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Adipose tissue plays a crucial role in maintaining metabolic homeostasis by storing lipids and glucose from circulation as intracellular fat. As peripheral tissues like adipose tissue become insulin resistant, decompensation of blood glucose levels occurs causing type 2 diabetes (T2D). Currently, modulating the glycocalyx, a layer of cell-surface glycans, is an underexplored pharmacological treatment strategy to improve glucose homeostasis in T2D patients. Here, we show a novel role for cell-surface heparan sulfate (HS) in establishing glucose uptake capacity and metabolic utilization in differentiated adipocytes. Using a combination of chemical and genetic interventions, we identified that HS modulates this metabolic phenotype by attenuating levels of Wnt signaling during adipogenesis. By engineering, the glycocalyx of pre-adipocytes with exogenous synthetic HS mimetics, we were able to enhance glucose clearance capacity after differentiation through modulation of Wnt ligand availability. These findings establish the cellular glycocalyx as a possible new target for therapeutic intervention in T2D patients by enhancing glucose clearance capacity independent of insulin secretion.

Life adapts to daily environmental changes through circadian rhythms, exhibiting spontaneous oscillations of biological processes. These daily functional oscillations must match the metabolic requirements responding to the time of the day. We focus on the molecular mechanism of how the circadian clock regulates glucose, the primary resource for energy production and other biosynthetic pathways. The complex regulation of the circadian rhythm includes many proteins that control this process at the transcriptional and translational levels and by protein-protein interactions. We have investigated the action of one of these proteins, cryptochrome (CRY), whose elevated mRNA and protein levels repress the function of an activator in the transcription-translation feedback loop, and this activator causes elevated Cry1 mRNA. We used a genome-edited cell line model to investigate downstream genes affected explicitly by the repressor CRY. We found that CRY can repress glycolytic genes, particularly that of the gatekeeper, pyruvate dehydro-genase kinase 1 (Pdk1), decreasing lactate accumulation and glucose utilization. CRY1-mediated decrease of Pdk1 expression can also be observed in a breast cancer cell line MDA-MB-231, whose glycolysis is associated with Pdk1 expression. We also found that exogenous expression of CRY1 in the MDA-MB-231 decreases glucose usage and growth rate. Furthermore, reduced CRY1 levels and the increased phosphorylation of PDK1 substrate were observed when cells were grown in suspension compared to cells grown in adhesion. Our data supports a model that the transcription-translation feedback loop can regulate the glucose metabolic pathway through Pdk1 gene expression according to the time of the day. [ABSTRACT FROM AUTHOR]

In the mammalian retina, a metabolic ecosystem exists in which photoreceptors acquire glucose from the choriocapillaris with the help of the retinal pigment epithelium (RPE). While the photoreceptor cells are primarily glycolytic, exhibiting Warburg-like metabolism, the RPE is reliant on mitochondrial respiration. However, the ways in which mitochondrial metabolism affect RPE cellular functions are not clear. We first used the human RPE cell line, ARPE-19, to examine mitochondrial metabolism in the context of cellular differentiation. We show that nicotinamide induced rapid differentiation of ARPE-19 cells, which was reversed by removal of supplemental nicotinamide. During the nicotinamide-induced differentiation, we observed using quantitative PCR, Western blotting, electron microscopy, and metabolic respiration and tracing assays that (1) mitochondrial gene and protein expression increased, (2) mitochondria became larger with more tightly folded cristae, and (3) mitochondrial metabolism was enhanced. In addition, we show that primary cultures of human fetal RPE cells responded similarly in the presence of nicotinamide. Furthermore, disruption of mitochondrial oxidation of pyruvate attenuated the nicotinamide-induced differentiation of the RPE cells. Together, our results demonstrate a remarkable effect of nicotinamide on RPE metabolism. We also identify mitochondrial respiration as a key contributor to the differentiated state of the RPE and thus to many of the RPE functions that are essential for retinal health and photoreception.

