidate the mechanism that regulates NNMT expression in adipocytes. METHODS: 3T3-L1 adipocytes were cultured in media with varying glucose concentrations or activators and inhibitors of intracellular pathways. NNMT mRNA and protein levels were measured with quantitative polymerase chain reaction and Western blotting. RESULTS: Glucose deprivation of 3T3-L1 adipocytes induced a 2-fold increase in NNMT mRNA and protein expression. This effect was mimicked by inhibition of glucose transport with phloretin, and by inhibition of glycolysis with the phosphoglucose isomerase inhibitor 2-deoxyglucose. Conversely, inhibition of the pentose phosphate pathway did not affect NNMT expression. Pharmacological activation of the cellular energy sensor AMP-activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin (mTOR) pathway caused an increase in NNMT levels that was similar to the effect of glucose deprivation. Activation of mTOR with MHY1485 prevented the effect of glucose deprivation on NNMT expression. Furthermore, upregulation of NNMT levels depended on functional autophagy and protein translation. CONCLUSION: Glucose availability regulates NNMT expression via an mTOR-dependent mechanism.

Low-concentration exposure to organochlorine pesticides (OCPs) in L6 myotubes and RIN-m5F pancreatic beta cells induces disorders of glucose metabolism.

We selected five substances among the organochlorine pesticides (OCPs), chlordane, heptachlor, p,p'-DDT, ß-HCH, and hexachlorobenzene, and investigated whether low-concentration exposure to the OCP compounds affected glucose metabolism. The exposure of L6 myotubes to the OCP compounds (1 or 5 µM) for 24 and 48 h significantly inhibited glucose uptake with an excessive production of intracellular reactive oxygen species (ROS) and peroxynitrite anions (ONOO-) compared to control cells. In contrast, the production of nitric oxide was highly reduced by exposure to the OCP compounds. The protein expression of glucose transporter 4 (GLUT4) in the L6 myotubes was significantly suppressed by exposure to the OCP compounds. In addition, exposure to the OCP compounds for 1 h in RIN-m5F pancreatic beta cells remarkably suppressed insulin secretion but the ability to secrete insulin recovered to control levels after 24 h exposure to the OCP compounds. The abundant ROS generated by 1 h exposure to OCP compounds was inversely related to insulin secretion in RIN-m5F pancreatic beta cells. Therefore, these results suggest that low-concenration exposure of skeletal muscle and pancreatic beta cells to OCP compounds may affect insulin secretion and insulin-dependent glucose uptake through extreme oxidative stress and inactivation of the glucose transport protein.

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

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

GLUT4 expression and glucose transport in human induced pluripotent stem cell-derived cardiomyocytes.

Induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) have the potential to transform regenerative cardiac medicine and the modelling of cardiac disease. This is of particular importance in the context of diabetic cardiomyopathy where diabetic individuals exhibit reduced cardiac diastolic contractile performance in the absence of vascular disease, significantly contributing towards high cardiovascular morbidity. In this study, the capacity of iPSC-CM to act as a novel cellular model of cardiomyocytes was assessed. The diabetic phenotype is characterised by insulin resistance, therefore there was a specific focus upon metabolic parameters. Despite expressing crucial insulin signalling intermediates and relevant trafficking proteins, it was identified that iPSC-CM do not exhibit insulin-stimulated glucose uptake. iPSC-CM are spontaneously contractile however contraction mediated uptake was not found to mask any insulin response. The fundamental limitation identified in these cells was a critical lack of expression of the insulin sensitive glucose transporter GLUT4. Using comparative immunoblot analysis and the GLUT-selective inhibitor BAY-876 to quantify expression of these transporters, we show that iPSC-CM express high levels of GLUT1 and low levels of GLUT4 compared to primary cardiomyocytes and cultured adipocytes. Interventions to overcome this limitation were unsuccessful. We suggest that the utility of iPSC-CMs to study cardiac metabolic disorders may be limited by their apparent foetal-like phenotype.

Structure-Based Docking Studies of GLUT4 Towards Exploring Selected Phytochemicals from Solanum xanthocarpum as a Therapeutic Target for the Treatment of Cancer.

