Glucose Signaling Pathways Research Articles

Cooked Black Turtle Beans Ameliorate Insulin Resistance and Restore Gut Microbiota in C57BL/6J Mice on High-Fat Diets.

Colored common beans are associated with health promoting and chronic disease prevention effects. Male C57BL/6J mice were fed high-fat (HF) diets supplemented with cooked black turtle beans (HFB) to prevent obesity related insulin resistance. Mice on both HF and HFB were obese compared to mice fed a low-fat (LF) diet. Plasma low density lipoprotein (LDL) and triglyceride concentrations of mice fed HFB diet were 28% and 36.6% lower than those on HF diet. Homeostatic model assessment of insulin resistance (HOMA-IR) index of mice fed HFB diet was 87% lower than that of mice fed HF diet. Diabetes related biomarkers, gastric inhibitory polypeptide (GIP), leptin, glucagon, and inflammatory cytokines interleukin 4 (IL-4) and IL-5, 10 and 12, IFN-g and TNF-α were significantly affected by HFB diet. Pparα, Cyp7a1 and Fasn were down-regulated by HFB diet while LDL-R, Srebp-2, Adipoq and Slc2a4 were up-regulated by HFB diet. The ratio of Firmicutes/Bacteroidetes (F/B) was also decreased 64.1% by HFB diet compared to HF diet. The results indicated that cooked black turtle bean consumption could ameliorate insulin resistance and lower plasma LDL in mice fed HF diet through glucose signaling pathway and JNK/c-Jun pathway. Meanwhile, cooked black turtle bean consumption restored the gut microbiome.

Relación entre Porphyromonas gingivalis y diabetes mellitus tipo 2: revisión sistemática exploratoria

Objetivo: analizar la relación entre Porphyromonas gingivalis y diabetes mellitus tipo 2, mediante una revisión sistemática exploratoria de la literatura científica publicada entre los años 2000 y 2019. Métodos: se utilizaron los siguientes términos MeSH: Porphyromonas gingivalis, diabetes mellitus type 2, periodontal disease, non insulin dependent diabetes. Se obtuvieron 346 resultados, de los cuales se seleccionaron 41 por título, se excluyeron 11 posterior a la lectura del abstract e introducción y 19 después de la lectura del texto completo. Finalmente, se incluyeron 11 artículos. Resultados: el lipopolisacárido de Porphyromonas gingivalis y su fimbria tipo II se relacionan con una mayor producción de citoquinas proinflamatorias como IL-6 y TNF-α, las cuales afectan las vías de señalización de la glucosa y se relacionan con insulinoresistencia. La dipeptidil peptidasa 4 de Porphyromonas gingivalis puede participar en la degradación de incretinas, lo cual afecta la producción de insulina en el huésped y promueve estados de hiperglicemia. El interactoma de Porphyromonas gingivalis puede superponerse con genes involucrados en resistencia a la insulina y diabetes mellitus tipo 2. Conclusión: según la evidencia científica publicada existen factores de virulencia y mecanismos por los cuales la Porphyromonas gingivalis influye en el desarrollo de insulinorresistencia y diabetes mellitus tipo 2.

Snd3 controls nucleus-vacuole junctions in response to glucose signaling

Membrane contact sites facilitate the exchange of metabolites between organelles to support interorganellar communication. The nucleus-vacuole junctions (NVJs) establish physical contact between the perinuclear endoplasmic reticulum (ER) and the vacuole. Although the NVJ tethers are known, how NVJ abundance and composition are controlled in response to metabolic cues remains elusive. Here, we identify the ER protein Snd3 as central factor for NVJ formation. Snd3 interacts with NVJ tethers, supports their targeting to the contacts, and is essential for NVJ formation. Upon glucose exhaustion, Snd3 relocalizes from the ER to NVJs and promotes contact expansion regulated by central glucose signaling pathways. Glucose replenishment induces the rapid dissociation of Snd3 from the NVJs, preceding the slow disassembly of the junctions. In sum, this study identifies a key factor required for formation and regulation of NVJs and provides a paradigm for metabolic control of membrane contact sites.

Improving Xylose Fermentation in Saccharomyces cerevisiae by Expressing Nuclear-Localized Hexokinase 2.