9-cis-retinoic acid (9cRA) binds retinoic acid receptors (RAR) and retinoid X receptors (RXR) with nanomolar affinities, in contrast to all-trans-retinoic acid (atRA), which binds only RAR with nanomolar affinities. RXR heterodimerize with type II nuclear receptors, including RAR, to regulate a vast gene array. Despite much effort, 9cRA has not been identified as an endogenous retinoid, other than in pancreas. By revising tissue analysis methods, 9cRA quantification by liquid chromatography-tandem mass spectrometry becomes possible in all mouse tissues analyzed. 9cRAoccurs in concentrations similar to or greater than atRA. Fasting increases 9cRA in white and brown adipose, brain and pancreas, while increasing atRA in white adipose, liver and pancreas. 9cRA supports FoxO1 actions in pancreas ß-cells and counteracts glucose actions that lead to glucotoxicity; in part by inducing Atg7mRNA, which encodes the key enzyme essential for autophagy. Glucose suppresses 9cRA biosynthesis in the ß-cell lines 832/13 and MIN6. Glucose reduces 9cRA biosynthesis in 832/13 cells by inhibiting Rdh5 transcription, unconnected to insulin, through cAMP and Akt, and inhibiting FoxO1. Through adapting tissue specifically to fasting, 9cRA would act independent of atRA. Widespread occurrence of 9cRAin vivo, and its selfsufficient adaptation to energy status, provides new perspectives into regulation of energy balance, attenuation of insulin and glucose actions, regulation of type II nuclear receptors, and retinoid biology. [ABSTRACT FROM AUTHOR]

The area postrema (AP) of the brain is exposed to circulating metabolites and hormones. However, whether AP detects glucose changes to exert biological responses remains unknown. Its neighboring nuclei, the nucleus tractus solitarius (NTS), responds to acute glucose infusion by inhibiting hepatic glucose production, but the mechanism also remains elusive. Herein, we characterized AP and NTS glucose-sensing mechanisms. Infusion of glucose into the AP, like the NTS, of chow rats suppressed glucose production during the pancreatic (basal insulin)-euglycemic clamps. Glucose transporter 1 or pyruvate kinase lentiviral-mediated knockdown in the AP negated AP glucose infusion to lower glucose production, while the glucoregulatory effect of NTS glucose infusion was also negated by knocking down glucose transporter 1 or pyruvate kinase in the NTS. Furthermore, we determined that high-fat (HF) feeding disrupts glucose infusion to lower glucose production in association with a modest reduction in the expression of glucose transporter 1, but not pyruvate kinase, in the AP and NTS. However, pyruvate dehydrogenase activator dichloroacetate infusion into the AP or NTS that enhanced downstream pyruvate metabolism and recapitulated the glucoregulatory effect of glucose in chow rats still failed to lower glucose production in HF rats. We discovered that a glucose transporter 1- and pyruvate kinase-dependent glucose-sensing mechanism in the AP (as well as the NTS) lowers glucose production in chow rats and that HF disrupts the glucosesensing mechanism that is downstream of pyruvate metabolism in the AP and NTS. These findings highlight the role of AP and NTS in mediating glucose to regulate hepatic glucose production. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) plays a dual role in regulating DNA damage response (DDR) signaling in pancreatic β-cells. As a genotoxic agent, NO activates two types of DDR signaling; however, when produced at micromolar levels by the inducible isoform of NO synthase, NO inhibits DDR signaling and DDRinduced apoptosis in a β-cell-selective manner. DDR signaling inhibition by NO correlates with mitochondrial oxidative metabolism inhibition and decreases in ATP and NAD+. Unlike most cell types, β-cells do not compensate for impaired mitochondrial oxidation by increasing glycolytic flux, and this metabolic inflexibility leads to a decrease in ATP and NAD+. Here, we used multiple analytical approaches to determine changes in intermediary metabolites in β-cells and non-β-cells treated with NO or complex I inhibitor rotenone. In addition to ATP and NAD+, glycolytic and tricarboxylic acid cycle intermediates as well as NADPH are significantly decreased in β-cells treated with NO or rotenone. Consistent with glucose-6-phosphate residing at the metabolic branchpoint for glycolysis and the pentose phosphate pathway (NADPH), we show that mitochondrial oxidation inhibitors limit glucose uptake in a β-cell-selective manner. Our findings indicate that the β-cell-selective inhibition of DDR signaling by NO is associated with a decrease in ATP to levels that fall significantly below the KM for ATP of glucokinase (glucose uptake) and suggest that this action places the β-cell in a state of suspended animation where it is metabolically inert until NO is removed, and metabolic function can be restored. [ABSTRACT FROM AUTHOR]

