Metabolites from Aerial Parts of Glycyrrhiza foetida as Modulators of Targets Related to Metabolic Syndrome.

A detailed phytochemical investigation has been carried out on the aerial parts of G. foetida leading to the isolation of 29 pure compounds, mainly belonging to the amorfrutin and polyphenol classes. Among them, the new amorfrutin N (5) and exiguaflavone L (21) were isolated and their structures elucidated by means of HR-ESIMS and NMR. All the isolated compounds were investigated for modulation of mitochondrial activity and stimulation of glucose uptake via GLUT transporters, two metabolic processes involved in intracellular glucose homeostasis, which, therefore, correlate with the incidence of metabolic syndrome. These experiments revealed that amorfrutins were active on both targets, with amorfrutin M (17) and decarboxyamorfrutin A (2) emerging as mitochondrial stimulators, and amorfrutin 2 (12) as a glucose uptake promoter. However, members of the rich chalcone/flavonoid fraction also proved to contribute to this activity.

Saturated fatty acids inhibit unsaturated fatty acid induced glucose uptake involving GLUT10 and aerobic glycolysis in bovine granulosa cells.

Fatty acids have been shown to modulate glucose metabolism in vitro and in vivo. However, there is still a need for substantial evidence and mechanistic understanding in many cell types whether both saturated and unsaturated fatty acids (SFAs and UFAs) pose a similar effect and, if not, what determines the net effect of fatty acid mixes on glucose metabolism. In the present study, we asked these questions by treating granulosa cells (GCs) with the most abundant non-esterified fatty acid species in bovine follicular fluid. Results revealed that oleic and alpha-linolenic acids (UFAs) significantly increased glucose consumption compared to palmitic and stearic acids (SFAs). A significant increase in lactate production, extracellular acidification rate, and decreased mitochondrial activity indicate glucose channeling through aerobic glycolysis in UFA treated GCs. We show that insulin independent glucose transporter GLUT10 is essential for UFA driven glucose consumption, and the induction of AKT and ERK signaling pathways necessary for GLUT10 expression. To mimic the physiological conditions, we co-treated GCs with mixes of SFAs and UFAs. Interestingly, co-treatments abolished the UFA induced glucose uptake and metabolism by inhibiting AKT and ERK phosphorylation and GLUT10 expression. These data suggest that the net effect of fatty acid induced glucose uptake in GCs is determined by SFAs under physiological conditions.

Opisthorchis viverrini excretory-secretory products suppress GLUT8 of cholangiocytes.

Opisthorchis viverrini infection and the subsequent bile duct cancer it induces remains a significant public health problem in Southeast Asia. Opisthorchiasis has been reported to cause reduced plasma glucose levels among infected patients. The underlying mechanism for this phenomenon is unclear. In the present study, evidence is presented to support the hypothesis that O. viverrini exploits host cholangiocyte glucose transporters (GLUTs) in a similar manner to that of rodent intestinal nematodes, to feed on unabsorbed glucose in the bile for survival. GLUT levels in a cholangiocyte H69 cell line co-cultured with excretory-secretory products of O. viverrini were examined using qPCR and immunoblotting. GLUT 8 mRNA and expressed proteins were found to be downregulated in H69 cells in the presence of O. viverrini. This suggests that O. viverrini alters glucose metabolism in cells within its vicinity by limiting transporter expression resulting in increased bile glucose that it can utilize and potentially explains the previously reported anti-insulin effect of opisthorchiasis.

The determinants of metabolic discrepancies in aerobic glycolysis: Providing potential targets for breast cancer treatment.

Altered aerobic glycolysis is the robust mechanism to support cancer cell survival and proliferation beyond the maintenance of cellular energy metabolism. Several investigators portrayed the important role of deregulated glycolysis in different cancers, including breast cancer. Breast cancer is the most ubiquitous form of cancer and the primary cause of cancer death in women worldwide. Breast cancer with increased glycolytic flux is hampered to eradicate with current therapies and can result in tumor recurrence. In spite of the low order efficiency of ATP production, cancer cells are highly addicted to glycolysis. The glycolytic dependency of cancer cells provides potential therapeutic strategies to preferentially kill cancer cells by inhibiting glycolysis using antiglycolytic agents. The present review emphasizes the most recent research on the implication of glycolytic enzymes, including glucose transporters (GLUTs), hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase-A (LDHA), associated signalling pathways and transcription factors, as well as the antiglycolytic agents that target key glycolytic enzymes in breast cancer. The potential activity of glycolytic inhibitors impinges cancer prevalence and cellular resistance to conventional drugs even under worse physiological conditions such as hypoxia. As a single agent or in combination with other chemotherapeutic drugs, it provides the feasibility of new therapeutic modalities against a wide spectrum of human cancers.