Understanding the relationship between xylose and the metabolic regulatory systems is a prerequisite to enhance xylose utilization in recombinant S. cerevisiae strains. Hexokinase 2 (Hxk2p) is an intracellular glucose sensor that localizes to the cytoplasm or the nucleus depending on the carbon source. Hxk2p interacts with Mig1p to regulate gene transcription in the nucleus. Here, we investigated the effect of nucleus-localized Hxk2p and Mig1p on xylose fermentation. The results show that the expression of HXK2S14A, which encodes a constitutively nucleus-localized Hxk2p, increased the xylose consumption rate, the ethanol production rate, and the ethanol yield of the engineered yeast strain by 23.5%, 78.6% and 42.6%, respectively. The deletion of MIG1 decreased xylose utilization and eliminated the positive effect of Hxk2p. We then performed RNA-seq and found that the targets of Hxk2pS14A on xylose were mainly genes that encode RNA-binding proteins. This is very different from the known targets of Mig1p and supports the notion that the Hxk2p-Mig1p interaction is abolished in the presence of xylose. These results will improve our understanding of the interrelation between the Snf1p-Mig1p-Hxk2p glucose signaling pathway and xylose utilization in S. cerevisiae and suggests that the expression of HXK2S14A could be a viable strategy to improve xylose utilization.

Exposure of Pancreatic β-Cells to Excess Glucose Results in Bimodal Activation of mTORC1 and mTOR-Dependent Metabolic Acceleration.

SummaryChronic exposure of pancreatic β-cells to excess glucose can lead to metabolic acceleration and loss of stimulus-secretion coupling. Here, we examined how exposure to excess glucose (defined here as concentrations above 5 mM) affects mTORC1 signaling and the metabolism of β-cells. Acute exposure to excess glucose stimulated glycolysis-dependent mTORC1 signaling, without changes in the PI3K or AMPK pathways. Prolonged exposure to excess glucose led to hyperactivation of mTORC1 and metabolic acceleration, characterized by higher basal respiration and maximal respiratory capacity, increased energy demand, and enhanced flux through mitochondrial pyruvate metabolism. Inhibition of pyruvate transport to the mitochondria decelerated the metabolism of β-cells chronically exposed to excess glucose and re-established glucose-dependent mTORC1 signaling, disrupting a positive feedback loop for mTORC1 hyperactivation. mTOR inhibition had positive and negative impacts on various metabolic pathways and insulin secretion, demonstrating a role for mTOR signaling in the long-term metabolic adaptation of β-cells to excess glucose.

Xylose assimilation enhances the production of isobutanol in engineered Saccharomyces cerevisiae.

Bioconversion of xylose-the second most abundant sugar in nature-into high-value fuels and chemicals by engineered Saccharomyces cerevisiae has been a long-term goal of the metabolic engineering community. Although most efforts have heavily focused on the production of ethanol by engineered S. cerevisiae, yields and productivities of ethanol produced from xylose have remained inferior as compared with ethanol produced from glucose. However, this entrenched focus on ethanol has concealed the fact that many aspects of xylose metabolism favor the production of nonethanol products. Through reduced overall metabolic flux, a more respiratory nature of consumption, and evading glucose signaling pathways, the bioconversion of xylose can be more amenable to redirecting flux away from ethanol towards the desired target product. In this report, we show that coupling xylose consumption via the oxidoreductive pathway with a mitochondrially-targeted isobutanol biosynthesis pathway leads to enhanced product yields and titers as compared to cultures utilizing glucose or galactose as a carbon source. Through the optimization of culture conditions, we achieve 2.6 g/L of isobutanol in the fed-batch flask and bioreactor fermentations. These results suggest that there may be synergistic benefits of coupling xylose assimilation with the production of nonethanol value-added products.

Allopregnanolone Reverses Bioenergetic Deficits in Female Triple Transgenic Alzheimer's Mouse Model.