Mouse studies linking adropin, a peptide hormone encoded by the energy homeostasis-associated (ENHO) gene, to biological clocks and to glucose and lipid metabolism suggest a potential therapeutic target for managing diseases of metabolism. However, adropin's roles in human metabolism are unclear. In silico expression profiling in a nonhuman primate diurnal transcriptome atlas (GSE98965) revealed a dynamic and diurnal pattern of ENHO expression. ENHO expression is abundant in brain, including ventromedial and lateral hypothalamic nuclei regulating appetite and autonomic function. Lower ENHO expression is present in liver, lung, kidney, ileum, and some endocrine glands. Hepatic ENHO expression associates with genes involved in glucose and lipid metabolism. Unsupervised hierarchical clustering identified 426 genes co-regulated with ENHO in liver, ileum, kidney medulla, and lung. Gene Ontology analysis of this cluster revealed enrichment for epigenetic silencing by histone H3K27 trimethylation and biological processes related to neural function. Dietary intervention experiments with 59 adult male rhesus macaques indicated low plasma adropin concentrations were positively correlated with fasting glucose, plasma leptin, and apolipoprotein C3 (APOC3) concentrations. During consumption of a high-sugar (fructose) diet, which induced 10% weight gain, animals with low adropin had larger increases of plasma leptin and more severe hyperglycemia. Declining adropin concentrations were correlated with increases of plasma APOC3 and triglycerides. In summary, peripheral ENHO expression associates with pathways related to epigenetic and neural functions, and carbohydrate and lipid metabolism, suggesting co-regulation in nonhuman primates. Low circulating adropin predicts increased weight gain and metabolic dysregulation during consumption of a high-sugar diet.

Mutations in genes involved in mitochondrial proline catabolism lead to the rare genetic disorder hyperprolinemia in humans. We have previously reported that mutations of proline catabolic genes in Caenorhabditis elegans impair mitochondrial homeostasis and shorten life span, and that these effects surprisingly occur in a diet type-dependent manner. Therefore, we speculated that a specific dietary component may mitigate the adverse effects of defective proline catabolism. Here, we discovered that high dietary glucose, which is generally detrimental to health, actually improves mitochondrial homeostasis and life span in C. elegans with faulty proline catabolism. Mechanistically, defective proline catabolism results in a shift of glucose catabolism toward the pentose phosphate pathway, which is crucial for cellular redox balance. This shift helps to maintain mitochondrial reactive oxygen species homeostasis and to extend life span, as suppression of the pentose phosphate pathway enzyme GSPD-1 prevents the favorable effects of high glucose. In addition, we demonstrate that this crosstalk between proline and glucose catabolism is mediated by the transcription factor DAF-16. Altogether, these findings suggest that a glucose-rich diet may be advantageous in certain situations and might represent a potentially viable treatment strategy for disorders involving impaired proline catabolism. [ABSTRACT FROM AUTHOR]

Glucose is a critical nutrient for cell proliferation. However, the molecular pathways that regulate glucose metabolism are still elusive. We discovered that co-activator-associated arginine methyltransferase 1 (CARM1) suppresses glucose metabolism toward serine biosynthesis. By tracing the 13C-labeled glucose, we found that Carm1 knockout mouse embryonic fibroblasts exhibit significantly increased de novo serine synthesis than WT cells. This is caused, at least in part, by the reduced pyruvate kinase (PK) activity in these cells. The M2 isoform of PK (PKM2) is arginine-methylated by CARM1, and methylation enhances its activity. Mechanistically, CARM1 methylates PKM2 at arginines 445 and 447, which enhances PKM2 tetramer formation. Consequently, Carm1 knockout cells exhibit significant survival advantages over WT cells when extracellular serine is limited, likely due to their enhanced de novo serine synthesis capacity. Altogether, we identified CARM1 as an important regulator of glucose metabolism and serine synthesis.