Revisiting the roles of glucose transporters in skeletal muscle physiology: is GLUT10 a novel player?

Skeletal muscle is the largest metabolic tissue responsible for systemic glucose handling. Glucose uptake into skeletal tissue is highly dynamic and delicately regulated, in part through the controlled expression and subcellular trafficking of multiple types of glucose transporters. Although the roles of GLUT4 in skeletal muscle metabolism are well established, the physiological significance of other, seemingly redundant, glucose transporters remain incompletely understood. Nonetheless, recent studies have shed light on the roles of several glucose transporters, such as GLUT1 and GLUT10, in skeletal muscle. Mice experiments suggest that GLUT10 could be a novel player in skeletal muscle metabolism in the context of mechanical overload, which is in line with the meta-analytical results of gene expression changes after resistance exercise in humans. Herein we discuss the knowns, unknowns, and implications of these recent findings.

Glucose transporters (GLUTs): Underreported yet crucial molecules in unraveling testicular toxicity.

Glucose transporters (GLUTs) are crucial in maintaining glucose homeostasis and supporting energy production in various tissues, including the testes. This review article delves into the distribution and function of GLUTs in distinct testicular cell types, namely Leydig cells, Sertoli cells, germ cells, and spermatozoa, shedding light on their significance in the context of male reproductive health-an issue of mounting global concern. Furthermore, this article examines the implications of GLUT dysregulation in testicular dysfunction. Altered GLUT expression has been associated with impaired steroidogenesis, spermatogenesis, sperm count, and motility in various animal models. Lastly, the article underscores the potential therapeutic implications of targeting GLUTs concerning testicular toxicity. Insights gleaned from studies in diabetes and cancer suggest that modulating GLUT expression and translocation could present novel strategies for mitigating testicular dysfunction and safeguarding male fertility. In summary, the intricate interplay between GLUTs, glucose metabolism, and testicular health underscores the significance of sustaining testicular glucose homeostasis for male reproductive health. Manipulating GLUTs presents an innovative avenue to address testicular dysfunction, potentially revolutionizing therapeutic strategies to restore male fertility and overall reproductive well-being. Future research in this field holds great promise for advancing male fertility treatments and reproductive health interventions.

SGLT2 inhibitor dapagliflozin protects the kidney in a murine model of Balkan nephropathy.

Proximal tubular uptake of aristolochic acid (AA) forms aristolactam (AL)-DNA adducts, which cause a p53/p21-mediated DNA damage response and acute tubular injury. Recurrent AA exposure causes kidney function loss and fibrosis in humans (Balkan endemic nephropathy) and mice and is a model of (acute kidney injury) AKI to chronic kidney disease (CKD) transition. Inhibitors of the proximal tubule sodium-glucose transporter SGLT2 can protect against CKD progression, but their effect on AA-induced kidney injury remains unknown. C57BL/6J mice (15-wk-old) were administered vehicle or AA every 3 days for 3 wk (10 and 3 mg/kg ip in females and males, respectively). Dapagliflozin (dapa, 0.01 g/kg diet) or vehicle was initiated 7 days prior to AA injections. All dapa effects were sex independent, including a robust glycosuria. Dapa lowered urinary kidney-injury molecule 1 (KIM-1) and albumin (both normalized to creatinine) after the last AA injection and kidney mRNA expression of early DNA damage response markers (p53 and p21) 3 wk later at the study end. Dapa also attenuated AA-induced increases in plasma creatinine as well as AA-induced up-regulation of renal pro-senescence, pro-inflammatory and pro-fibrotic genes, and kidney collagen staining. When assessed 1 day after a single AA injection, dapa pretreatment attenuated AL-DNA adduct formation by 10 and 20% in kidney and liver, respectively, associated with reduced p21 expression. Initiating dapa application after the last AA injection also improved kidney outcome but in a less robust manner. In conclusion, the first evidence is presented that pretreatment with an SGLT2 inhibitor can attenuate the AA-induced DNA damage response and subsequent nephropathy.NEW & NOTEWORTHY Recurrent exposure to aristolochic acid (AA) causes kidney function loss and fibrosis in mice and in humans, e.g., in the form of the endemic Balkan nephropathy. Inhibitors of the proximal tubule sodium-glucose transporter SGLT2 can protect against CKD progression, but their effect on AA-induced kidney injury remains unknown. Here we provide the first evidence in a murine model that pretreatment with an SGLT2 inhibitor can attenuate the AA-induced DNA damage response and subsequent nephropathy.

