Expression Of Glucose Transporter GLUT1 Research Articles

MTOR signaling is required for phagocyte free radical production, GLUT1 expression, and control of Staphylococcus aureus infection.

Mammalian target of rapamycin (mTOR) is a key regulator of metabolism in the mammalian cell. Here, we show the essential role for mTOR signaling in the immune response to bacterial infection. Inhibition of mTOR during infection with Staphylococcus aureus revealed that mTOR signaling is required for bactericidal free radical production by phagocytes. Mechanistically, mTOR supported glucose transporter GLUT1 expression, potentially through hypoxia-inducible factor 1α, upon phagocyte activation. Cytokine and chemokine signaling, inducible nitric oxide synthase, and p65 nuclear translocation were present at similar levels during mTOR suppression, suggesting an NF-κB-independent role for mTOR signaling in the immune response during bacterial infection. We propose that mTOR signaling primarily mediates the metabolic requirements necessary for phagocyte bactericidal free radical production. This study has important implications for the metabolic requirements of innate immune cells during bacterial infection as well as the clinical use of mTOR inhibitors.IMPORTANCESirolimus, everolimus, temsirolimus, and similar are a class of pharmaceutics commonly used in the clinical treatment of cancer and the anti-rejection of transplanted organs. Each of these agents suppresses the activity of the mammalian target of rapamycin (mTOR), a master regulator of metabolism in human cells. Activation of mTOR is also involved in the immune response to bacterial infection, and treatments that inhibit mTOR are associated with increased susceptibility to bacterial infections in the skin and soft tissue. Infections caused by Staphylococcus aureus are among the most common and severe. Our study shows that this susceptibility to S. aureus infection during mTOR suppression is due to an impaired function of phagocytic immune cells responsible for controlling bacterial infections. Specifically, we observed that mTOR activity is required for phagocytes to produce antimicrobial free radicals. These results have important implications for immune responses during clinical treatments and in disease states where mTOR is suppressed.

Suprachiasmatic nucleus promotes hyperglycemia induced by sleep delay

Short sleep is linked to disturbances in glucose metabolism and may induce a prediabetic condition. The biological clock in the suprachiasmatic nucleus (SCN) regulates the glucose rhythm in the circulation and the sleep-wake cycle. SCN vasopressin neurons (SCNVP) control daily glycemia by regulating the entrance of glucose into the arcuate nucleus (ARC). Thus, we hypothesized that sleep delay may influence SCN neuronal activity. We, therefore, investigated the role of SCNVP when sleep is disrupted by forced locomotor activity. After 2h of sleep delay, rats exhibited decreased SCNVP neuronal activity, a decrease in the glucose transporter GLUT1 expression in tanycytes lining the third ventricle, lowered glucose entrance into the ARC, and developed hyperglycemia. The association between reduced SCNVP neuronal activity and hyperglycemia in sleep-delayed rats was evidenced by injecting intracerebroventricular vasopressin; this increased GLUT1 immunoreactivity in tanycytes, thus promoting normoglycemia. Following sleep recovery, glucose levels decreased, whereas SCNVP neuronal activity increased. These results imply that sleep-delay-induced changes in SCNVP activity lead to glycemic impairment, inferring that disruption of biological clock function might represent a critical step in developing type 2 diabetes.

Antimutagenic 1,4-Dihydropyridine AV-153 Normalizes Expression of GLUT1, GLUT4, INOS, PARP1, and Gamma H2AX Histone in Myocardium of Rats with Streptozotocin Model of Diabetes Mellitus

Abstract Diabetic cardiomyopathy increases the risk of heart failure and worsens prognosis for diabetes mellitus (DM) patients. Its development depends on many factors, including modification of nitric oxide production and impaired DNA repair. The goal of the present work was to study in vivo effects of a 1,4-dihydropyridine AV-153, known as antimutagen and DNA-binder, on DNA integrity, and on the expression of several proteins involved in glucose transport, nitric oxide metabolism, and DNA repair in myocardium in diabetic rats. DM was induced in rats by streptozotocin (STZ) injection. Expression of proteins was studied by means of immunohistochemistry. Development of the STZ-induced DM significantly induced PARP1 and gamma H2AX histone, markers of DNA breakage, protein expression in heart tissue, while AV-153 administration decreased PARP1 and H2AX protein expression. In this model of diabetes, myocardial expression of iNOS was also significantly increased, but administration of AV-153 reduced it to normal levels. AV-153 also upregulated the expression of insulin-dependent GLUT4 and insulin-independent GLUT1 glucose transporters up to normal level in diabetic rats. Thus, AV-153 appears to be prospective for creation of a remedy for prevention of diabetic cardiomyopathy.