Higher demand for nutrients including glucose is characteristic of cancer. "Starving cancer" has been pursued to curb tumor progression. An intriguing regime is to inhibit glucose transporter GLUT1 in cancer cells. In addition, during cancer progression, cancer cells may suffer from insufficient glucose supply. Yet, cancer cells can somehow tolerate glucose starvation. Uncovering the underlying mechanisms shall shed insight into cancer progression and benefit cancer therapy. TFE3 is a transcription factor known to activate autophagic genes. Physiological TFE3 activity is regulated by phosphorylation-triggered translocation responsive to nutrient status. We recently reported TFE3 constitutively localizes to the cell nucleus and promotes cell proliferation in kidney cancer even under nutrient replete condition. It remains unclear whether and how TFE3 responds to glucose starvation. In this study, we show TFE3 promotes kidney cancer cell resistance to glucose starvation by exposing cells to physiologically relevant glucose concentration. We find glucose starvation triggers TFE3 protein stabilization through increasing its O-GlcNAcylation. Furthermore, through an unbiased functional genomic study, we identify SLC36A1, a lysosomal amino acid transporter, as a TFE3 target gene sensitive to TFE3 protein level. We find SLC36A1 is overexpressed in kidney cancer, which promotes mTOR activity and kidney cancer cell proliferation. Importantly, SLC36A1 level is induced by glucose starvation through TFE3, which enhances cellular resistance to glucose starvation. Suppressing TFE3 or SLC36A1 significantly increases cellular sensitivity to GLUT1 inhibitor in kidney cancer cells. Collectively, we uncover a functional TFE3-SLC36A1 axis that responds to glucose starvation and enhances starvation tolerance in kidney cancer., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Eukaryotic elongation factor 1A1 (EEF1A1) is canonically involved in protein synthesis but also has noncanonical functions in diverse cellular processes. Previously, we identified EEF1A1 as a mediator of lipotoxicity and demonstrated that chemical inhibition of EEF1A1 activity reduced mouse liver lipid accumulation. These findings suggested a link between EEF1A1 and metabolism. Therefore, we investigated its role in regulating metabolic substrate preference. EEF1A1-deficient Chinese hamster ovary (2E2) cells displayed reduced media lactate accumulation. These effects were also observed with EEF1A1 knockdown in human hepatocyte-like HepG2 cells and in WT Chinese hamster ovary and HepG2 cells treated with selective EEF1A inhibitors, didemnin B, or plitidepsin. Extracellular flux analyses revealed decreased glycolytic ATP production and increased mitochondrial-to-glycolytic ATP production ratio in 2E2 cells, suggesting a more oxidative metabolic phenotype. Correspondingly, fatty acid oxidation was increased in 2E2 cells. Both 2E2 cells and HepG2 cells treated with didemnin B exhibited increased neutral lipid content, which may be required to support elevated oxidative metabolism. RNA-seq revealed a >90-fold downregulation of a rate-limiting glycolytic enzyme, hexokinase 2, which we confirmed through immunoblotting and enzyme activity assays. Pathway enrichment analysis identified downregulations in TNFA signaling via NFKB and MYC targets. Correspondingly, nuclear abundances of RELB and MYC were reduced in 2E2 cells. Thus, EEF1A1 deficiency may perturb glycolysis by limiting NFKB- and MYC-mediated gene expression, leading to decreased hexokinase expression and activity. This is the first evidence of a role for a translation elongation factor, EEF1A1, in regulating metabolic substrate utilization in mammalian cells., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Invasive candidiasis poses a major healthcare threat. The human opportunistic fungal pathogen Candida glabrata, which causes mucosal and deep-seated infections, is armed with distinct virulence attributes, including a family of 11 glycosylphosphatidylinositol-linked aspartyl proteases, CgYapsins. Here, we have profiled total membrane proteomes of the C. glabrata wildtype and 11 proteases-deficient strain, Cgyps1-11Δ, by mass spectrometry analysis and uncovered a novel role for fungal yapsins in glucose sensing and homeostasis. Furthermore, through label-free quantitative membrane proteome analysis, we showed differential abundance of 42% of identified membrane proteins, with electron transport chain and glycolysis proteins displaying lower and higher abundance in Cgyps1-11Δ cells, compared with wildtype cells, respectively. We also demonstrated elevated glucose uptake and upregulation of genes that code for the low-glucose sensor CgSnf3, transcriptional regulators CgMig1 and CgRgt1, and hexose transporter CgHxt2/10 in the Cgyps1-11Δ mutant. We further elucidated a potential underlying mechanism through genetic and transcript measurement analysis under low- and high-glucose conditions and found CgSNF3 deletion to rescue high glucose uptake and attenuated growth of the Cgyps1-11Δ mutant in YPD medium, thereby linking CgYapsins with regulation of the CgSnf3-dependent low-glucose sensing pathway. Last, high ethanol production, diminished mitochondrial membrane potential, and elevated susceptibility to oxidative phosphorylation inhibitors point toward increased fermentative and decreased respiratory metabolism in the Cgyps1-11Δ mutant. Altogether, our findings revealed new possible glucose metabolism-regulatory roles for putative cell surface-associated CgYapsins and advanced our understanding of fungal carbohydrate homeostasis mechanisms. [ABSTRACT FROM AUTHOR]