BACKGROUND: In recent years, there has been an exponential increase in the global burden of cancer which has been associated with several factors including environmental influence, aging, diet, infectious agents, hormonal imbalance and chronic inflammation, among others. Cancerous cells utilize more glucose for its proliferation and survival than normal cells. Thus, the regulation of glucose consumption of cancerous cells through the inhibition of glucose transporter-4-protein (GLUT4) encoded by solute carrier family-2-member-4-gene (Slc2a4) by selected phytochemicals from Solanum xanthocarpum may serve as a new therapeutic candidate for the treatment of cancer. METHODS: The seven identified potential inhibitors of GLUT4 from Solanum xanthocarpum were retrieved from PubChem database. Examination of their drug-likeness, toxicity prediction and molecular docking studies of these compounds with GLUT4 were carried out using online tools such as Molinspiration, PreADMET V.2.0 and Patchdock server. RESULTS: The findings revealed that, five out of the seven compounds fulfil oral drugability of Lipinski's rule of five (RO5) while two slightly meet the criteria of RO5. Conversely, five of the compounds are predicted to be mutagen while the remaining two are predicted to be safe for the body. Additionally, stigmasterol glucoside has higher binding-affinity (7590) with GLUT4 when compared to doxorubicin (6600) the control. CONCLUSION: These findings suggest that stigmasterol glucoside from Solanum xanthocarpum could be a promising therapeutic agent with better therapeutic efficacy than doxorubicin in the treatment of cancer via the inhibition of GLUT4.

High Glucose Promotes Epithelial-Mesenchymal Transition of Uterus Endometrial Cancer Cells by Increasing ER/GLUT4-Mediated VEGF Secretion.

BACKGROUND/AIMS: Uterus endometrial cancer (UEC) is the common malignancy among gynecologic cancers, and most of them are type I estrogen-dependent UEC. Diabetes is well-known risk factor for the development of UEC. However, the underlying link between high glucose (HG) and the estrogen receptor in UEC remains unclear. Epithelial-mesenchymal transition (EMT) has also been shown to occur during the initiation of metastasis in cancer progression. The aim of this study was to determine the relationships and roles of HG, estrogen receptor and EMT in the growth and migration of UEC. METHODS: The expression of glucose transport protein 4 (GLUT4) in the control endometrium and UEC tissues was detected by immunohistochemistry (IHC); the cell viability and invasion were analyzed through CCK-8 and Matrigel invasion assays; the transcriptional level of EMT-related genes was evaluated through real-time PCR; and the effect of HG and / or GLUT4 on estrogen receptors, vascular endothelial growth factor (VEGF) and its receptor VEGFR was analyzed through western blotting, ELISA and flow cytometry (FCM) assay, respectively. In addition, Ishikawa-xenografted nude mice were constructed and were used to analyze the effect of estrogen and GLUT4 on the growth of UEC in vivo. RESULTS: Here, we found that exposure to HG led to a high level of viability and invasion of UEC cell lines (UECC, Ishikawa and RL95-2 cells). Compared with the normal endometrium, a higher level of GLUT4 was observed in UEC tissues. Silencing GLUT4 obviously inhibited the HG-promoted viability, invasion and expression of EMT-related genes (TWIST, SNAIL and CTNNB1) of UECC promoted by HG. Further analysis showed that HG and GLUT4 promoted the secretion of VEGF and expression of VEGFR in UECC. Treatment with HG led to the increase of estrogen receptor α (ERα) and ß (ERß) in UECC, blocking ERα or ERß resulted in the decreases in GLUT4 expression, TWIST, SNAIL and CTNNB1 transcription, and VEGF and VEGFR expression in UECC. Treatment with anti-human VEGF neutralizing antibody restricted the viability and invasion of UECC that was induced by HG and estrogen. Exposure to estrogen accelerated growth, VEGF production, and TWIST and CTNNB1 expression in UEC in Ishikawa-xenografted nude mice, and silencing GLUT4 restricted these effects. CONCLUSION: These data suggest that HG increases GLUT4 and VEGF/VEGFR expression, further promotes EMT process and accelerates the development of UEC by up-regulating ER.

Anti-adipogenic effects of KD025 (SLx-2119), a ROCK2-specific inhibitor, in 3T3-L1 cells.