Accumulation of branched-chain amino acids reprograms glucose metabolism in CD8+ T cells with enhanced effector function and anti-tumor response

Branched-chain amino acids (BCAAs) provide nutrient signals for cell survival and growth. How BCAAs affect CD8+ Tcell functions remains unexplored. Herein, we report that accumulation of BCAAs in CD8+ Tcells due to the impairment of BCAA degradation in 2C-type serine/threonine protein phosphatase (PP2Cm)-deficient mice leads to hyper-activity of CD8+ Tcells and enhanced anti-tumor immunity. CD8+ Tcells from PP2Cm-/- mice upregulate glucose transporter Glut1 expression in a FoxO1-dependent manner with more glucose uptake, as well as increased glycolysis and oxidative phosphorylation. Moreover, BCAA supplementation recapitulates CD8+ Tcell hyper-functions and synergizes with anti-PD-1, in line with a better prognosis in NSCLC patients containing high BCAAs when receiving anti-PD-1 therapy. Our finding thus reveals that accumulation of BCAAs promotes effector function and anti-tumor immunity of CD8+ Tcells through reprogramming glucose metabolism, making BCAAs alternative supplementary components to increase the clinical efficacy of anti-PD-1 immunotherapy against tumors.

Cystathionine γ-lyase and hydrogen sulfide modulates glucose transporter Glut1 expression via NF-κB and PI3k/Akt in macrophages during inflammation.

Macrophages play a crucial role in inflammation, a defense mechanism of the innate immune system. Metabolic function powered by glucose transporter isoform 1 (Glut1) is necessary for macrophage activity during inflammation. The present study investigated the roles of cystathionine-γ-lyase (CSE) and its byproduct, hydrogen sulfide (H2S), in macrophage glucose metabolism to explore the mechanism by which H2S acts as an inflammatory regulator in lipopolysaccharide- (LPS) induced macrophages. Our results demonstrated that LPS-treated macrophages increased Glut1 expression. LPS-induced Glut1 expression is regulated via nuclear factor (NF)-κB activation and is associated with phosphatidylinositol-3-kinase PI3k activation. Small interfering (si) RNA-mediated silencing of CSE decreased the LPS-induced NF-κB activation and Glut1 expression, suggesting a role for H2S in metabolic function in macrophages during pro-inflammatory response. Confoundingly, treatment with GYY4137, an H2S-donor molecule, also displayed inhibitory effects upon LPS-induced NF-κB activation and Glut1 expression. Moreover, GYY4137 treatment increased Akt activation, suggesting a role in promoting resolution of inflammation. Our study provides evidence that the source of H2S, either endogenous (via CSE) or exogenous (via GYY4137), supports or inhibits the LPS-induced NF-κB activity and Glut1 expression, respectively. Therefore, H2S may influence metabolic programming in immune cells to alter glucose substrate availability that impacts the immune response.

Glucose transporter GLUT1 expression is important for oriental river prawn (Macrobrachium nipponense) hemocyte adaptation to hypoxic conditions

Crustaceans have an open vascular system in which hemocytes freely circulate in hemolymph. Hemocytes are rich in hemocyanin, a specific oxygen-transport protein in crustaceans; therefore, understanding the response of hemocytes to hypoxia is crucial. Although hemocytes take up glucose during hypoxia, the molecular mechanism of glucose uptake in crustaceans remains unclear. Herein, we identified two highly conserved glucose transporters (GLUT1 and GLUT2) in Macrobrachium nipponense (oriental river prawn) and analyzed their tissue-specific expression patterns. Our immunofluorescence assays showed that GLUT1 and GLUT2 are located on the cell membrane, with a strong GLUT1 signal in primary hemocytes under hypoxia. We found that during acute hypoxia, hypoxia-inducible factor-1α-related metabolic alterations result in decreased mitochondrial cytochrome c oxidase activity, implying a classic glycolytic mechanism. As a proof of concept, we replicated these findings in insect S2 cells. Acute hypoxia significantly induced hypoxia-inducible factor-1α, GLUT1, and pyruvate dehydrogenase kinase isozyme 1 expression in primary hemocytes, and hypoxia-induced increases in glucose uptake and lactate secretion were observed. GLUT1 knockdown induced intracellular reactive oxygen species generation and apoptosis invitro and invivo, resulting in increased prawn mortality and more apoptotic cells in their brains, implying a vital function of GLUT1 in hypoxia adaptation. Taken together, our results suggest a close relationship between hypoxia-mediated glycolysis and GLUT1 in hemocytes. These results demonstrated that in crustaceans, adaptation to hypoxia involves glucose metabolic plasticity.