The maintenance of optimal membrane composition under basal and stress conditions is critical for the survival of an organism. High-glucose stress has been shown to perturb membrane properties by decreasing membrane fluidity, and the membrane sensor PAQR-2 is required to restore membrane integrity. However, the mechanisms required to respond to elevated dietary glucose are not fully established. In this study, we used a 13C stable isotope-enriched diet and mass spectrometry to better understand the impact of glucose on fatty acid dynamics in the membrane of Caenorhabditis elegans. We found a novel role for monomethyl branched-chain fatty acids (mmBCFAs) in mediating the ability of the nematodes to survive conditions of elevated dietary glucose. This requirement of mmBCFAs is unique to glucose stress and was not observed when the nematode was fed elevated dietary saturated fatty acid. In addition, when worms deficient in elo-5, the major biosynthesis enzyme of mmBCFAs, were fed Bacillus subtilis (a bacteria strain rich in mmBCFAs) in combination with high glucose, their survival rates were rescued to wild-type levels. Finally, the results suggest that mmBCFAs are part of the PAQR-2 signaling response during glucose stress. Taken together, we have identified a novel role for mmBCFAs in stress response in nematodes and have established these fatty acids as critical for adapting to elevated glucose. [ABSTRACT FROM AUTHOR]

We report that intra-islet glucagon secreted from α-cells signals through β-cell glucagon and GLP-1 receptors (GcgR and GLP-1R), thereby conferring to rat islets their competence to exhibit first-phase glucose-stimulated insulin secretion (GSIS). Thus, in islets not treated with exogenous glucagon or GLP-1, first-phase GSIS is abolished by a GcgR antagonist (LY2786890) or a GLP-1R antagonist (Ex[9-39]). Mechanistically, glucose competence in response to intra-islet glucagon is conditional on β-cell cAMP signaling because it is blocked by the cAMP antagonist prodrug Rp-8-Br-cAMPS-pAB. In its role as a paracrine hormone, intra-islet glucagon binds with high affinity to the GcgR, while also exerting a "spillover" effect to bind with low affinity to the GLP-1R. This produces a right shift of the concentration-response relationship for the potentiation of GSIS by exogenous glucagon. Thus, 0.3 nM glucagon fails to potentiate GSIS, as expected if similar concentrations of intra-islet glucagon already occupy the GcgR. However, 10 to 30 nM glucagon effectively engages the β-cell GLP-1R to potentiate GSIS, an action blocked by Ex[9-39] but not LY2786890. Finally, we report that the action of intra-islet glucagon to support insulin secretion requires a step-wise increase of glucose concentration to trigger first-phase GSIS. It is not measurable when GSIS is stimulated by a gradient of increasing glucose concentrations, as occurs during an oral glucose tolerance test in vivo. Collectively, such findings are understandable if defective intra-islet glucagon action contributes to the characteristic loss of first-phase GSIS in an intravenous glucose tolerance test that is diagnostic of type 2 diabetes in the clinical setting. [ABSTRACT FROM AUTHOR]