Hypoxia promotes the growth and metastasis of ovarian cancer cells by suppressing ferroptosis via upregulating SLC2A12.

BACKGROUND: Ovarian cancer has been a worldwide health burden for women and its progression is highly hypoxia-independent. Here, we investigated the exact mechanisms by which hypoxia contributes to the malignant progression of ovarian cancer. METHOD: MTT, transwell, colony formation, and scratch wound healing assays were carried out for cellular functions. The underlying mechanism by which hypoxia functions was explored by RNA-seq, enrichment analysis, western blotting, qRT-PCR, flow cytometry, ChIP, luciferase reporter, and ELISA. Finally, animal experiments including the xenograft model and tumor metastasis model were constructed to validate the role of SLC2A12 in vivo. RESULTS: Hypoxia treatment promoted the cell proliferation, mobility, and colony growth abilities of the two ovarian cancer cell lines HO-8910 and A2780. RNA-seq and enrichment analysis showed that SLC2A12 was hyper-expressed under hypoxia condition and it may be related to glutathione and lipid metabolism. Besides, the expression of SLC2A12 was negatively correlated with overall survival. Hypoxia suppressed ferroptosis by SLC2A12 because silencing SLC2A12 declined the cell viability of HO-8910 and A2780 cells under hypoxia conditions, while the ferroptosis inhibitor ferrostatin-1 (Fer-1) breached that result and upregulated the expression of glutathione peroxidase 4 (GPX4). Moreover, hypoxia increased the expression of hypoxia inducible factor 1 A (HIF-1A), and the accumulated HIF-1A binds to hypoxia inducible factor 1 B (HIF1B) to form HIF-1 complex, then promoted the binding of hypoxic response elements (HRE) to SLC2A12 promoter by HIF-1/HRE signal. Subsequently, SLC2A12 regulated glutathione metabolism and in turn inhibited ferroptosis. The animal experiments indicated that silencing SLC2A12 could significantly inhibit tumor growth and metastasis in vivo. CONCLUSION: Hypoxia promoted ovarian cancer progression by upregulating SLC2A12 and then regulating glutathione metabolism to inhibit ferroptosis.

Glucose Transport and Utilization in the Hippocampus: From Neurophysiology to Diabetes-Related Development of Dementia.

The association of diabetes with cognitive dysfunction has at least 60 years of history, which started with the observation that children with type 1 diabetes mellitus (T1D), who had recurrent episodes of hypoglycemia and consequently low glucose supply to the brain, showed a deficit of cognitive capacity. Later, the growing incidence of type 2 diabetes mellitus (T2D) and dementia in aged populations revealed their high association, in which a reduced neuronal glucose supply has also been considered as a key mechanism, despite hyperglycemia. Here, we discuss the role of glucose in neuronal functioning/preservation, and how peripheral blood glucose accesses the neuronal intracellular compartment, including the exquisite glucose flux across the blood-brain barrier (BBB) and the complex network of glucose transporters, in dementia-related areas such as the hippocampus. In addition, insulin resistance-induced abnormalities in the hippocampus of obese/T2D patients, such as inflammatory stress, oxidative stress, and mitochondrial stress, increased generation of advanced glycated end products and BBB dysfunction, as well as their association with dementia/Alzheimer's disease, are addressed. Finally, we discuss how these abnormalities are accompained by the reduction in the expression and translocation of the high capacity insulin-sensitive glucose transporter GLUT4 in hippocampal neurons, which leads to neurocytoglycopenia and eventually to cognitive dysfunction. This knowledge should further encourage investigations into the beneficial effects of promising therapeutic approaches which could improve central insulin sensitivity and GLUT4 expression, to fight diabetes-related cognitive dysfunctions.

