Glucose Transporter GLUT4 Research Articles

Impact of pathogenic mutations of the GLUT1 glucose transporter on solute carrier dynamics using ComDYN enhanced sampling

Background: The solute carrier (SLC) family of membrane proteins is a large class of transporters for many small molecules that are vital for cellular function. Several pathogenic mutations are reported in the glucose transporter subfamily SLC2, causing Glut1-deficiency syndrome (GLUT1DS1, GLUT1DS2), epilepsy (EIG2) and cryohydrocytosis with neurological defects (Dystonia-9). Understanding the link between these mutations and transporter dynamics is crucial to elucidate their role in the dysfunction of the underlying transport mechanism, which we investigate using molecular dynamics simulations. Methods: We studied pathogenic and non-pathogenic mutations, using a newly developed coarse-grained simulation approach ‘ComDYN’, which captures the ‘COMmon constraints DYNamics’ between both states of the solute carrier protein. To guarantee the sampling of large conformational changes, we only include common constraints of the elastic network introduced upon coarse-graining, which showed similar reference distances between both conformational states (≤1 Å difference). Results: ComDYN is computationally efficient and sufficiently sensitive to capture effects of different mutations. Our results clearly indicate that the pathogenic mutation in GLUT1, G91D, situated at the highly conserved RXGRR motif between helices 2 and 3, has a strong impact on transporter function, as it blocks the protein from sampling both conformational states. In comparison, predictions from SIFT and PolyPhen only provided an impression of the impact upon mutation in the highly conserved RXGRR motifs, but yielded no clear differentiation between pathogenic and non-pathogenic mutations. Conclusions: Using our approach, we can explain the pathogenicity of the mutation G91D and some of the effects of other known pathogenic mutations, when we observe the configurations of the transmembrane helices, suggesting that their relative position is crucial for the correct functioning of the GLUT1 protein. To fully understand the impact of other mutations in the future, it is necessary to consider the effect of ligands, e.g., glucose, within the transport mechanism.

Taurine Ameliorates Streptozotocin-Induced Diabetes by Modulating Hepatic Glucose Metabolism and Oxidative Stress in Mice.

Taurine is a sulfated amino acid derivative that plays an important role in maintaining the cell function of the living body. Although taurine has been shown to ameliorate diabetes, its mechanism of action has not yet been fully elucidated. The present study investigated the effects of taurine on diabetes focusing on glucose metabolism and oxidative stress. Type 1 diabetes was induced by the administration of streptozotocin (STZ) to male C57BL/6J mice. Taurine was dissolved in drinking water at 3% (w/v) and allowed to be freely ingested by diabetic mice. The weight and blood glucose levels were measured weekly. After nine weeks, mice were sacrificed and their serum, liver, and kidney were removed and used for biochemical and histological analyses. A microarray analysis was also performed in normal mice. Taurine alleviated STZ-induced hyperglycemia and hyperketonemia, accompanied by the suppression of the decrease in hepatic glycogen and upregulation of the mRNA expression of hepatic glucose transporter GLUT-2. Furthermore, STZ-induced elevation of oxidative stress in the liver and kidney was suppressed by taurine treatment. These results showed that taurine ameliorated diabetes and diabetic complications by improving hepatic glucose metabolism and reducing oxidative stress.

MODL-23. The identification and validation of plasma membrane proteins as potential therapeutic targets for histone H3 mutant paediatric high-grade gliomas.

Abstract Paediatric high grade gliomas (pHGG) are the leading cause of cancer-related death in children. Histone H3.3 mutations are an important hallmark of pHGG, but their underlying oncogenic mechanisms are incompletely understood. Towards identifying molecular effectors that may be therapeutically effective, we employed proteomic profiling and determined plasma membrane proteins of the surfactome that are differentially expressed on the surface of H3.3 mutant (G34R, G34V) compared to H3.3-wildtype human glioma cells in culture. Profiling of plasma membrane-enriched fractions identified 34 membrane proteins exclusively detected from H3 G34V mutant cells compared to H3 wild-type cells, and 10 membrane proteins exclusively detected from H3 G34R mutant cells, with 39 membrane proteins shared between both H3 G34R/V genotypes. One of these shared membrane proteins, GLUT1 (glucose transporter), has very recently been reported to be over-expressed and linked to tumour phenotypes in mouse glioma cells manipulated to carry the most common DIPG-associated H3.3-K27M mutation [Miya et al. Neuro-Oncology Advances 2021]. Here, we explore the functional significance of GLUT1 differential expression in H3.3 mutant (G34R, G34V) human glioma cells. Collective observations support a model where different H3.3 mutations converge on changes in GLUT1 expression and reinforce GLUT1 as a potential therapeutic target in paediatric high-grade gliomas.