Recent evidence suggests an important role for outer retinal cells in the pathogenesis of diabetic retinopathy (DR). Here we investigated the effect of the visual cycle inhibitor retinylamine (Ret-NH2) on the development of early DR lesions. Wild-type (WT) C57BL/6J mice (male, 2 months old when diabetes was induced) were made diabetic with streptozotocin, and some were given Ret-NH2 once per week. Lecithin-retinol acyltransferase (LRAT)-deficient mice and P23H mutant mice were similarly studied. Mice were euthanized after 2 (WT and Lrat(-/-)) and 8 months (WT) of study to assess vascular histopathology, accumulation of albumin, visual function, and biochemical and physiological abnormalities in the retina. Non-retinal effects of Ret-NH2 were examined in leukocytes treated in vivo. Superoxide generation and expression of inflammatory proteins were significantly increased in retinas of mice diabetic for 2 or 8 months, and the number of degenerate retinal capillaries and accumulation of albumin in neural retina were significantly increased in mice diabetic for 8 months compared with nondiabetic controls. Administration of Ret-NH2 once per week inhibited capillary degeneration and accumulation of albumin in the neural retina, significantly reducing diabetes-induced retinal superoxide and expression of inflammatory proteins. Superoxide generation also was suppressed in Lrat(-/-) diabetic mice. Leukocytes isolated from diabetic mice treated with Ret-NH2 caused significantly less cytotoxicity to retinal endothelial cells ex vivo than did leukocytes from control diabetics. Administration of Ret-NH2 once per week significantly inhibited the pathogenesis of lesions characteristic of early DR in diabetic mice. The visual cycle constitutes a novel target for inhibition of DR.

Sex as a physiologic factor has a strong association with the features of metabolic syndrome. Our previous study showed that loss of the voltage-gated proton channel Hv1 inhibits insulin secretion and leads to hyperglycemia and glucose intolerance in male mice. However, there are significant differences in blood glucose between male and female Hv1-knockout (KO) mice. Here, we investigated the differences in glucose metabolism and insulin sensitivity between male and female KO mice and how sex steroids contribute to these differences. We found that the fasting blood glucose in female KO mice was visibly lower than that in male KO mice, which was accompanied by hypotestosteronemia. KO mice in both sexes exhibited higher expression of gluconeogenesis-related genes in liver compared with WT mice. Also, the livers from KO males displayed a decrease in glycolysis-related gene expression and an increase in gluconeogenesis-related gene expression compared with KO females. Furthermore, exogenous testosterone supplementation decreased blood glucose levels in male KO mice, as well as enhancing insulin signaling. Taken together, our data demonstrate that knockout of Hv1 results in higher blood glucose levels in male than female mice, despite a decreased insulin secretion in both sexes. This sex-related difference in glucose homeostasis is associated with the glucose metabolism in liver tissue, likely due to the physiological levels of testosterone in KO male mice. [ABSTRACT FROM AUTHOR]

Glucose-mediated signaling regulates the expression of a limited number of genes in human pancreatic ß-cells at the transcriptional level. However, it is unclear whether glucose plays a role in posttranscriptional RNA processing or translational control of gene expression. Here, we asked whether glucose affects posttranscriptional steps and regulates protein synthesis in human ß-cell lines. We first showed the involvement of the mTOR pathway in glucose-related signaling. We also used the surface sensing of translation technique, based on puromycin incorporation into newly translated proteins, to demonstrate that glucose treatment increased protein translation. Among the list of glucose-induced proteins, we identified the proconvertase PCSK1, an enzyme involved in the proteolytic conversion of proinsulin to insulin, whose translation was induced within minutes following glucose treatment. We finally performed global proteomic analysis by mass spectrometry to characterize newly translated proteins upon glucose treatment. We found enrichment in proteins involved in translation, glycolysis, TCA metabolism, and insulin secretion. Taken together, our study demonstrates that, although glucose minorly affects gene transcription in human ß-cells, it plays a major role at the translational level. [ABSTRACT FROM AUTHOR]

The altered metabolism in most tumor cells consists of elevated glucose uptake and increased glycolysis even in the presence of high oxygen tension. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an obligatory enzyme in glycolysis. Here, we report that acetylation at lysine 254 (K254) increases GAPDH activity in response to glucose. Furthermore, acetylation of GAPDH (K254) is reversibly regulated by the acetyltransferase PCAF and the deacetylase HDAC5. Substitution of K254 to glutamine compromises the ability of GAPDH to support cell proliferation and tumor growth. Our study reveals a mechanism of GAPDH enzyme activity regulation by acetylation and its critical role in cellular regulation.