Adipose tissue is a specialized organ that synthesizes and stores fat. During adipogenesis, Rho and Rho-associated kinase (ROCK) 2 are inactivated, which enhances the expression of pro-adipogenic genes and induces the loss of actin stress fibers. Furthermore, pan ROCK inhibitors enhance adipogenesis in 3T3-L1 cells. Here, we show that KD025 (formerly known as SLx-2119), a ROCK2-specific inhibitor, suppresses adipogenesis in 3T3-L1 cells partially through a ROCK2-independent mechanism. KD025 downregulated the expression of key adipogenic transcription factors PPARγ and C/EBPα during adipogenesis in addition to lipogenic factors FABP4 and Glut4. Interestingly, adipogenesis was blocked by KD025 during days 1~3 of differentiation; after differentiation terminated, lipid accumulation was unaffected. Clonal expansion occurred normally in KD025-treated cells. These results suggest that KD025 could function during the intermediate stage after clonal expansion. Data from depletion of ROCKs showed that KD025 suppressed cell differentiation partially independent of ROCK's activity. Furthermore, no further loss of actin stress fibers emerged in KD025-treated cells during and after differentiation compared to control cells. These results indicate that in contrast to the pro-adipogenic effect of pan-inhibitors, KD025 suppresses adipogenesis in 3T3-L1 cells by regulating key pro-adipogenic factors. This outcome further implies that KD025 could be a potential anti-adipogenic/obesity agent.

Insulin modulates hippocampally-mediated spatial working memory via glucose transporter-4.

The insulin-regulated glucose transporter, GluT4, is a key molecule in peripheral insulin signaling. Although GluT4 is abundantly expressed in neurons of specific brain regions such as the hippocampus, the functional role of neuronal GluT4 is unclear. Here, we used pharmacological inhibition of GluT4-mediated glucose uptake to determine whether GluT4 mediates insulin-mediated glucose uptake in the hippocampus. Consistent with previous reports, we found that glucose utilization increased in the dorsal hippocampus of male rats during spontaneous alternation (SA), a hippocampally-mediated spatial working memory task. We previously showed that insulin signaling within the hippocampus is required for processing this task, and that administration of exogenous insulin enhances performance. At baseline levels of hippocampal insulin, inhibition of GluT4-mediated glucose uptake did not affect SA performance. However, inhibition of an upstream regulator of GluT4, Akt, did impair SA performance. Conversely, when a memory-enhancing dose of insulin was delivered to the hippocampus prior to SA-testing, inhibition of GluT4-mediated glucose transport prevented cognitive enhancement. These data suggest that baseline hippocampal cognitive processing does not require functional hippocampal GluT4, but that cognitive enhancement by supra-baseline insulin does. Consistent with these findings, we found that in neuronal cell culture, insulin increases glucose utilization in a GluT4-dependent manner. Collectively, these data demonstrate a key role for GluT4 in transducing the procognitive effects of elevated hippocampal insulin.

Suppression of adipocyte differentiation and lipid accumulation by stearidonic acid (SDA) in 3T3-L1 cells.

BACKGROUND: Increased consumption of omega-3 (ω-3) fatty acids found in cold-water fish and fish oil has been reported to protect against obesity. A potential mechanism may be through reduction in adipocyte differentiation. Stearidonic acid (SDA), a plant-based ω-3 fatty acid, has been targeted as a potential surrogate for fish-based fatty acids; however, its role in adipocyte differentiation is unknown. This study was designed to evaluate the effects of SDA on adipocyte differentiation in 3T3-L1 cells. METHODS: 3T3-L1 preadipocytes were differentiated in the presence of SDA or vehicle-control. Cell viability assay was conducted to determine potential toxicity of SDA. Lipid accumulation was measured by Oil Red O staining and triglyceride (TG) quantification in differentiated 3T3-L1 adipocytes. Adipocyte differentiation was evaluated by adipogenic transcription factors and lipid accumulation gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Fatty acid analysis was conducted by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). RESULTS: 3T3-L1 cells treated with SDA were viable at concentrations used for all studies. SDA treatment reduced lipid accumulation in 3T3-L1 adipocytes. This anti-adipogenic effect by SDA was a result of down-regulation of mRNA levels of the adipogenic transcription factors CCAAT/enhancer-binding proteins alpha and beta (C/EBPα, C/EBPß), peroxisome proliferator-activated receptor gamma (PPARγ), and sterol-regulatory element binding protein-1c (SREBP-1c). SDA treatment resulted in decreased expression of the lipid accumulation genes adipocyte fatty-acid binding protein (AP2), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD-1), lipoprotein lipase (LPL), glucose transporter 4 (GLUT4) and phosphoenolpyruvate carboxykinase (PEPCK). The transcriptional activity of PPARγ was found to be decreased with SDA treatment. SDA treatment led to significant EPA enrichment in 3T3-L1 adipocytes compared to vehicle-control. CONCLUSION: These results demonstrated that SDA can suppress adipocyte differentiation and lipid accumulation in 3T3-L1 cells through down-regulation of adipogenic transcription factors and genes associated with lipid accumulation. This study suggests the use of SDA as a dietary treatment for obesity.