1285-P: Differentiating between Diabetic and Idiopathic Gastroparesis: A Genetic Perspective

Gastroparesis is a serious medical condition characterized by delayed gastric emptying and symptoms of nausea, vomiting, bloating, fullness after meals, and abdominal pain. An innate immune dysregulation and injury to the interstitial cells of Cajal and other components of the enteric nervous system is likely central to the pathogenesis of gastroparesis. Most common etiologies include diabetes, post-surgical but most cases are idiopathic. Thus far, little is known about the underlying genetic risk factors for gastroparesis. We have done a genome-wide association study comparing idiopathic and diabetic cases with controls as well as contrasting idiopathic versus diabetic cases. We report a novel genetic association of SLC15A4 locus with idiopathic gastroparesis. This signal is driven by multiple variants with top missense variant SLC15A4:NM145648:exon2:c.T716C:p.V239A, rs33990080. Altogether, among EUR cohort we report 69 carriers out of 214 versus controls 165 out of 896 (MAF: cases 0.18; MAF: controls 0.09) and OR: 1.9 (p-value<10^-6) . We furthermore differentiate idiopathic from diabetic gastroparesis with an established diabetes genetic risk score, - result that is statistically significant (p-value<0.0004) . We also delineate a number of significant loci implicated in diabetes risk including (HLA-DQB1,HLA-DQA2, EXOC6B) in the idiopathic versus diabetic gastroparesis analysis. Of particular interest are the Exocyst complex component, 6 (EXOC6/6B) with lead variant rs41420. previously identified to play a role in insulin signaling through its interaction with the glucose transporter GLUT4. Current genetic findings suggest that a mechanism directly connected to macrophage polarization may be implicated in the etiology gastroparesis. The GWAS picture that is emerging implicates novels loci implicated in the pathophysiology of gastroparesis differentiating those of diabetic versus idiopathic etiology. Disclosure S.P.Smieszek: None.

1320-P: Insulin Receptors and Their Signal Transduction Pathways in Choroid Plexus

The insulin receptor (IR) is a tyrosine kinase receptor present in cell membranes. It is highly expressed in hepatocytes (where it promotes glycogenesis and inhibits gluconeogenesis) , and skeletal muscle and fat (where it facilitates glucose uptake by translocation of the glucose transporter GLUT4) . IR expression is also found throughout the brain, both in neurons and all glial cell types, and for decades impaired insulin signaling has been implicated in the onset and development of Alzheimer’s disease. IR expression pattern in the brain parenchyma is heterogeneous, with highest levels of expression found in olfactory bulb, hypothalamus, and hippocampus. However, the choroid plexus (CP) , a structure present in all 4 ventricles of the brain and responsible for the secretion of cerebrospinal fluid, has the highest affinity for insulin binding compared to any other brain region. This work aims to characterize the insulin signaling in the epithelial cells of the choroid plexus (EChP) and its possible implications in diseases of the brain. Relative expression levels of specific splicing variants of the Insr gene in dissected CP of adult mice were evaluated by qPCR and revealed predominant expression of the variant coding for isoform IR-A when compared to isoform IR-B. In vitro stimulation of EChP with insulin elicits a classical tyrosine kinase cascade, with activation of both the mitogenic (ERK) and metabolic (Akt) arms of the insulin signaling pathway, in a time-and concentration-dependent manner. Using pharmacological (S961, an insulin receptor antagonist) and genetic (insulin receptor shRNA, and Insr cre-loxP) approaches, we show that downregulation of IR in these cells not only impairs insulin signaling, but it also decreases IGF2’s ability to activate ERK and Akt, even when IGF1R levels are unaltered. Finally, using a viral vector coding for an IR shRNA, we show that downregulation of Insr expression in CP of adult mice has a profound effect on its structure, as demonstrated by MRI and histological technique. Disclosure C.Mazucanti: None. V.L.Kennedy: None. A.Cho: None. Q.Liu: None. J.F.O’connell: None. S.Camandola: None. J.M.Egan: None.