Huangqin Decoction Exerts Beneficial Effects on Rotenone-Induced Rat Model of Parkinson's Disease by Improving Mitochondrial Dysfunction and Alleviating Metabolic Abnormality of Mitochondria.

Parkinson's disease (PD) is a common neurodegenerative disease, and the pathogenesis of PD is closely related to mitochondrial dysfunction. Previous studies have indicated that traditional Chinese medicine composition of Huangqin Decoction (HQD), including Scutellariae Radix, licorice, and Paeoniae Radix Alba, has therapeutic effects on PD, but whether HQD has a therapeutic effect on PD has not been reported. In this study, the protective effects of HQD on rotenone-induced PD rats were evaluated by behavioral assays (open field, rotating rod, suspension, gait, inclined plate, and grid) and immunohistochemistry. The mechanisms of HQD on attenuation of mitochondrial dysfunction were detected by biochemical assays and mitochondrial metabolomics. The results showed that HQD (20 g/kg) can protect rats with PD by improving motor coordination and muscle strength, increasing the number of tyrosine hydroxylase (TH)-positive neurons in rats with PD. Besides, HQD can improve mitochondrial dysfunction by increasing the content of adenosine triphosphate (ATP) and mitochondrial complex I. Mitochondrial metabolomics analysis revealed that the ketone body of acetoacetic acid (AcAc) in the rotenone group was significantly higher than that of the control group. Ketone bodies have been known to be used as an alternative energy source to provide energy to the brain when glucose was deficient. Further studies demonstrated that HQD could increase the expression of glucose transporter GLUT1, the content of tricarboxylic acid cycle rate-limiting enzyme citrate synthase (CS), and the level of hexokinase (HK) in rats with PD but could decrease the content of ketone bodies [AcAc and β-hydroxybutyric acid (β-HB)] and the expression of their transporters (MCT1). Our study revealed that the decrease of glucose metabolism in the rotenone group was parallel to the increase of substitute substrates (ketone bodies) and related transporters, and HQD could improve PD symptoms by activating the aerobic glycolysis pathway.

Abstract 1089: Targeting amino acid uptake in lung squamous cell carcinomas to improve response to targeted therapies

Abstract Lung squamous cell carcinomas (LUSC) are a subtype of non-small cell lung cancer characterized by poor overall survival. LUSC are highly aggressive in part due to their high metabolic activity, with uptake of high levels of glucose supporting their growth and proliferation. The main driver of high rate of glycolysis in LUSC is elevated expression of glucose transporter GLUT1, whose levels and membrane localization are regulated by activated PI3K-AKT-mTOR pathway. The Cancer Genome Atlas revealed that LUSC have high tumor mutational burden with varied genetic profile that includes amplification of receptor tyrosine kinases (RTKs), PI3KCA, and a loss of PTEN. These alterations lead to increased activity of mTOR pathway, one of the major pathways in cancer cells that drives cell proliferation and metabolism. We have recently demonstrated that catalytic mTOR kinase inhibitor TAK228 (MLN128) inhibits glycolysis in LUSC cell lines in vitro, in xenografts of human LUSC lines, in patient derived xenografts (PDXs) and genetically engineered mouse models. However, despite lowered glucose uptake measured by 18F-FDG signal LUSC tumors were able to maintain high levels of proliferation. Tumors were able to adapt to prolonged treatment with TAK228 by relying on glutaminolysis. Increased glutaminolysis was due to increased uptake of glutamine as well as increased levels of glutaminase (GLS), a key enzyme that converts glutamine to glutamate. Inhibiting both mTOR (with TAK228) and GLS (with CB-839) resulted in reduced proliferation index and lower tumor volume. However, while combination therapy halted tumor growth, there was a lack of tumor regression in xenografts and PDXs of LUSC. Therefore, tumors are poised to further adapt to combination therapy through metabolism of additional amino acids beyond glutamine. Here we show that macropinocytosis, a form of endocytosis that allows cancer cells to uptake extracellular fluids and further process solutes and macromolecules in lysosomes leading to the release of amino acids inside the cytoplasm, is upregulated in LUSC tumors that are resistant to TAK228. Macropinocytosis is induced by active RTKs, PI3KCA, Ras, loss of PTEN, and active NRF2 pathway - oncogenic alterations commonly found in LUSC tumors. This suggests that macropinocytosis might be a mechanism that supports amino acid metabolism utilized by LUSC in order to escape mTOR therapy. Basal levels of macropinocytosis in LUSC appear low, however, upon treatment with mTOR inhibitors, lysosomal catabolism of macropinocytosed proteins increases, allowing tumor cells to increase their proliferation. Resistance to targeted therapies is a major hurdle to achieving effective therapies in multiple cancer types. Identifying ways to overcome resistance to targeted therapies would allow for longer therapeutic response for larger number of patients, ultimately leading to longer survival and better quality of life. Citation Format: Milica Momcilovic, David Shackelford. Targeting amino acid uptake in lung squamous cell carcinomas to improve response to targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1089.