Development of GLUT4-selective antagonists for multiple myeloma therapy.

Cancer cells consume more glucose to fuel metabolic programs fundamental to sustaining their survival, growth and proliferation. Among the fourteen SLC2A family members, GLUTs 1 and 4 are high-affinity glucose transporters. GLUT4 (SLC2A4) is highly expressed in muscle and adipose tissue. Basally retained within the cell, GLUT4 traffics to the plasma membrane (PM) in response to insulin and exercise-stimulation. The plasma cell malignancy multiple myeloma (MM) exhibits increased constitutive expression of GLUT4 on the PM, co-opting use of GLUT4 for survival and proliferation. GLUT4 inhibition by knockdown or treatment with the FDA-approved HIV protease inhibitor ritonavir leads to cytostatic and/or cytotoxic and chemosensitizing effects in tumor cells both in vitro and in vivo. We recently reported our generation of GLUT4 homology models and virtual high-throughput screening (vHTS) to identify multiple series of novel GLUT4 antagonists. In this report, we describe our initial hit-to-lead optimization to synthesize new analogs with improved potency and selectivity for GLUT4, and the biological characterization of these compounds in a variety of assays. We show that our lead compound (compound 20) decreases glucose uptake and cell proliferation as well as inhibits the expression of pro-survival MCL-1 in MM similar to the effect observed via knockdown of GLUT4 expression. Compound 20 is also effective at chemosensitizing multiple myeloma cell lines and patient samples to venetoclax, dexamethasone and melphalan. In sum, we report development of selective GLUT4 inhibitors lacking inhibitory activity against GLUT1 and GLUT8. We show that selective pharmacological inhibition of GLUT4 is feasible and this may represent a novel strategy for the treatment and chemosensitization of multiple myeloma to standard therapeutics.

PTEN, Insulin Resistance and Cancer.

BACKGROUND: The tumor suppressor PTEN serves as a negative regulator of PI3K/PTEN/Akt signaling pathway that regulates cellular functions such as cell growth, differentiation, proliferation and migration. The PI3K/PTEN/Akt signaling cascades might also have effect on glucose uptake via translocation of GLUT-4. Insulin controls energy storage and the whole body glucose homeostasis. Its binding to insulin receptor on the surface of diverse cells allows glucose entry into cells, and activates a variety of cellular actions. Insulin resistance is a common metabolic feature and established risk factor of many diseases. Its fundamental principle is inability of insulin to exert its normal metabolic effects, and nutrient imbalance and abnormal lipid accumulation in skeletal muscle, liver and adipose tissues. METHODS: We review the literature on the structure and function of PTEN and its involvement in insulin resistance and tumor regulation, and summarized the detailed scientific achievements on this topic. RESULTS: Suppressing PTEN expression plays a role in pro- or anti-inflammatory state during insulin resistance associated with obesity. Selective disruption of PTEN in pancreatic α-cells demonstrates that a lack of PTEN reduces circulating glucagon levels and protects against hyperglycemia and insulin resistance in high-fat diet-fed mice. Loss-of-function PTEN mutations in adipose tissue results in systemic glucose tolerance and insulin sensitivity improvement because of ascended recruitment of the GLUT-4 towards the membrane. Targeting tissuespecific PTEN deletion improves insulin sensitivity and protects from systemic insulin resistance. PTEN, as an important tumor suppressor gene, is frequently deleted or mutated in a variety of human tumors. Inactivation of PTEN by loss-of-function mutations leads to deregulated hyperproliferation of cells, leading to oncogenic transformation. CONCLUSION: Considering PTEN's important role in insulin resistance and tumor regulation, targeting the PTEN gene and/or protein will likely provide an efficient strategy for therapeutic intervention in cancer and metabolic diseases like type 2 diabetes mellitus, obesity, and cardiovascular dysfunction.