313-OR: A Cytoskeleton-Based Mechanism Regulates Lipid Metabolism and Energy Homeostasis in Brown Adipose Tissue

Genetic variants in ANK2, encoding ankyrin-B (AnkB) , increase risk of obesity and type 2 diabetes in humans. We previously concluded that AnkB deficiency in white (WAT) and brown (BAT) adipose tissue (AT) of mice (AT-AnkB KO) led to lipotoxicity, WAT hypertrophy, and insulin resistance associated with age and consumption of a high-fat diet. These metabolic deficits were caused by impairments in glucose transporter GLUT4 endocytosis in WAT due to AnkB deficiency. Additionally, we found that AT-AnkB KO mice had decreased energy expenditure and lipid accumulation in BAT prior to the onset of obesity suggesting a potential role of AnkB in BAT. However, the role of AnkB in BAT and its contribution to metabolic homeostasis through BAT has not been elucidated. We investigated the role of AnkB in mice conditionally lacking AnkB in BAT (BAT-AnkB KO) . Based on histological and transmission electron microscopy analysis, we found that young (4-month-old) BAT-AnkB KO mice have enlarged lipid droplets, lipid accumulation, and decreased mitochondrial content in BAT with no changes in BAT size, body weight, or body composition. Consistent with these findings, primary brown adipocytes from BAT-AnkB KO mice exhibited lipid accumulation suggesting that AnkB may regulate lipid homeostasis in BAT in a cell-autonomous manner. Similar to our previous findings, young BAT-AnkB KO mice also had decreases in oxygen consumption and energy expenditure. These mice also exhibit minimal changes in glucose handling and insulin tolerance at 4-months of age. Proteomic-MS analysis was performed on AnkB associated proteins from BAT which revealed that GLUT4 as well as a number of critical regulators of lipid metabolism form complexes with AnkB in BAT. In combination with this data, we will present our latest findings in gene expression of genes regulating lipid metabolism and mitochondrial function as well as cellular assays in brown adipocytes that may highlight metabolic pathways regulated by AnkB in BAT. Disclosure A.Aguillard: None. J.Arricastres: None. J.Tzeng: None. D.Lorenzo: None. Funding American Diabetes Association (1-19-JDF-081)

203-LB: The Role of the Exocyst Trafficking Complex in CD36 Fatty Acid Translocase Trafficking in Skeletal Muscle Cells

Skeletal muscle free fatty acid (FFA) uptake correlates with the amount of fatty acid translocase CD36 on the cell surface. In obesity, chronic membrane-localization of CD36, but not other FFA transporters, augments FFA uptake and intracellular lipid accumulation. This ectopic lipid accumulation then causes insulin resistance (IR) by inhibiting GLUT4 glucose transporter trafficking and glucose uptake in response to insulin. GLUT4 and CD36 cell-surface delivery is stimulated by insulin and contraction signaling, sharing conserved downstream effectors. The eight-protein exocyst complex is responsible for the targeted docking of membrane-bound vesicles, a process underlying cell-surface delivery of fuel transporters. The exocyst indeed regulates insulin-induced glucose uptake via GLUT4 trafficking to the membrane in adipocytes and skeletal muscle. In addition, it controls lipid uptake in adipocytes, but we do not know which of the FFA-transporting proteins the exocyst targets during this process. Based on the high degree of conservation of the fuel transporter trafficking mechanisms in adipose and skeletal muscle tissue, we hypothesized that the exocyst contributes to lipid uptake in skeletal muscle via the targeted trafficking of CD36. Our data shows that the exocyst plays a role in FFA uptake in skeletal muscle cells. Using an L6 GLUT4myc myoblast model we demonstrate that the exocyst is recruited to CD36 vesicles upon insulin and contraction signaling. We also show that disrupted exocyst activity leads to an impaired CD36 surface delivery and decreased FFA uptake in skeletal myoblasts. We are the first to demonstrate that the exocyst is necessary for insulin- and contraction-induced CD36 membrane trafficking and FFA metabolism in muscle cells. Further studies will continue to investigate the mechanism of exocyst-regulated FFA uptake and lipid metabolism in skeletal muscle to help our understanding of ectopic lipid accumulation leading to IR. Disclosure N. K. Nakamura: None. D. S. Tokunaga: None. H. Y. J. Ha: None. N. Polgar: None.