Dihydroartemisinin and artesunate inhibit aerobic glycolysis via suppressing c-Myc signaling in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) cells exhibit aberrant metabolism characterized by high glycolysis even in the presence of abundant oxygen. Inhibition of aerobic glycolysis remains challenging when identifying potential cancer-specific inhibitors while maintaining or even boosting the anti-cancer immunity. Artemisinin derivatives DHA and AS have shown excellent anti-tumor and immunoenhancing roles in numerous malignancies, but the molecular mechanism of DHA and AS in regulating cancer glucose metabolism is largely unknown. In this study, we proved that DHA and AS inhibit NSCLC growth via prohibiting cancer cell aerobic glycolysis through ERK/c-Myc pathway. First, we proved that DHA and AS have comparable anti-cancer growth roles in both NSCLC cell lines and mouse Lewis Lung Cancer model. Then, our data clearly showed that DHA and AS dose- and time-dependently reduce the uptake of glucose, the production of ATP, and the secretion of lactate, the expression of glucose transporter GLUT1 and two key glycolysis-related enzymes hexokinase and lactate dehydrogenase, as well as the level of c-Myc. Finally, we generated c-Mychigh stable-expressing NSCLC cell line and treated it with DHA or AS, respectively. Our data clearly showed that c-Myc overexpression can partially reverse the glycolysis-repressing role of DHA and AS which strongly supported our proposal that AS and DHA suppress aerobic glycolysis in a c-Myc-dependent manner in NSCLC cells. This study extends our knowledge of artemisinin derivatives in regulating tumor glucose metabolism and provides potential strategy in the therapy of lung cancer.

Glucose-assisted trophic conversion of Chlamydomonas reinhardtii by expression of glucose transporter GLUT1

The use of genetic engineering techniques to alter the nutritional patterns of microalgae is a key means of enhancing microalgal biomass, as it enables the highly efficient heterotrophic growth of microalgae via the utilisation of organic carbon sources, especially glucose. To investigate the effects of the expression of the glucose transporter on the growth of the model alga, Chlamydomonas reinhardtii, we transformed C. reinhardtii using the human GLUT1 gene, regulated by the Hsp70, PsaD, and Arg7 promoters, and obtained engineered algae after screening. The engineered algae showed better growth than the control algae under both heterotrophic and mixotrophic growth conditions. The engineered alga in which the GLUT1 gene was regulated by the Arg7 promoter exhibited the best growth. Although the engineered algae exhibited heterotrophic growth in complete darkness by using glucose as the carbon source, their growth rates and biomass were relatively low compared to those of the control. Analysis of the glucose transport ability of the engineered algae revealed that glucose was successfully transported to the cytoplasm via the glucose transporters, but was unable to enter the chloroplasts, which could explain the slow heterotrophic growth of the engineered algae.

Treatment resistance analysis reveals GLUT-1-mediated glucose uptake as a major target of synthetic rocaglates in cancer cells.