The role of salt bridge networks in the stability of the yeast hexose transporter 1.

BACKGROUND: The yeast S. cerevisiae preferably metabolizes glucose through aerobic glycolysis. Glucose transport is facilitated by multiple hexose transporters (Hxts), and their expression and activity are tightly regulated by multiple mechanisms. However, detailed structural and functional analyses of Hxts remain limited, largely due to the lack of crystal structure. METHODS: Homology modeling was used to build a 3D structural model for the yeast glucose transporter Hxt1 and investigate the effects of site directed mutations on Hxt1 stability and glucose transport activity. RESULTS: The conserved salt bridge-forming residues observed in the human Glut4 and the yeast glucose receptor Rgt2 were identified within and between the two 6-transmembrane spanning segments of Hxt1. Most of the RGT2 mutations that disrupt the salt bridge networks were known to cause constitutive signal generation, whereas the corresponding substitutions in HXT1 were shown to decrease Hxt1 stability. While substitutions of the two residues in the salt bridge 2 in Glut4-E329Q and E393D-were reported to abolish glucose transport, the equivalent substitutions in Hxt1 (D382Q and E454D) did not affect Hxt1 glucose transport activity. CONCLUSIONS: Substitutions of equivalent salt bridge-forming residues in Hxt1, Rgt2, and Glut4 are predicted to lock them in an inward-facing conformation but lead to different functional consequences. GENERAL SIGNIFICANCE: The salt bridge networks in yeast and human glucose transporters and yeast glucose receptors may play different roles in maintaining their structural and functional integrity.

The glucose transporter 2 regulates CD8+ T cell function via environment sensing.

T cell activation is associated with a profound and rapid metabolic response to meet increased energy demands for cell division, differentiation and development of effector function. Glucose uptake and engagement of the glycolytic pathway are major checkpoints for this event. Here we show that the low-affinity, concentration-dependent glucose transporter 2 (Glut2) regulates the development of CD8+ T cell effector responses in mice by promoting glucose uptake, glycolysis and glucose storage. Expression of Glut2 is modulated by environmental factors including glucose and oxygen availability and extracellular acidification. Glut2 is highly expressed by circulating, recently primed T cells, allowing efficient glucose uptake and storage. In glucose-deprived inflammatory environments, Glut2 becomes downregulated, thus preventing passive loss of intracellular glucose. Mechanistically, Glut2 expression is regulated by a combination of molecular interactions involving hypoxia-inducible factor-1 alpha, galectin-9 and stomatin. Finally, we show that human T cells also rely on this glucose transporter, thus providing a potential target for therapeutic immunomodulation.

Interaction of amisulpride with GLUT1 at the blood-brain barrier. Relevance to Alzheimer's disease.

Blood-brain barrier (BBB) dysfunction may be involved in the increased sensitivity of Alzheimer's disease (AD) patients to antipsychotics, including amisulpride. Studies indicate that antipsychotics interact with facilitated glucose transporters (GLUT), including GLUT1, and that GLUT1 BBB expression decreases in AD. We tested the hypotheses that amisulpride (charge: +1) interacts with GLUT1, and that BBB transport of amisulpride is compromised in AD. GLUT1 substrates, GLUT1 inhibitors and GLUT-interacting antipsychotics were identified by literature review and their physicochemical characteristics summarised. Interactions between amisulpride and GLUT1 were studied using in silico approaches and the human cerebral endothelial cell line, hCMEC/D3. Brain distribution of [3H]amisulpride was determined using in situ perfusion in wild type (WT) and 5xFamilial AD (5xFAD) mice. With transmission electron microscopy (TEM) we investigated brain capillary degeneration in WT mice, 5xFAD mice and human samples. Western blots determined BBB transporter expression in mouse and human. Literature review revealed that, although D-glucose has no charge, charged molecules can interact with GLUT1. GLUT1 substrates are smaller (184.95±6.45g/mol) than inhibitors (325.50±14.40g/mol) and GLUT-interacting antipsychotics (369.38±16.04). Molecular docking showed beta-D-glucose (free energy binding: -15.39kcal/mol) and amisulpride (-29.04kcal/mol) interact with GLUT1. Amisulpride did not affect [14C]D-glucose hCMEC/D3 accumulation. [3H]amisulpride uptake into the brain (except supernatant) of 5xFAD mice compared to WT remained unchanged. TEM revealed brain capillary degeneration in human AD. There was no difference in GLUT1 or P-glycoprotein BBB expression between WT and 5xFAD mice. In contrast, caudate P-glycoprotein, but not GLUT1, expression was decreased in human AD capillaries versus controls. This study provides new details about the BBB transport of amisulpride, evidence that amisulpride interacts with GLUT1 and that BBB transporter expression is altered in AD. This suggests that antipsychotics could potentially exacerbate the cerebral hypometabolism in AD. Further research into the mechanism of amisulpride transport by GLUT1 is important for improving antipsychotics safety.