Quantitative characterization of the path of glucose diffusion facilitated by human glucose transporter 1

Glucose transporter GLUT1 is ubiquitously expressed in the human body from the red cells to the blood-brain barrier to the skeletal muscles. It is physiologically relevant to understand how GLUT1 facilitates diffusion of glucose across the cell membrane. It is also pathologically relevant because GLUT1 deficiency causes neurological disorders and anemia and because GLUT1 overexpression fuels the abnormal growth of cancer cells. This article presents a quantitative investigation of GLUT1 based on all-atom molecular-dynamics (MD) simulations of the transporter embedded in lipid bilayers of asymmetric inner-and-outer-leaflet lipid compositions, subject to asymmetric intra-and-extra-cellular environments. This is in contrast with the current literature of MD studies that have not considered both of the aforementioned asymmetries of the cell membrane. The equilibrium (unbiased) dynamics of GLUT1 shows that it can facilitate glucose diffusion across the cell membrane without undergoing large-scale conformational motions. The Gibbs free-energy profile, which is still lacking in the current literature of GLUT1, quantitatively characterizes the diffusion path of glucose from the periplasm, through an extracellular gate of GLUT1, on to the binding site, and off to the cytoplasm. This transport mechanism is validated by the experimental data that GLUT1 has low water-permeability, uptake-efflux symmetry, and 10 kcal/mol Arrhenius activation barrier around 37 °C.

Knockout of TIGAR enhances myocardial phosphofructokinase activity and preserves diastolic function in heart failure.

Hypertension is an important risk factor in the pathogenesis of diastolic dysfunction. Growing evidence indicates that glucose metabolism plays an essential role in diastolic dysfunction. TP53-induced glycolysis and apoptosis regulator (TIGAR) has been shown to regulate glucose metabolism and heart failure (HF). In the present study, we investigated the role of TIGAR in diastolic function and cardiac fibrosis during pressure overload (PO)-induced HF. WT mice subjected to transverse aortic constriction (TAC), a commonly used method to induce diastolic dysfunction, exhibited diastolic dysfunction as evidenced by increased E/A ratio and E/E' ratio when compared to its sham controls. This was accompanied by increased cardiac interstitial fibrosis. In contrast, the knockout of TIGAR attenuated PO-induced diastolic dysfunction and interstitial fibrosis. Mechanistically, the levels of glucose transporter Glut-1, Glut-4, and key glycolytic enzyme phosphofructokinase 1 (PFK-1) were significantly elevated in TIGAR KO subjected to TAC as compared to that of WT mice. Knockout of TIGAR significantly increased fructose 2,6-bisphosphate levels and phosphofructokinase activity in mouse hearts. In addition, PO resulted in a significant increase in perivascular fibrosis and endothelial activation in the WT mice, but not in the TIGAR KO mice. Our present study suggests a necessary role of TIGAR-mediated glucose metabolism in PO-induced cardiac fibrosis and diastolic dysfunction.

Impaired glucose metabolism underlies articular cartilage degeneration in osteoarthritis.

Osteoarthritis (OA) is the leading joint disease characterized by cartilage destruction and loss of mobility. Accumulating evidence indicates that the incidence and severity of OA increases with diabetes, implicating systemic glucose metabolism in joint health. However, a definitive link between cellular metabolism in articular cartilage and OA pathogenesis is not yet established. Here, we report that in mice surgically induced to develop knee OA through destabilization of medial meniscus (DMM), expression of the main glucose transporter Glut1 is notably reduced in joint cartilage. Inducible deletion of Glut1 specifically in the Prg4-expressing articular cartilage accelerates cartilage loss in DMM-induced OA. Conversely, forced expression of Glut1 protects against cartilage destruction following DMM. Moreover, in mice with type I diabetes, both Glut1 expression and the rate of glycolysis are diminished in the articular cartilage, and the diabetic mice exhibit more severe cartilage destruction than their nondiabetic counterparts following DMM. The results provide proof of concept that boosting glucose metabolism in articular chondrocytes may ameliorate cartilage degeneration in OA.

Cryo-EM structure of human glucose transporter GLUT4

GLUT4 is the primary glucose transporter in adipose and skeletal muscle tissues. Its cellular trafficking is regulated by insulin signaling. Failed or reduced plasma membrane localization of GLUT4 is associated with diabetes. Here, we report the cryo-EM structures of human GLUT4 bound to a small molecule inhibitor cytochalasin B (CCB) at resolutions of 3.3 Å in both detergent micelles and lipid nanodiscs. CCB-bound GLUT4 exhibits an inward-open conformation. Despite the nearly identical conformation of the transmembrane domain to GLUT1, the cryo-EM structure reveals an extracellular glycosylation site and an intracellular helix that is invisible in the crystal structure of GLUT1. The structural study presented here lays the foundation for further mechanistic investigation of the modulation of GLUT4 trafficking. Our methods for cryo-EM analysis of GLUT4 will also facilitate structural determination of many other small size solute carriers.