Rocaglates are natural compounds that have been extensively studied for their ability to inhibit translation initiation. Rocaglates represent promising drug candidates for tumor treatment due to their growth‐inhibitory effects on neoplastic cells. In contrast to natural rocaglates, synthetic analogues of rocaglates have been less comprehensively characterized, but were also shown to have similar effects on the process of protein translation. Here, we demonstrate an enhanced growth‐inhibitory effect of synthetic rocaglates when combined with glucose anti‐metabolite 2‐deoxy‐D‐glucose (2DG) in different cancer cell lines. Moreover, we unravel a new aspect in the mechanism of action of synthetic rocaglates involving reduction of glucose uptake mediated by downregulation or abrogation of glucose transporter GLUT‐1 expression. Importantly, cells with genetically induced resistance to synthetic rocaglates showed substantially less pronounced treatment effect on glucose metabolism and did not demonstrate GLUT‐1 downregulation, pointing at the crucial role of this mechanism for the anti‐tumor activity of the synthetic rocaglates. Transcriptome profiling revealed glycolysis as one of the major pathways differentially regulated in sensitive and resistant cells. Analysis of synthetic rocaglate efficacy in a 3D tissue context with a co‐culture of tumor and normal cells demonstrated a selective effect on tumor cells and substantiated the mechanistic observations obtained in cancer cell lines. Increased glucose uptake and metabolism is a universal feature across different tumor types. Therefore, targeting this feature by synthetic rocaglates could represent a promising direction for exploitation of rocaglates in novel anti‐tumor therapies.

LncRNA GAS6-AS1 inhibits progression and glucose metabolism reprogramming in LUAD via repressing E2F1-mediated transcription of GLUT1

Glucose metabolism reprogramming is one of the hallmarks of cancer cells, although functional and regulatory mechanisms of long noncoding RNA (lncRNA) in the contribution of glucose metabolism in lung adenocarcinoma (LUAD) remain incompletely understood. The aim of this study was to uncover the role of GAS6-AS1 in the regulation of progression and glucose metabolism in LUAD. We discovered that overexpression of GAS6-AS1 suppressed tumor progression of LUAD both in vitro and in vivo. Metabolism-related assays revealed that GAS6-AS1 inhibited glucose metabolism reprogramming. Mechanically, GAS6-AS1 was found to repress the expression of glucose transporter GLUT1, a key regulator of glucose metabolism. Ectopic expression of GLUT1 restored the inhibition effect of GAS6-AS1 on cancer progression and glucose metabolism reprogramming. Further investigation identified that GAS6-AS1 directly interacted with transcription factor E2F1 and suppressed E2F1-mediated transcription of GLUT1, and GAS6-AS1 was downregulated in LUAD tissues and correlated with clinicopathological characteristics and survival of patients. Taken together, our results identified GAS6-AS1 as a novel tumor suppressor in LUAD and unraveled its underlying molecular mechanism in reprogramming glucose metabolism. GAS6-AS1 potentially may serve as a prognostic marker and therapeutic target in LUAD.

Inhibition of eIF5A hypusination reprogrammes metabolism and glucose handling in mouse kidney

Inhibition of the eukaryotic initiation factor 5A activation by the spermidine analogue GC7 has been shown to protect proximal cells and whole kidneys against an acute episode of ischaemia. The highlighted mechanism involves a metabolic switch from oxidative phosphorylation toward glycolysis allowing cells to be transiently independent of oxygen supply. Here we show that GC7 decreases protein expression of the renal GLUT1 glucose transporter leading to a decrease in transcellular glucose flux. At the same time, GC7 modifies the native energy source of the proximal cells from glutamine toward glucose use. Thus, GC7 acutely and reversibly reprogrammes function and metabolism of kidney cells to make glucose its single substrate, and thus allowing cells to be oxygen independent through anaerobic glycolysis. The physiological consequences are an increase in the renal excretion of glucose and lactate reflecting a decrease in glucose reabsorption and an increased glycolysis. Such a reversible reprogramming of glucose handling and oxygen dependence of kidney cells by GC7 represents a pharmacological opportunity in ischaemic as well as hyperglycaemia-associated pathologies from renal origin.

LncRNA HOTAIR regulates glucose transporter Glut1 expression and glucose uptake in macrophages during inflammation