Previously, we reported that the neurosteroid allopregnanolone (Allo) promoted neural stem cell regeneration, restored cognitive function, and reduced Alzheimer's Disease (AD) pathology in the triple transgenic Alzheimer's mouse model (3xTgAD). To investigate the underlying systems biology of Allo action in AD models in vivo, we assessed the regulation of Allo on the bioenergetic system of the brain. Outcomes of these analysis indicated that Allo significantly reversed deficits in mitochondrial respiration and biogenesis and key mitochondrial enzyme activity and reduced lipid peroxidation in the 3xTgAD mice in vivo. To explore the mechanisms by which Allo regulates the brain metabolism, we conducted targeted transcriptome analysis. These data further confirmed that Allo upregulated genes involved in glucose metabolism, mitochondrial bioenergetics, and signaling pathways while simultaneously downregulating genes involved in Alzheimer's pathology, fatty acid metabolism, and mitochondrial uncoupling and dynamics. Upstream regulatory pathway analysis predicted that Allo induced peroxisome proliferator-activated receptor gamma (PPARG) and coactivator 1-alpha (PPARGC1A) pathways while simultaneously inhibiting the presenilin 1 (PSEN 1), phosphatase and tensin homolog (PTEN), and tumor necrosis factor (TNF) pathways to reduce AD pathology. Collectively, these data indicate that Allo functions as a systems biology regulator of bioenergetics, cholesterol homeostasis, and β-amyloid reduction in the brain. These systems are critical to neurological health, thus providing a plausible mechanistic rationale for Allo as a therapeutic to promote neural cell function and reduce the burden of AD pathology.

Deregulation of phytoene-\u03b2-carotene synthase results in derepression of astaxanthin synthesis at high glucose concentration in Phaffia rhodozyma astaxanthin-overproducing strain MK19

BackgroundA major obstacle to industrial-scale astaxanthin production by the yeast Phaffia rhodozyma is the strong inhibitory effect of high glucose concentration on astaxanthin synthesis. We investigated, for the first time, the mechanism of the regulatory effect of high glucose (> 100 g/L) at the metabolite and transcription levels.ResultsTotal carotenoid, β-carotene, and astaxanthin contents were greatly reduced in wild-type JCM9042 at high (110 g/L) glucose; in particular, β-carotene content at 24–72 h was only 14–17% of that at low (40 g/L) glucose. The inhibitory effect of high glucose on astaxanthin synthesis appeared to be due mainly to repression of lycopene-to-β-carotene and β-carotene-to-astaxanthin steps in the pathway. Expression of carotenogenic genes crtE, pbs, and ast was also strongly inhibited by high glucose; such inhibition was mediated by creA, a global negative regulator of carotenogenic genes which is strongly induced by glucose. In contrast, astaxanthin-overproducing, glucose metabolic derepression mutant strain MK19 displayed de-inhibition of astaxanthin synthesis at 110 g/L glucose; this de-inhibition was due mainly to deregulation of pbs and ast expression, which in turn resulted from low creA expression. Failure of glucose to induce the genes reg1 and hxk2, which maintain CreA activity, also accounts for the fact that astaxanthin synthesis in MK19 was not repressed at high glucose.ConclusionWe conclude that astaxanthin synthesis in MK19 at high glucose is enhanced primarily through derepression of carotenogenic genes (particularly pbs), and that this process is mediated by CreA, Reg1, and Hxk2 in the glucose signaling pathway.

The hypoxic expression of the glucose transporter RAG1 reveals the role of the bHLH transcription factor Sck1 as a novel hypoxic modulator in Kluyveromyces lactis

Glucose is the preferred nutrient for most living cells and is also a signaling molecule that modulates several cellular processes. Glucose regulates the expression of glucose permease genes in yeasts through signaling pathways dependent on plasma membrane glucose sensors. In the yeast Kluyveromyces lactis, sufficient levels of glucose induction of the low-affinity glucose transporter RAG1 gene also depends on a functional glycolysis, suggesting additional intracellular signaling. We have found that the expression of RAG1 gene is also induced by hypoxia in the presence of glucose, indicating that glucose and oxygen signaling pathways are interconnected. In this study we investigated the molecular mechanisms underlying this crosstalk. By analyzing RAG1 expression in various K. lactis mutants, we found that the bHLH transcriptional activator Sck1 is required for the hypoxic induction of RAG1 gene. The RAG1 promoter region essential for its hypoxic induction was identified by promoter deletion experiments. Taken together, these results show that the RAG1 glucose permease gene is synergistically induced by hypoxia and glucose and highlighted a novel role for the transcriptional activator Sck1 as a key mediator in this mechanism.