Alcohol-Induced Glycolytic Shift in Alveolar Macrophages Is Mediated by Hypoxia-Inducible Factor-1 Alpha.

Excessive alcohol use increases the risk of developing respiratory infections partially due to impaired alveolar macrophage (AM) phagocytic capacity. Previously, we showed that chronic ethanol (EtOH) exposure led to mitochondrial derangements and diminished oxidative phosphorylation in AM. Since oxidative phosphorylation is needed to meet the energy demands of phagocytosis, EtOH mediated decreases in oxidative phosphorylation likely contribute to impaired AM phagocytosis. Treatment with the peroxisome proliferator-activated receptor gamma (PPARγ) ligand, pioglitazone (PIO), improved EtOH-mediated decreases in oxidative phosphorylation. In other models, hypoxia-inducible factor-1 alpha (HIF-1α) has been shown to mediate the switch from oxidative phosphorylation to glycolysis; however, the role of HIF-1α in chronic EtOH mediated derangements in AM has not been explored. We hypothesize that AM undergo a metabolic shift from oxidative phosphorylation to a glycolytic phenotype in response to chronic EtOH exposure. Further, we speculate that HIF-1α is a critical mediator of this metabolic switch. To test these hypotheses, primary mouse AM (mAM) were isolated from a mouse model of chronic EtOH consumption and a mouse AM cell line (MH-S) were exposed to EtOH in vitro. Expression of HIF-1α, glucose transporters (Glut1 and 4), and components of the glycolytic pathway (Pfkfb3 and PKM2), were measured by qRT-PCR and western blot. Lactate levels (lactate assay), cell energy phenotype (extracellular flux analyzer), glycolysis stress tests (extracellular flux analyzer), and phagocytic function (fluorescent microscopy) were conducted. EtOH exposure increased expression of HIF-1α, Glut1, Glut4, Pfkfb3, and PKM2 and shifted AM to a glycolytic phenotype. Pharmacological stabilization of HIF-1α via cobalt chloride treatment in vitro mimicked EtOH-induced AM derangements (increased glycolysis and diminished phagocytic capacity). Further, PIO treatment diminished HIF-1α levels and reversed glycolytic shift following EtOH exposure. These studies support a critical role for HIF-1α in mediating the glycolytic shift in energy metabolism of AM during excessive alcohol use.

A Fatty Diet Induces a Jejunal Ketogenesis Which Inhibits Local SGLT1-Based Glucose Transport via an Acetylation Mechanism-Results from a Randomized Cross-Over Study between Iso-Caloric High-Fat versus High-Carbohydrate Diets in Healthy Volunteers.

Background and aims: Insights into the nature of gut adaptation after different diets enhance the understanding of how food modifications can be used to treat type 2 diabetes and obesity. The aim was to understand how diets, enriched in fat or carbohydrates, affect glucose absorption in the human healthy jejunum, and what mechanisms are involved. Methods: Fifteen healthy subjects received, in randomised order and a crossover study design, two weeks of iso-caloric high-fat diet (HFD) and high-carbohydrate diet (HCD). Following each dietary period, jejunal mucosa samples were retrieved and assessed for protein expression using immunofluorescence and western blotting. Functional characterisation of epithelial glucose transport was assessed ex vivo using Ussing chambers. Regulation of SGLT1 through histone acetylation was studied in vitro in Caco-2 and human jejunal enteroid monolayer cultures. Results: HFD, compared to HCD, decreased jejunal Ussing chamber epithelial glucose transport and the expression of apical transporters for glucose (SGLT1) and fructose (GLUT5), while expression of the basolateral glucose transporter GLUT2 was increased. HFD also increased protein expression of the ketogenesis rate-limiting enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2) and decreased the acetylation of histone 3 at lysine 9 (H3K9ac). Studies in Caco-2 and human jejunal enteroid monolayer cultures indicated a ketogenesis-induced activation of sirtuins, in turn decreasing SGLT1 expression. Conclusion: Jejunal glucose absorption is decreased by a fat-enriched diet, via a ketogenesis-induced alteration of histone acetylation responsible for the silencing of SGLT1 transcription. The work relates to a secondary outcome in ClinicalTrials.gov (NCT02088853).