Inflammation plays central roles in the immune response. Inflammatory response normally requires higher energy and therefore is associated with glucose metabolism. Our recent study demonstrates that lncRNA HOTAIR plays key roles in NF-kB activation, cytokine expression, and inflammation. Here, we investigated if HOTAIR plays any role in the regulation of glucose metabolism in immune cells during inflammation. Our results demonstrate that LPS-induced inflammation induces the expression of glucose transporter isoform 1 (Glut1) which controls the glucose uptake in macrophages. LPS-induced Glut1 expression is regulated via NF-kB activation. Importantly, siRNA-mediated knockdown of HOTAIR suppressed the LPS-induced expression of Glut1 suggesting key roles of HOTAIR in LPS-induced Glut1 expression in macrophage. HOTAIR induces NF-kB activation, which in turn increases Glut1 expression in response to LPS. We also found that HOTAIR regulates glucose uptake in macrophages during LPS-induced inflammation and its knockdown decreases LPS-induced increased glucose uptake. HOTAIR also regulates other upstream regulators of glucose metabolism such as PTEN and HIF1α, suggesting its multimodal functions in glucose metabolism. Overall, our study demonstrated that lncRNA HOTAIR plays key roles in LPS-induced Glut1 expression and glucose uptake by activating NF-kB and hence HOTAIR regulates metabolic programming in immune cells potentially to meet the energy needs during the immune response.

The Effect of Inorganic and Organic Zinc Supplementation on Growth Performance, Mineral Profile and Gene Expression Pattern of GLUT1 in Malabari Kids.

The objective of this experiment was to study and compare the effects of dietary supplementation of organic and inorganic zinc (Zn) on growth performance, nutrient utilisation and gene expression pattern of glucose transporter protein in peripheral blood mononuclear cells (PBMC) in Malabari kids. Fifteen, 3-4-month-old goat kids were divided into three groups uniformly by using completely randomised design (CRD). Group G1 was fed on basal diet as per NRC requirement, and G2 and G3 were fed on basal diet + 40ppm Zn as inorganic zinc sulphate (ZnSO4) and 40ppm Zn as organic Zn methionine, respectively, for a period of 91days. Supplementation of inorganic and organic Zn had no significant effect on dry matter (DM) intake. The digestibility of crude protein (CP), ether extract (EE), neutral detergent fibre (NDF), hemicellulose and cellulose was significantly more in the organic Zn-supplemented group. The average daily gain and feed:gain ratio were significantly (p < 0.05) better in group G3 in comparison to G1 and G2, while the nitrogen retention was found to be (p < 0.01) higher in group G3 than in group G1. Zinc balance was found to be significantly (p < 0.01) increased in both supplemented groups with respect to unsupplemented group G1. The blood glucose level was (p < 0.01) lower in group G3 compared to group G1 suggesting the insulin-like activity of Zn. Serum Zn concentration was significantly (p < 0.01) increased in both Zn-supplemented groups. There was a significant (p < 0.05) rise in glucose transporter GLUT1 expression in groups G2 and G3 when compared to control group G1. Moreover, GLUT1 expression was found to be higher (p < 0.05) in group G3 as against the animals of group G2. Lowered blood glucose level might have stimulated more glucose transporter GLUT1 expression in PBMC. Organic Zn supplemented at 40ppm level resulted in better growth performance, nutrient digestibility and nitrogen as well as Zn retention in goat kids. There was better absorption, and hence, less amount of Zn got excreted in the organic Zn-supplemented group.

N-Methyl-D-Aspartate Receptor Antibodies in Autoimmune Encephalopathy Alter Oligodendrocyte Function.

Antibodies against neuronal N-methyl-D-aspartate receptors (NMDARs) in patients with anti-NMDAR encephalitis alter neuronal synaptic function and plasticity, but the effects on other cells of the nervous system are unknown. Cerebrospinal fluid (CSF) of patients with anti-NMDAR encephalitis (preabsorbed or not with GluN1) and a human NMDAR-specific monoclonal antibody (SSM5) derived from plasma cells of a patient, along the corresponding controls, were used in the studies. To evaluate the activity of oligodendrocyte NMDARs and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in vitro after exposure to patients' CSF antibodies or SSM5, we used a functional assay based on cytosolic Ca2+ imaging. Expression of the glucose transporter (GLUT1) in oligodendrocytes was assessed by immunocytochemistry. NMDAR agonist responses were robustly reduced after preincubation of oligodendrocytes with patients' CSF or SSM5 but remained largely unaltered with the corresponding controls. These effects were NMDAR specific, as patients' CSF did not alter responses to AMPA receptor agonists and was abrogated by preabsorption of CSF with HEK cells expressing GluN1 subunit. Patients' CSF also reduced oligodendrocyte expression of glucose transporter GLUT1 induced by NMDAR activity. Antibodies from patients with anti-NMDAR encephalitis specifically alter the function of NMDARs in oligodendrocytes, causing a decrease of expression of GLUT1. Considering that normal GLUT1 expression in oligodendrocytes and myelin is needed to metabolically support axonal function, the findings suggest a link between antibody-mediated dysfunction of NMDARs in oligodendrocytes and the white matter alterations reported in patients with this disorder. ANN NEUROL 2020;87:670-676.