Less is more or more is less: Implications of glucose metabolism in the regulation of the reproductive potential and total lifespan of the Saccharomyces cerevisiae yeast.

Carbohydrates are dietary nutrients that have an influence on cells physiology, cell reproductive capacity and, consequently, the lifespan of organisms. They are used in cellular processes after conversion to glucose, which is the primary source of energy and carbon skeleton for biosynthetic processes. Studies of the influence of glucose on cellular parameters and lifespan of organisms are primarily concerned with the effect of low glucose concentration defined as calorie restriction conditions. However, the effect of high glucose concentration on cell physiology is also very important. Thus, a comparative analysis of the effects of low and high glucose concentration conditions on cell efficiency was proposed with regard to reproductive capacity and total lifespan of the cell. Glucose concentration determines the type of metabolism and biosynthetic capabilities, which in turn, through the regulation on the cell size, may affect the reproductive capacity of cells. This study was conducted on yeast cells of wild-type and mutant strains Δgpa2 and Δgpr1 with glucose signalling pathway impairment. Such an experimental model enabled testing both the role of glucose concentration in the regulation of metabolic changes and the extent to which these changes depend on the extracellular or intracellular glucose concentrations. It has been shown here that calorie/glucose excess connected with changes in cell metabolic fluxes increases biosynthetic capabilities of yeast cells. This leads to an increase in cell dry weight accompanied by the increase in cell size and a simultaneous decrease in the reproductive potential and the overall length of cell life.

Membrane raft-mediated regulation of glucose signaling pathway leading to acrosome reaction in chicken sperm†.

Despite knowledge that glucose metabolism is essential for the regulation of signaling cascades in the sperm that are pre-assembled into specific areas and function at multistage for fertilization, the physiological roles of glucose in avian sperm are poorly understood. Accumulated results of studies conducted in our laboratory and others indicate that sperm possess membrane microdomains, or membrane rafts, which play important roles in several processes, including the induction of acrosome reaction (AR). When characterizing proteomes associated with chicken sperm rafts, we observed marked enrichment of glucose transporter 3 (GLUT3). Here we show that glucose uptake is mediated by membrane rafts and stimulates AR induction by activating AMP-activated protein kinase (AMPK). Using a specific antibody, we observed that GLUT3 is localized to the entire flagellum and acrosome region and highly associated with membrane rafts. The addition of glucose stimulated AR in a dose-dependent manner without affecting sperm motility. AR and glucose uptake assays were performed using both inhibitors and activators, and demonstrated that glucose-dependent AR results from the activity of a glucose transporter located in membrane rafts and associated with AMPK. To better understand the mechanism of AMPK activation by glucose, we evaluated localization and phosphorylation status of AMPKα, showing that glucose uptake stimulates AMPKα phosphorylation, leading to its complete activation. Together, these results lead us to propose a novel mechanism by which glucose uptake stimulates the AMPK signaling pathway via membrane rafts, resulting in maximal acrosomal responsiveness in avian sperm as migrating upward to a fertilization site.

Maternal obesity has sex-dependent effects on insulin, glucose and lipid metabolism and the liver transcriptome in young adult rat offspring.

Maternal high-fat diet consumption predisposes to metabolic dysfunction in male and female offspring at young adulthood. Maternal obesity programs non-alcoholic fatty liver disease (NAFLD) in a sex-dependent manner. We demonstrate sex-dependent liver transcriptome profiles in rat offspring of obese mothers. In this study, we focused on pathways related to insulin, glucose and lipid signalling. These results improve understanding of the mechanisms by which a maternal high-fat diet affects the offspring. Maternal obesity (MO) predisposes offspring (F1) to obesity, insulin resistance (IR) and non-alcoholic fatty liver disease (NAFLD). MO's effects on the F1 liver transcriptome are poorly understood. We used RNA-seqto determine the liver transcriptome of male and female F1 of MO and control-fed mothers. We hypothesized that MO-F1 are predisposed to sex-dependent adult liver dysfunction. Female Wistar rat mothers ate a control (C) or obesogenic (MO) diet from the time they were weaned through breeding at postnatal day (PND) 120, delivery and lactation. After weaning, all male and female F1 ate a control diet. At PND 110, F1 serum, liver and fat were collected to analyse metabolites, histology and liver differentially expressed genes. Male and female MO-F1 showed increased adiposity index, triglycerides, insulin and homeostatic model assessment vs. C-F1 with similar body weight and glucose serum concentrations. MO-F1 males presented greater physiological and histological NAFLD characteristics than MO-F1 females. RNA-seq revealed 1365 genes significantly changed in male MO-F1 liver and only 70 genes in female MO-F1 compared with controls. GO and KEGG analysis identified differentially expressed genes related to metabolic processes. Male MO-F1 liver showed the following altered pathways: insulin signalling (22 genes), phospholipase D signalling (14 genes), NAFLD (13 genes) and glycolysis/gluconeogenesis (7 genes). In contrast, few genes were altered in these pathways in MO-F1 females. In summary, MO programs sex-dependent F1 changes in insulin, glucose and lipid signalling pathways, leading to liver dysfunction and insulin resistance.