Blood-brain barrier targeted delivery of lacosamide-conjugated gold nanoparticles: Improving outcomes in absence seizures

ObjectiveMost currently available antiepileptics are not fully effective in the prevention of seizures in absence epilepsy owing to the presence of blood-brain barrier (BBB). We aimed to test whether binding an antiepileptic drug, lacosamide (LCM), to glucose-coated gold nanoparticles (GNPs) enables efficient brain drug delivery to suppress the epileptic activity in WAG/Rij rats with absence epilepsy. MethodsIn these animals, intracranial-EEG recording, behavioral test, in vivo imaging of LCM and LCM-GNP conjugate distribution in the brain, inductively coupled plasma mass spectrometry analysis, immunofluorescence staining of glucose transporter (Glut)− 1, glial fibrillary acidic protein (GFAP), and p-glycoprotein (P-gp) and electron microscopy were performed. ResultsLacosamide-GNP conjugates decreased the amplitude and frequency of spike-wave-like discharges (SWDs) and alleviated the anxiety-like behavior as assessed by EEG and elevated plus-maze test, respectively (p < 0.01). The in vivo imaging system results showed higher levels of fluorescein dye tagged to LCM-GNP in the brain during the 5-day injection period (p < 0.01). Immunofluorescence staining displayed decreased P-gp, Glut-1, and GFAP expression by LCM-GNP conjugate treatment predominantly in the cerebral cortex suggesting a potential functionality of this brain region in the modulation of neuronal activity in our experimental setting (p < 0.01). SignificanceWe suggest that the conjugation of LCM to GNPs may provide a novel approach for efficient brain drug delivery in light of the effectiveness of our strategy not only in suppressing the seizure activity but also in decreasing the need to use high dosages of the antiepileptics to reduce the frequently encountered side effects in drug-resistant epilepsy.

Cardiac glycosides stimulate endocytosis of GLUT1 via intracellular Na+ ,K+ -ATPase α3-isoform in human cancer cells.

Glucose transporter GLUT1 plays a primary role in the glucose metabolism of cancer cells. Here, we found that cardiac glycosides (CGs) such as ouabain, oleandrin, and digoxin, which are Na+ ,K+ -ATPase inhibitors, decreased the GLUT1 expression in the plasma membrane of human cancer cells (liver cancer HepG2, colon cancer HT-29, gastric cancer MKN45, and oral cancer KB cells). The effective concentration of ouabain was lower than that for inhibiting the activity of Na+ ,K+ -ATPase α1-isoform (α1NaK) in the plasma membrane. The CGs also inhibited [3 H]2-deoxy- d-glucose uptake, lactate secretion, and proliferation of the cancer cells. In intracellular vesicles of human cancer cells, Na+ ,K+ -ATPase α3-isoform (α3NaK) is abnormally expressed. Here, a low concentration of ouabain inhibited the activity of α3NaK. Knockdown of α3NaK significantly inhibited the ouabain-decreased GLUT1 expression in HepG2 cells, while the α1NaK knockdown did not. Consistent with the results in human cancer cells, CGs had no effect on GLUT1 expression in rat liver cancer dRLh-84 cells where α3NaK was not endogenously expressed. Interestingly, CGs decreased GLUT expression in the dRLh-84 cells exogenously expressing α3NaK. In HepG2 cells, α3NaK was found to be colocalized with TPC1, a Ca2+ -releasing channel activated by nicotinic acid adenine dinucleotide phosphate (NAADP). The CGs-decreased GLUT1 expression was significantly inhibited by a Ca2+ chelator, a Ca2+ -ATPase inhibitor, and a NAADP antagonist. The GLUT1 decrease was also attenuated by inhibitors of dynamin and phosphatidylinositol-3 kinases (PI3Ks). In conclusion, the binding of CGs to intracellular α3NaK elicits the NAADP-mediated Ca2+ mobilization followed by the dynamin-dependent GLUT1 endocytosis in human cancer cells.