Vasoactive Intestinal Peptide induces glucose and neutral amino acid uptake through mTOR signalling in human cytotrophoblast cells

The transport of nutrients across the placenta involves trophoblast cell specific transporters modulated through the mammalian target of rapamycin (mTOR). The vasoactive intestinal peptide (VIP) has embryotrophic effects in mice and regulates human cytotrophoblast cell migration and invasion. Here we explored the effect of VIP on glucose and System A amino acid uptake by human trophoblast-derived cells (Swan 71 and BeWo cell lines). VIP activated D-glucose specific uptake in single cytotrophoblast cells in a concentration-dependent manner through PKA, MAPK, PI3K and mTOR signalling pathways. Glucose uptake was reduced in VIP-knocked down cytotrophoblast cells. Also, VIP stimulated System A amino acid uptake and the expression of GLUT1 glucose transporter and SNAT1 neutral amino acid transporter. VIP increased mTOR expression and mTOR/S6 phosphorylation whereas VIP silencing reduced mTOR mRNA and protein expression. Inhibition of mTOR signalling with rapamycin reduced the expression of endogenous VIP and of VIP-induced S6 phosphorylation. Our findings support a role of VIP in the transport of glucose and neutral amino acids in cytotrophoblast cells through mTOR-regulated pathways and they are instrumental for understanding the physiological regulation of nutrient sensing by endogenous VIP at the maternal-foetal interface.

Heat shock induces the cellular antioxidant defenses peroxiredoxin, glutathione and glucose 6-phosphate dehydrogenase through Nrf2

Hyperthermia is a promising anticancer treatment used in combination with radiotherapy and/or chemotherapy. Heat (42–45 °C) can kill cancer cells. Low doses of heat at milder temperatures (39–41 °C) induce thermotolerance, an adaptive survival response that upregulates defense molecules to protect cells against subsequent exposure to toxic stress. Although hyperthermia has proven effective in clinical trials, there is still much to learn about its cellular mechanisms. This study aims to understand the role of reactive oxygen species (ROS), antioxidants and the antioxidant transcription factor Nrf2 in cellular stress responses to mild and lethal heat shock. Mild thermotolerance (40 °C) and hyperthermia (42–43 °C) caused increased expression of the antioxidants peroxiredoxin-3 (Prx3) and Prx2, and its hyperoxidized form Prx-SO3. Cellular levels of superoxide and peroxides increased at 40 °C and 42 °C. Heat shock (42 °C)-induced increases in Prx3 and Prx-SO3 were inhibited by antioxidants (PEG-catalase, MnTBAP) and a Nrf2 shRNA. Glucose metabolism by the pentose phosphate pathway produces NADPH, which maintains the antioxidant glutathione in its reduced form, GSH. Heat shock (40°C–42 °C) increased GSH levels, expression of glucose transporter GLUT1, and enzymatic activity and expression of glucose 6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose cycle. Heat-induced increases in GSH levels and G6PD expression were inhibited by antioxidants and Nrf2 knockdown. These results suggest that heat shock-generated ROS were involved in induction of cellular defense molecules Prxs, GSH and G6PD through Nrf2 activation. Our study sheds new light on the role of Nrf2 and antioxidants in cellular responses to heat shock at mild and lethal temperatures.

Human placental GLUT1 glucose transporter expression and the fetal insulin-like growth factor axis in pregnancies complicated by diabetes

We have previously described regulation of syncytial GLUT1 glucose transporters by IGF-I. Despite this, it is not clear what signal regulates transplacental glucose transport. In this report we asked whether changes in GLUT1 expression and glucose transport activity in diabetic pregnancies were associated with alterations in the fetal IGF axis. Cord blood samples and paired syncytial microvillous and basal membranes were isolated from normal term pregnancies and pregnancies characterized by gestational diabetes type A2 (GDM A2) and pre-existing insulin-dependent diabetes mellitus (IDDM). Circulating IGF-I, basal membrane GLUT1 expression and glucose transporter activity were correlated with birth weight, but only in control, not diabetic groups. Basal membrane GLUT1 and transporter activity were correlated with IGF-I concentrations in control, but not diabetic groups. IGF binding protein (IGFBP) binding capacity showed a ≥50% reduction in the diabetic groups compared to control; both showed a higher level of free IGF-I. The absence of a correlation between birth weight and factors such as fetal IGF-I or GLUT1 expression in the diabetic groups suggests that IGF-I-stimulated effects may have reached a limiting threshold, such that further increases in IGF-I (or GLUT1) are without effect. These data support that fetal IGF-I acts as a fetal nutritional signal, modulating placental GLUT1 expression and birth weight via altered levels of fetal circulating IGFBPs. Diabetes appears to exert its effects on fetal and placental factors prior to the third trimester and, despite good glycemic control immediately prior to, and in the third trimester, these effects persist to term.