The Small Yeast GTPase Rho5 and Its Dimeric GEF Dck1/Lmo1 Respond to Glucose Starvation

Rho5 is a small GTPase of Saccharomyces cerevisiae and a homolog of mammalian Rac1. The latter regulates glucose metabolism and actin cytoskeleton dynamics, and its misregulation causes cancer and a variety of other diseases. In yeast, Rho5 has been implicated in different signal transduction pathways, governing cell wall integrity and the responses to high medium osmolarity and oxidative stress. It has also been proposed to affect mitophagy and apoptosis. Here, we demonstrate that Rho5 rapidly relocates from the plasma membrane to mitochondria upon glucose starvation, mediated by its dimeric GDP/GTP exchange factor (GEF) Dck1/Lmo1. A function in response to glucose availability is also suggested by synthetic genetic phenotypes of a rho5 deletion with gpr1, gpa2, and sch9 null mutants. On the other hand, the role of mammalian Rac1 in regulating the action cytoskeleton does not seem to be strongly conserved in S. cerevisiae Rho5. We propose that Rho5 serves as a central hub in integrating various stress conditions, including a crosstalk with the cAMP/PKA (cyclic AMP activating protein kinase A) and Sch9 branches of glucose signaling pathways.

T3 and Glucose Increase Expression of ChREBPß to Induce Expression of Phosphoenolpyruvate Carboxykinase (Pck1) Leading to Increased ß-Cell Proliferation

Thyroid hormone (T3) is required for islet development and function. Both T3 and high glucose concentrations are critical components of differentiation protocols of stem cells to β-cells. We previously demonstrated that in brown adipose tissue, T3 and glucose synergistically regulate carbohydrate response element binding protein (ChREBP), which in turn upregulates Ucp1, Glut4 and Fasn, resulting in increased thermogenesis, decreased body weight and improved glycemic levels. In the present study, we asked whether T3 and glucose signaling pathways can coordinately regulate transcription of genes important for β-cell function and mass. RNA-seq analysis was performed on cadaveric human islets from five different donors in response to low and high glucose concentrations (6 and 20 mM, respectively) and in the presence or absence of T3 (10 nM). We found that T3 and glucose coordinately regulate the expression of ChREBPβ and Pck1 (phosphoenolpyruvate carboxykinase-1). Typically, it is thought that Pck1 is not expressed in pancreatic β-cells, but it is a known target of T3 in hepatocytes. However, in FACS sorted adult human β-cells, we found that high concentrations of glucose (20 mM) and T3 (10 nM) induced the expression of Pck1 by 17.2±1.3 fold for mRNA levels. In addition, we found that overexpression of Pck1 together with its substrate dimethyl malate (DMM) increased β- cell proliferation in human islets (0.49±0.03% for human islets overexpressing Pck1, treated with DMM compared with 0.01±0.01% for control islets, n=4). Finally, using a sh-RNA knockdown approach, we demonstrated that in Ins-1 cells, ChREBPβ is necessary for Pck1 dependent β-cell proliferation. We conclude that T3 and glucose act together to regulate ChREBPβ which in turn upregulates expression of Pck1. In turn, Pck1 activity leads to increased β-cell mass. Disclosure L.S. Katz: None. D. Scott: None.

The Role of Dehydroepiandrosterone (DHEA) in Skeletal Muscle.