Subcellular Recycling of CD36 as Target to Rescue Lipid Overload‐induced Myocardial Contractile Dysfunction

BackgroundDisturbances in cardiac metabolism are increasingly being recognized as crucial drivers of the development and progression of heart disease and ageing. In particular, lipid overload due to Western life style or obesity eventually elicits cardiac insulin resistance and contractile dysfunction (referred to as diabetic cardiomyopathy). As a result, there is an urgent unmet clinical need for treatments that can directly target metabolic defects in heart failure. The transmembrane protein CD36 both facilitates myocardial fatty acid uptake and regulates this process by its subcellular recycling between endosomes and the sarcolemma. In turn, CD36 recycling was recently found to be regulated by instantaneous changes in activity of vacuolar‐type H+‐ATPase (v‐ATPase) in the endosomes, as controlled by rapid assembly and disassembly of V0 and V1 subunits of v‐ATPase.HypothesisRegulation of subcellular recycling of CD36 by v‐ATPase is an effective target for metabolic modulation therapy in diabetic cardiomyopathy.ObjectiveTo investigate whether manipulating v‐ATPase activity, and thereby CD36 recycling, rescues insulin resistance and contractile dysfunction of the diabetic heart.MethodsIsolated rat cardiomyocytes and human (iPSC‐derived and differentiated) cardiomyocytes were treated with bafilomycin (v‐ATPase inhibitor) or energy substrates, i.e., fatty acids, glucose, amino acids (AAs) or b‐hydroxy butyrate (HB). In addition, rats in vivo were treated with AAs. v‐ATPase activity (assembly status), substrate transporter localization (CD36 and glucose transporter GLUT4) and fatty acid and glucose uptake rates (in absence or presence of insulin) were measured.ResultsIn rat and human cardiomyocytes both bafilomycin, excess palmitate and HB inhibited v‐ATPase activity, increased sarcolemmal CD36 content, increased fatty acid uptake and lipid accumulation, and elicited contractile dysfunction. However, the latter could be rescued by excess glucose or AAs, concomitant with in each case a similar increase of v‐ATPase activity and CD36 reinternalization, while glucose uptake was increased. Hence, v‐ATPase activity/assembly is required for retention of CD36 in endosomes. In case of AAs, v‐ATPase (re)assembly was dependent on mTORC1 activation. In high fat diet‐fed rats, AAs had a similar beneficial action at the myocellular level.ConclusionsThese novel data suggest a pivotal role for endosomal v‐ATPase activity and subcellular CD36 recycling in determining myocardial substrate preference, in particular fatty acids versus glucose. The molecular mechanism involves assembly/disassembly of the V0 and V1 subunits of v‐ATPase, allowing a rapid change in activity. Lipid accumulation and contractile dysfunction in diabetic cardiomyopathy is aggravated by HB but resolved by AAs.PerspectiveSpecific amino acids acting through v‐ATPase reassembly may be an effective nutraceutical therapy in diabetic cardiomyopathy, but ketone bodies should not be used as these may aggravate lipid accumulation and contractile dysfunction.

Medium‐chain fatty acids inhibit jejunal SGLT1 activity in vitro and suppress glucose absorption in vivo in mice

Medium‐chain triglycerides (MCT) in diet improves glucose tolerance and insulin sensitivity, compared to long‐chain triglycerides (LCT). However, the direct effects of medium‐chain fatty acids (MCFA) on glucose transport have not been studied. Luminal glucose is absorbed in the small intestine through the electrogenic sodium‐dependent glucose transporter SGLT1 and electroneutral glucose transporter GLUT2. We thus examined the effects of luminal MCFA on electrogenic SGLT1 activity and glucose absorption in mice.SGLT1 activity was assessed in the Ussing chambered, muscle‐stripped jejunal mucosa by measuring phlorizin (Phz)‐sensitive short‐circuit current (Isc) evoked by luminal addition of glucose (10 mM). Sodium propionate (C3), caproate (C6), caprylate (C8), caprate (C10), laurate (C12) and myristate (C14) were luminally added before or after glucose application. Furthermore, the effects of C10 on glucose absorption were assessed by oral glucose tolerance test (OGTT, glucose 2g/kg body weight, ig) with or without C10 treatment (1.94g/kg, ig) in conscious mice. Moreover, the portal venous (PV) blood glucose levels were measured though the PV cannulation, followed by intraduodenal bolus injection of glucose solution (100 mM) with or without C10 (10 mM) in the anesthetized mice.Luminal pretreatment of C10 and C12 dose‐dependently inhibited the glucose‐induced Isc increase at IC50 = 1.02 mM and 1.06 mM, respectively. C8 also inhibited the glucose‐induced Isc increase, but less effective (IC50 8.4 mM), whereas C3, C6 and C14 had little effect. C10 (10 mM) also inhibited methyl α‐D‐glucopyranoside (αMG)‐induced Isc increase. Post‐treatment of C10 and C12 (10 mM) rapidly reduced the increased Isc by glucose, mimicking the inhibitory effect of Phz (1 mM). OGTT showed that glucose levels reached the peak at 30 min, while C10 pretreatment remarkably inhibited glucose increase. Furthermore, intraduodenal injection of glucose solution increased the PV glucose levels at 10 – 20 min, while the addition of C10 in the glucose solution abolished the response.These data suggest that C10 and C12 act as the SGLT1 inhibitor. Since MCT releases C10 and C12 via digestion by pancreatic lipase, slower than glucose release by maltase/sucrase, free C10/C12 solution may be more effective therapeutics for the treatment of type 2 diabetes.