Skeletal muscle is responsible for the majority of glucose disposal following meals, and this is achieved by insulimmediated trafficking of glucose transporter type 4 (GLUT4) to the cell membrane. The eight-protein exocyst trafficking complex facilitates targeted docking of membrane-bound vesicles, a process underlying the regulated delivery of fuel transporters. We previously demonstrated the role of exocyst subunit EXOC5 in insulin-stimulated GLUT4 exocytosis and glucose uptake in cultured rat skeletal myoblasts. However, the in vivo role of EXOC5 in skeletal muscle remains unclear. Using mice with inducible, skeletal-muscle-specific knockout of exocyst subunit EXOC5 (Exoc5-SMKO), we examined how muscle-specific disruption of the exocyst would affect glucose homeostasis in vivo. We found that both male and female Exoc5-SMKO mice displayed elevated fasting glucose levels. Additionally, male Exoc5-SMKO mice had impaired glucose tolerance and lower serum insulin levels. Using indirect calorimetry, we observed that male Exoc5-SMKO mice have a reduced respiratory exchange ratio during the light period and lower energy expenditure. Using the hyperinsulinemic-euglycemic clamp method, we further showed that insulin-stimulated skeletal muscle glucose uptake is reduced in Exoc5-SMKO males compared with wildtype controls. Overall, our findings indicate that EXOC5 and the exocyst are necessary for insulin-stimulated glucose uptake in skeletal muscle and regulate glucose homeostasis in vivo. [ABSTRACT FROM AUTHOR]

Traditionally, lipolysis has been regarded as an enzymatic activity that liberates fatty acids as metabolic fuel. However, recent work has shown that novel substrates, including a variety of lipid compounds such as fatty acids and their derivatives, release lipolysis products that act as signaling molecules and transcriptional modulators. While these studies have expanded the role of lipolysis, the mechanisms underpinning lipolysis signaling are not fully defined. Here, we uncover a new mechanism regulating glucose uptake, whereby activation of lipolysis, in response to elevated cAMP, leads to the stimulation of thioredoxin-interacting protein (TXNIP) degradation. This, in turn, selectively induces glucose transporter 1 surface localization and glucose uptake in 3T3-L1 adipocytes and increases lactate production. Interestingly, cAMP-induced glucose uptake via degradation of TXNIP is largely dependent upon adipose triglyceride lipase (ATGL) and not hormone-sensitive lipase or monoacylglycerol lipase. Pharmacological inhibition or knockdown of ATGL alone prevents cAMP-dependent TXNIP degradation and thus significantly decreases glucose uptake and lactate secretion. Conversely, overexpression of ATGL amplifies the cAMP response, yielding increased glucose uptake and lactate production. Similarly, knockdown of TXNIP elicits enhanced basal glucose uptake and lactate secretion, and increased cAMP further amplifies this phenotype. Overexpression of TXNIP reduces basal and cAMP-stimulated glucose uptake and lactate secretion. As a proof of concept, we replicated these findings in human primary adipocytes and observed TXNIP degradation and increased glucose uptake and lactate secretion upon elevated cAMP signaling. Taken together, our results suggest a crosstalk between ATGL-mediated lipolysis and glucose uptake. [ABSTRACT FROM AUTHOR]