Dehydroepiandrosterone (DHEA) is a precursor of sex steroid hormones and is converted to testosterone and estradiol. Normally, androgens and estrogens produced adrenal cortex, testis, and ovary; however, recent studies revealed androgens and estrogens are synthesized by peripheral tissues such as brain, skin, liver, kidney, bone, etc. We found skeletal muscles are also capable of synthesizing androgens and estrogens from DHEA. Circulating DHEA provides substrates required for conversion into potent androgens and estrogens in peripheral tissues. Sex steroid hormone administration has important roles: one is that the enhancement of protein synthesis and anabolism, resulting in muscle growth and increased muscle strength. The other is improvement of hyperglycemia through the activation of glucose signaling pathway in skeletal muscle as well as acceleration of muscle lipid metabolism that increase peroxisome proliferator-activated receptor alpha (PPARα) and PPAR delta (PPARδ). We introduce the effect of DHEA and sex steroid hormones administration on muscle glucose and lipid metabolisms as well as the effect of sex steroid hormone on the muscle hypertrophy.

Consequences of calorie restriction and calorie excess for the physiological parameters of the yeast Saccharomyces cerevisiae cells.

Glucose plays an important role in cell metabolism and has an impact on cellular physiology. Changes in glucose availability may strongly influence growth rate of the cell size, cell metabolism and the rate of generation of cellular by-products, such as reactive oxygen species. The positive effect of low glucose concentration conditions-calorie restriction is observed in a wide range of species, including the Saccharomyces cerevisiae yeast, yet little is known about the effect of high glucose concentrations-calorie excess. Such analysis seems to be particularly important due to recently common problem of diabetes and obesity. The effect of glucose on morphological and physiological parameters of the yeast cell was conducted using genetic alteration (disruption of genes involved in glucose signalling) and calorie restriction and calorie excess conditions. The results show a significant relationship among extracellular glucose concentration, cell size and reactive oxygen species generation in yeast cells. Furthermore, the results obtained through the use of mutant strains with disorders in glucose signalling pathways suggest that the intracellular level of glucose is more important than its extracellular concentration. These data also suggest that the calorie excess as a factor, which has a significant impact on cell physiology, requires further comprehensive analyses.

An alternative model for studying age-associated metabolic complications: Senescence-accelerated mouse prone 8

Rodent animal models take at least 18months to develop aging phenotypes for researchers to investigate the mechanism of age-related metabolic complications. Senescence-accelerated mouse prone 8 (SAMP8) shortens the process of aging and may facilitate an alternative model for studying age-related insulin resistance. The short-lived strain SAMP8 and two long-lived strains SAM resistant 1 (SAMR1) mice and C57BL/6 mice at 12 (young) and 40weeks old (old) were used in the present study. Glucose tolerance test, histology and signaling pathways involved in lipid metabolism in adipose tissue and liver and key components of insulin signaling pathway in the skeletal muscle were determined in these three strains. We found that short-lived SAMP8 mice developed symptoms of insulin resistance including hyperglycemia, hyperinsulinemia, and impaired glucose tolerance in association with adipocyte hypertrophy and ectopic lipid accumulation in liver and muscle at 40-wk.-old. Significantly increased serum IL-6, leptin, and resistin levels and adipogenic transcription factor PPARγ and macrophage marker F4/80 mRNA expression in adipose tissues were observed in old SAMP8 mice, compared with that in young SAMP8 mice. Marked increases in SREBP1 and PPARγ and a decrease in PPARα at mRNA level in accordance with activation of mTOR/Akt pathway were contributed to hepatic lipid accumulation in old SAMP8 mice. Down-regulation of insulin signaling pathway including IRβ, IRS1, and AS160 at protein level in skeletal muscle was observed in old SAMP8 mice. At 40-wk.-old, both long-lived SAMR1 and C57BL/6 mice have not been fully developed age-related metabolic disorders including insulin resistance and visceral fat expansion in line with fewer defects in lipid metabolism and skeletal muscle insulin signaling pathway. In conclusion, our data suggest the suitability of the SAMP8 mice as a model for studying age-related metabolic complications.

Multilevel regulation of an α-arrestin by glucose depletion controls hexose transporter endocytosis.