RIP140 inhibits glycolysis-dependent proliferation of breast cancer cells by regulating GLUT3 expression through transcriptional crosstalk between hypoxia induced factor and p53

Glycolysis is essential to support cancer cell proliferation, even in the presence of oxygen. The transcriptional co-regulator RIP140 represses the activity of transcription factors that drive cell proliferation and metabolism and plays a role in mammary tumorigenesis. Here we use cell proliferation and metabolic assays to demonstrate that RIP140-deficiency causes a glycolysis-dependent increase in breast tumor growth. We further demonstrate that RIP140 reduces the transcription of the glucose transporter GLUT3 gene, by inhibiting the transcriptional activity of hypoxia inducible factor HIF-2α in cooperation with p53. Interestingly, RIP140 expression was significantly associated with good prognosis only for breast cancer patients with tumors expressing low GLUT3, low HIF-2α and high p53, thus confirming the mechanism of RIP140 anti-tumor activity provided by our experimental data. Overall, our work establishes RIP140 as a critical modulator of the p53/HIF cross-talk to inhibit breast cancer cell glycolysis and proliferation.

The clinical and biochemical hallmarks generally associated with GLUT1DS may be caused by defects in genes other than SLC2A1.

Glucose transporter 1 deficiency syndrome (GLUT1DS) is a neurometabolic disorder caused by haploinsufficiency of the GLUT1 glucose transporter (encoded by SLC2A1) leading to defective glucose transport across the blood–brain barrier. This work describes the genetic analysis of 56 patients with clinical or biochemical GLUT1DS hallmarks. 55.4% of these patients had a pathogenic variant of SLC2A1, and 23.2% had a variant in one of 13 different genes. No pathogenic variant was identified for the remaining patients. Expression analysis of SLC2A1 indicated a reduction in SLC2A1 mRNA in patients with pathogenic variants of this gene, as well as in one patient with a pathogenic variant in SLC9A6, and in three for whom no candidate variant was identified. Thus, the clinical and biochemical hallmarks generally associated with GLUT1DS may be caused by defects in genes other than SLC2A1.

Sustained endocrine and exocrine function in the pancreas of the Pacific spiny dogfish post-feeding.

Secretions of the exocrine pancreas contain digestive enzymes integral to the digestive process. The Pacific spiny dogfish (Squalus suckleyi) has a discrete pancreas, divided into two lobes termed the dorsal and ventral lobes. These lobes drain into the anterior intestine via a common duct to enable digestion. Previous studies have identified that the exocrine pancreas produces (co)lipases, chymotrypsin, carboxypeptidase, and low levels of chitinases; however, investigations into other digestive enzymes are limited. We detect the presence of lipase, trypsin, and carbohydrase and show that activities are equivalent between both lobes of the pancreas. Additionally, we sought to investigate the influence of a single feeding event (2% body weight ration of herring by gavage) on enzyme activities over an extended time course (0, 20, 48, 72, 168h) post-feeding. The results indicate that there are no differences in pancreatic tissue digestive enzyme activities between fed or fasted states. Analysis of acinar cell circumference post-feeding demonstrates a significant increase at 20 and 48h, that returns to fasting levels by 72h. No significant changes were observed regarding whole-tissue insulin or glucagon mRNA abundance or with glucose transporter (glut) 1 or 3. Yet, a significant and transient decrease in glut4 and sodium glucose-linked transporter mRNA abundance was found at 48h post-feeding. We propose that the constant enzyme activity across this relatively large organ, in combination with a relatively slow rate of digestion leads to an evenly distributed, sustained release of digestive enzymes regardless of digestive state.