Astragalus polysaccharide restores insulin secretion impaired by lipopolysaccharides through the protein kinase B /mammalian target of rapamycin/glucose transporter 2 pathway.

BACKGROUND: Lipopolysaccharide (LPS)-induced dysfunction of pancreatic ß-cells leads to impaired insulin (INS) secretion. Astragalus polysaccharide (APS) is a bioactive heteropolysaccharide extracted from Astragalus membranaceus and is a popular Chinese herbal medicine. This study aimed to elucidate the mechanisms by which APS affects INS secretion from ß-cells under LPS stress. METHODS: Rat insulinoma (INS-1) cells were treated with LPS at a low, medium, or high concentration of APS. Glucose-stimulated insulin secretion (GSIS) was evaluated using an enzyme-linked immunosorbent assay (ELISA). Transcriptome sequencing was used to assess genome-wide gene expression. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to determine the signaling pathways affected by APS. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to evaluate the gene expression of glucose transporter 2 (GLUT2), glucokinase (GCK), pancreatic duodenal homeobox-1 (PDX-1), and INS. Western blot analysis was used to detect the protein expression of phosphorylated protein kinase B (p-Akt), total Akt (t-Akt), phosphorylated mammalian target of rapamycin (p-mTOR), total mTOR (t-mTOR), and GLUT2. RESULTS: LPS decreased GLUT2, GCK, PDX-1, and INS expression and reduced GSIS. These LPS-induced decreases in gene expression and GSIS were restored by APS treatment. In addition, transcriptome sequencing in combination with KEGG enrichment analysis revealed changes in the INS signaling pathway following APS treatment. LPS decreased p-Akt and p-mTOR expression, which was restored by APS treatment. The restorative effects of APS on GSIS as well as on the expression of GLUT2, GCK, PDX-1, and INS were abolished by treatment with the Akt inhibitor MK2206 or the mTOR inhibitor rapamycin (RPM). CONCLUSIONS: APS restored GSIS in LPS-stimulated pancreatic ß-cells by activating the Akt/mTOR/GLUT2 signaling pathway.

The intracellular helical bundle of human glucose transporter GLUT4 is important for complex formation with ASPL.

Glucose transporters (GLUTs) are responsible for transporting hexose molecules across cellular membranes. In adipocytes, insulin stimulates glucose uptake by redistributing GLUT4 to the plasma membrane. In unstimulated adipose-like mouse cell lines, GLUT4 is known to be retained intracellularly by binding to TUG protein, while upon insulin stimulation, GLUT4 dissociates from TUG. Here, we report that the TUG homolog in human, ASPL, exerts similar properties, i.e., forms a complex with GLUT4. We describe the structural details of complex formation by combining biochemical assays with cross-linking mass spectrometry and computational modeling. Combined, the data suggest that the intracellular domain of GLUT4 binds to the helical lariat of ASPL and contributes to the regulation of GLUT4 trafficking by cooperative binding.

Role of lysosomes in insulin signaling and glucose uptake in cultured rat podocytes.

Podocytes are sensitive to insulin, which governs the functional and structural integrity of podocytes that are essential for proper function of the glomerular filtration barrier. Lysosomes are acidic organelles that are implicated in regulation of the insulin signaling pathway. Cathepsin D (CTPD) and lysosome-associated membrane protein 1 (LAMP1) are major lysosomal proteins that reflect the functional state of lysosomes. However, the effect of insulin on lysosome activity and role of lysosomes in the regulation of insulin-dependent glucose uptake in podocytes are unknown. Our studies showed that the short-term incubation of podocytes with insulin decreased LAMP1 and CTPD mRNA levels. Insulin and bafilomycin A1 reduced both the amounts of LAMP1 and CTPD proteins and activity of CTPD, which were associated with a decrease in the fluorescence intensity of lysosomes that were labeled with LysoTracker. Bafilomycin A1 inhibited insulin-dependent endocytosis of the insulin receptor and increased the amounts of the insulin receptor and glucose transporter 4 on the cell surface of podocytes. Bafilomycin A1 also inhibited insulin-dependent glucose uptake despite an increase in the amount of glucose transporter 4 in the plasma membrane of podocytes. These results suggest that lysosomes are signaling hubs that may be involved in the coupling of insulin signaling with the regulation of glucose uptake in podocytes. The dysregulation of this mechanism can lead to the dysfunction of podocytes and development of insulin resistance.