Nutrient availability controls the landscape of nutrient transporters present at the plasma membrane, notably by regulating their ubiquitylation and subsequent endocytosis. In yeast, this involves the Nedd4 ubiquitin ligase Rsp5 and arrestin-related trafficking adaptors (ARTs). ARTs are targeted by signaling pathways and warrant that cargo ubiquitylation and endocytosis appropriately respond to nutritional inputs. Here, we show that glucose deprivation regulates the ART protein Csr2/Art8 at multiple levels to trigger high-affinity glucose transporter endocytosis. Csr2 is transcriptionally induced in these conditions through the AMPK orthologue Snf1 and downstream transcriptional repressors. Upon synthesis, Csr2 becomes activated by ubiquitylation. In contrast, glucose replenishment induces CSR2 transcriptional shutdown and switches Csr2 to an inactive, deubiquitylated form. This glucose-induced deubiquitylation of Csr2 correlates with its phospho-dependent association with 14-3-3 proteins and involves protein kinase A. Thus, two glucose signaling pathways converge onto Csr2 to regulate hexose transporter endocytosis by glucose availability. These data illustrate novel mechanisms by which nutrients modulate ART activity and endocytosis.

Validation, Identification, and Biological Consequences of the Site-specific O-GlcNAcylation Dynamics of Carbohydrate-responsive Element-binding Protein (ChREBP).

O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP) is believed as an important modulator of ChREBP activities, however little direct evidence of O-GlcNAcylation on ChREBP and no exact O-GlcNAcylation sites have been reported so far. Here, we validate O-GlcNAcylation on ChREBP in cell-free coupled transcription/translation system and in cells by chemoenzymatic and metabolic labeling, respectively. Moreover, for the first time, we identify O-GlcNAcylation on Ser614 in the C-terminus of ChREBP by mass spectrometry and validate two important sites, Thr517 and Ser839 for O-GlcNAcylation and their function via molecular and chemical biological method. Under high glucose conditions, Ser514 phosphorylation enhances ChREBP O-GlcNAcylation, maintaining the transcriptional activity of ChREBP; Ser839 O-GlcNAcylation is essential for Mlx-heterodimerization and DNA-binding activity enhancement, consequently inducing transcriptional activity. Ser839 O-GlcNAcylation is also crucial for ChREBP nuclear export partially by strengthening interactions with CRM1 and 14-3-3. This work is a detailed study of ChREBP O-GlcNAcylation and highlights the biological consequences of the site-specific O-GlcNAcylation dynamics of ChREBP.

Metabolic fate of glucose and candidate signaling and excess-fuel detoxification pathways in pancreatic β-cells

Glucose metabolism promotes insulin secretion in β-cells via metabolic coupling factors that are incompletely defined. Moreover, chronically elevated glucose causes β-cell dysfunction, but little is known about how cells handle excess fuels to avoid toxicity. Here we sought to determine which among the candidate pathways and coupling factors best correlates with glucose-stimulated insulin secretion (GSIS), define the fate of glucose in the β-cell, and identify pathways possibly involved in excess-fuel detoxification. We exposed isolated rat islets for 1 h to increasing glucose concentrations and measured various pathways and metabolites. Glucose oxidation, oxygen consumption, and ATP production correlated well with GSIS and saturated at 16 mm glucose. However, glucose utilization, glycerol release, triglyceride and glycogen contents, free fatty acid (FFA) content and release, and cholesterol and cholesterol esters increased linearly up to 25 mm glucose. Besides being oxidized, glucose was mainly metabolized via glycerol production and release and lipid synthesis (particularly FFA, triglycerides, and cholesterol), whereas glycogen production was comparatively low. Using targeted metabolomics in INS-1(832/13) cells, we found that several metabolites correlated well with GSIS, in particular some Krebs cycle intermediates, malonyl-CoA, and lower ADP levels. Glucose dose-dependently increased the dihydroxyacetone phosphate/glycerol 3-phosphate ratio in INS-1(832/13) cells, indicating a more oxidized state of NAD in the cytosol upon glucose stimulation. Overall, the data support a role for accelerated oxidative mitochondrial metabolism, anaplerosis, and malonyl-CoA/lipid signaling in β-cell metabolic signaling and suggest that a decrease in ADP levels is important in GSIS. The results also suggest that excess-fuel detoxification pathways in β-cells possibly comprise glycerol and FFA formation and release extracellularly and the diversion of glucose carbons to triglycerides and cholesterol esters.