CD28 Induces Metabolic Fitness in Multiple Myeloma for ROS-Dependent Survival

Multiple myeloma (MM) is a hematological malignancy of bone marrow resident plasma cells. Although patient outcomes have improved with new classes of proteasome inhibitors, immunomodulatory drugs, and targeted monoclonal antibodies, the disease remains largely incurable. Understanding the mechanisms by which resistant myeloma cells are able to survive is critical for the development of therapeutic strategies to prevent patient relapse.We have published that expression of CD28, the canonical T cell co-stimulatory molecule, increases during myeloma disease progression, is associated with poor prognosis, and provides a pro-survival signal to myeloma cells in vitro and in vivo; however, the mechanistic basis for this is unclear. During T cell activation, CD28 induces glycolysis at the expense of mitochondrial respiration. In four myeloma cell lines tested (MM1S, U266, 8226 and KMS11,) CD28 is able to increase the expression of glucose transporter glut1. Upregulation of glut1 is PI3k dependent, as treatment with the PI3k inhibitor LY294002 prevents CD28-mediated upregulation of glut1. Interestingly, CD28 has minimal effect on the glycolytic rate, but does increase the glycolytic capacity and reserve, suggesting that CD28 enhances metabolic fitness in myeloma cells when nutrient availability is low.Recently published data demonstrate a critical role for the metabolic phenotype in driving myeloma resistance to treatment, and that disruption of mitochondrial biogenesis promotes myeloma disease progression. CD28 activation induces mitochondrial biogenesis in multiple myeloma, increasing both the basal oxygen consumption as well as the maximal respiratory capacity. Mechanistically, CD28 activation increases protein levels of Irf4 and its direct target Myc in myeloma cells, which has been demonstrated in the literature to regulate mitochondrial biogenesis.Both glycolysis-derived pyruvate and lipid based fatty acids (FA) can be used in mitochondrial respiration. To our great surprise, CD28-mediated mitochondrial respiration is dependent upon fatty acid uptake through the mitochondrial membrane resident FA transporter CPT1a rather than glucose-derived pyruvate. In the bone marrow microenvironment, myeloma cells may not be able to depend upon the extracellular availability of glucose or fatty acids for use in energy production. Interestingly, CD28 activation increases autophagy in myeloma cells, a process of cellular degradation that has the capacity to provide lipid precursors for use in fatty acid oxidation. Inhibition of autophagy depresses myeloma mitochondrial respiration, suggesting a model wherein CD28-mediated upregulation of Irf4-Myc drives mitochondrial biogenesis, and by inducing autophagy, CD28 provides a supply of fatty acid fuel for increased mitochondrial respiration.A major byproduct of mitochondrial respiration is the production of reactive oxygen species (ROS), known to regulate both cell signaling as well as DNA damage. Inhibition of ROS prevents CD28-mediated survival in multiple myeloma. ROS augment PI3k signaling in myeloma, as inhibition of ROS leads to decreased phosphorylation of Akt, critical for CD28-mediated survival signaling. Furthermore, ROS inhibition prevents CD28-mediated increases in mitochondrial biogenesis, mitochondrial respiration, and the maximal respiratory capacity. In this manner, byproducts of CD28-mediated metabolic fitness reinforce signaling for myeloma cell survival.Treatment with a sublethal dose of Bortezomib decreased glycolytic and mitochondrial metabolism in myeloma. This leads to an attractive clinical application of our findings, wherein blockade of CD28-mediated metabolic fitness with Abatacept, an FDA approved drug for rheumatoid arthritis, may augment the efficacy of Bortezomib and other therapeutic strategies that exploit the metabolic demands of multiple myeloma throughout disease progression and relapse. DisclosuresNo relevant conflicts of interest to declare.