Correlations between class I glucose transporter expression patterns and clinical outcomes in non-small cell lung cancer.

BACKGROUND: Glucose transporters (GLUTs) are highly expressed in various cancers. However, the implications of these variable expression patterns are unclear. This study aimed to clarify the correlation between class I GLUT expression patterns and clinical outcomes in non-small cell lung cancer (NSCLC), including their potential role in inflammatory signaling. METHODS: Biopsy tissues from 132 patients with NSCLC (92 adenocarcinomas [ADC] and 40 squamous cell carcinomas [SQCC]) were analyzed. mRNA expression levels of class I GLUTs (solute carrier 2A [SLC2A]1, SLC2A2, SLC2A3, and SLC2A4) and inflammation-related molecules (toll-like receptors TLR4, RelA/p65, and interleukins IL8 and IL6) were measured. Cellular localization of GLUT3 and GLUT4 was investigated using immunofluorescence. RESULTS: Single, combined, and negative GLUT (SLC2A) expression were observed in 27/92 (29.3%), 27/92 (29.3%), and 38/92 (41.3%, p < 0.001) of ADC and 8/40 (20.0%), 29/40 (72.5%, p < 0.001), and 3/40 (7.5%) of SQCC, respectively. In ADC, the single SLC2A3-expressed group had a significantly poorer prognosis, whereas the single SLC2A4-expressed group had a significantly better prognosis. The combined expression groups showed no significant difference. SLC2A expression was not correlated with SQCC prognosis. SLC2A4 expression correlated with lower IL8 expression. GLUT3 and GLUT4 expressions were localized in the tumor cytoplasm. CONCLUSIONS: In lung ADC, single SLC2A3 expression correlated with poor prognosis, whereas single SLC2A4 expression correlated with better prognosis and lower IL8 expression. GLUT3 expression, which is increased by IL8 overexpression, may be suppressed by increasing the expression of GLUT4 through decreased IL8 expression.

Review: A species comparison of the kinetic homogeneous and heterogeneous organization of sodium-dependent glucose transport systems along the intestine.

The targeted use of carbohydrates by feed and food industries to create balanced and cost-effective diets has generated a tremendous amount of research in carbohydrate digestion and absorption in different species. Specifically, this research has led us to a larger observation that identified different organizations of intestinal sodium-dependent glucose absorption across species, which has not been previously collated and reviewed. Thus, this review will compare the kinetic segregation of sodium-dependent glucose transport across the intestine of different species, which we have termed either homogeneous or heterogeneous systems. For instance, the pig follows a heterogeneous system of sodium-dependent glucose transport with a high-affinity, super-low-capacity (Ha/sLc) in the jejunum, and a high-affinity, super-high-capacity (Ha/sHc) in the ileum. This is achieved by multiple sodium-dependent glucose transporters contributing to each segment. In contrast, tilapia have a homogenous system characterized by high-affinity, high-capacity (Ha/Hc) throughout the intestine. Additionally, we are the first to report glucose transporter patterns across species presented from vertebrates to invertebrates. Finally, other kinetic transport systems are briefly covered to illustrate possible contributions/modulations to sodium-dependent glucose transporter organization. Overall, we present a new perspective on the organization of glucose absorption along the intestinal tract.

Determinants of sugar-induced influx in the mammalian fructose transporter GLUT5.

In mammals, glucose transporters (GLUT) control organism-wide blood-glucose homeostasis. In human, this is accomplished by 14 different GLUT isoforms, that transport glucose and other monosaccharides with varying substrate preferences and kinetics. Nevertheless, there is little difference between the sugar-coordinating residues in the GLUT proteins and even the malarial Plasmodium falciparum transporter PfHT1, which is uniquely able to transport a wide range of different sugars. PfHT1 was captured in an intermediate 'occluded' state, revealing how the extracellular gating helix TM7b has moved to break and occlude the sugar-binding site. Sequence difference and kinetics indicated that the TM7b gating helix dynamics and interactions likely evolved to enable substrate promiscuity in PfHT1, rather than the sugar-binding site itself. It was unclear, however, if the TM7b structural transitions observed in PfHT1 would be similar in the other GLUT proteins. Here, using enhanced sampling molecular dynamics simulations, we show that the fructose transporter GLUT5 spontaneously transitions through an occluded state that closely resembles PfHT1. The coordination of D-fructose lowers the energetic barriers between the outward- and inward-facing states, and the observed binding mode for D-fructose is consistent with biochemical analysis. Rather than a substrate-binding site that achieves strict specificity by having a high affinity for the substrate, we conclude GLUT proteins have allosterically coupled sugar binding with an extracellular gate that forms the high-affinity transition-state instead. This substrate-coupling pathway presumably enables the catalysis of fast sugar flux at physiological relevant blood-glucose concentrations.

Reconstructing the transport cycle in the sugar porter superfamily using coevolution-powered machine learning.

Sugar porters (SPs) represent the largest group of secondary-active transporters. Some members, such as the glucose transporters (GLUTs), are well known for their role in maintaining blood glucose homeostasis in mammals, with their expression upregulated in many types of cancers. Because only a few sugar porter structures have been determined, mechanistic models have been constructed by piecing together structural states of distantly related proteins. Current GLUT transport models are predominantly descriptive and oversimplified. Here, we have combined coevolution analysis and comparative modeling, to predict structures of the entire sugar porter superfamily in each state of the transport cycle. We have analyzed the state-specific contacts inferred from coevolving residue pairs and shown how this information can be used to rapidly generate free-energy landscapes consistent with experimental estimates, as illustrated here for the mammalian fructose transporter GLUT5. By comparing many different sugar porter models and scrutinizing their sequence, we have been able to define the molecular determinants of the transport cycle, which are conserved throughout the sugar porter superfamily. We have also been able to highlight differences leading to the emergence of proton-coupling, validating, and extending the previously proposed latch mechanism. Our computational approach is transferable to any transporter, and to other protein families in general.

Environmentally relevant exposure to cypermethrin aggravates diet-induced diabetic symptoms in mice: The interaction between environmental chemicals and diet.

Pyrethroids, a class of widely used insecticides, have been linked to diabetes. However, it remains unclear whether and how environmentally relevant exposure to pyrethroids aggravates diet-induced diabetic symptoms. In this study, we investigated the diabetogenic effects of exposure to environmentally relevant doses of cypermethrin (CP), one of the most commonly used pyrethroids, and a high calorie diet (HCD) in adult male mice. Notably, HCD consumption significantly facilitated the bioaccumulation of CP in the liver. CP exposure at the lowest dose in the range of human daily intake exacerbated HCD-induced insulin resistance. In HCD-fed mice, CP treatment significantly decreased hepatic glucose uptake by impairing the translocation of glucose transporter GLUT2. CP exposure regulated hepatic AKT2/GSK3ß/GYS2 pathway, thereby reducing glycogenesis and stimulating gluconeogenesis in the livers of HCD-fed mice. Hepatic transcriptome data showed that CP exposure of HCD-fed mice increased hepatic expression of thioredoxin-interacting protein (Txnip) and vanin-1 (VnnI) genes, which were involved in regulating GLUT2 translocation and AKT2/GSK3ß/GYS2 pathway activity, respectively. CP treatment significantly decreased hepatic glucose uptake in HCD-fed mice by impairing the translocation of glucose transporter GLUT2, which was modulated by upregulation of TXNIP. CP exposure regulated hepatic AKT2/GSK3ß/GYS2 pathway through upregulation of VNNI, thereby reducing glycogenesis and stimulating gluconeogenesis in the livers of HCD-fed mice. This is the first study to show that HCD led to an enrichment of lipophilic CP in the liver, which significantly disrupted glucose homeostasis and caused prediabetic phenotype. Our findings suggest that when assessing the health risks of lipophilic environmental chemicals, especially for metabolism-related outcomes, the interaction between contaminants and diet factors should be considered, otherwise the health risks may be underestimated.