The glycoside hydrolase family 55 (GH55) includes inverting exo-β-1,3-glucosidases and endo-β-1,3-glucanases, acting on laminarin, which is a β1-3/1-6-glucan consisting of a β1-3/1-6-linked main chain and β1-6-linked branches. Despite their different modes of action toward laminarin, endo-β-1,3-glucanases share with exo-β-1,3-glucosidases conserved residues that form the dead-end structure of subsite -1. Here, we investigated the mechanism of endo-type action on laminarin by GH55 endo-β-1,3-glucanase MnLam55A, identified from Microdochium nivale. MnLam55A, like other endo-β-1,3-glucanases, degraded internal β-d-glucosidic linkages of laminarin, producing more reducing sugars than the sum of d-glucose and gentiooligosaccharides detected. β1-3-Glucans lacking β1-6-linkages in the main chain were not hydrolyzed. NMR analysis of the initial degradation of laminarin revealed that MnLam55A preferentially cleaved the nonreducing terminal β1-3-linkage of the laminarioligosaccharide moiety at the reducing end side of the main chain β1-6-linkage. MnLam55A liberates d-glucose from laminaritriose and longer laminarioligosaccharides, but k cat /K m values to laminarioligosaccharides (≤4.21 s -1  mM -1 ) were much lower than to laminarin (5920 s -1  mM -1 ). These results indicate that β-glucan binding to the minus subsites of MnLam55A, including exclusive binding of the gentiobiosyl moiety to subsites -1 and -2, is required for high hydrolytic activity. A crystal structure of MnLam55A, determined at 2.4 Å resolution, showed that MnLam55A adopts an overall structure and catalytic site similar to those of exo-β-1,3-glucosidases. However, MnLam55A possesses an extended substrate-binding cleft that is expected to form the minus subsites. Sequence comparison suggested that other endo-type enzymes share the extended cleft. The specific hydrolysis of internal linkages in laminarin is presumably common to GH55 endo-β-1,3-glucanases., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

AMP-activated protein kinase, the "energy sensor of the cell," responds to low cellular energy stores by regulating enzymes and transcription factors that allow the cell to adapt to limiting nutritional conditions. Snf1, the yeast ortholog of AMP-activated protein kinase, has an essential role in respiratory metabolism in Saccharomyces cerevisiae that includes activating the transcription factor Adr1. How Snf1 regulates Adr1 activity is poorly understood. We used an analog-sensitive allele, SNF1(as)(I132G), that is inhibited by 2-naphthylmethyl pyrazolopyrimidine 1 to study the role of Snf1 in transcriptional regulation of glucose-repressible genes. When Snf1(as) was inhibited at the time of glucose depletion, there was a promoter-specific response with some Snf1-dependent genes being activated by low levels of inhibitor, whereas all Snf1-dependent genes were inhibited at high levels. Transcript accumulation was more sensitive to Snf1(as) inhibition than Adr1 or RNA polymerase (pol) II binding or acetylation of promoter nucleosomes. When Snf1(as) was inhibited after gene activation, Adr1 and RNA pol II remained at promoters, and RNA pol II remained in the ORF with associated nascent transcripts. However, cytoplasmic mRNAs were lost at a rapid rate compared with their decay following chemical or genetic inactivation of RNA pol II. In conclusion, Snf1 appears to affect multiple steps in gene regulation, including transcription factor binding, RNA polymerase II activity, and cytoplasmic mRNA stability.

The Staphylococcus epidermidis glucose/H1 symporter (GlcPSe) is a membrane transporter highly specific for glucose and a homolog of the human glucose transporters (GLUT, SLC2 family).Most GLUTs and their bacterial counterparts differ in the transport mechanism, adopting uniport and sugar/H1 symport, respectively. Unlike other bacterial GLUT homologs (for example, XylE), GlcPSe has a loose H1/sugar coupling. Asp22 is part of the proton-binding site of GlcPSe and crucial for the glucose/H1 co-transport mechanism. To determine how pH variations affect the proton site and the transporter, we performed surface-enhanced IR absorption spectroscopy on the immobilized GlcPSe. We found that Asp22 has a pKa of 8.5 6 0.1, a value consistent with that determined previously for glucose transport, confirming the central role of this residue for the transport mechanism of GlcPSe. A neutral replacement of the negatively charged Asp22 led to positive charge displacements over the entire pH range, suggesting that the polarity change of the WT reflects the protonation state of Asp22. We expected that the substitution of the residue Ile105 for a serine, located within hydrogen-bonding distance to Asp22, would change the microenvironment, but the pKa of Asp22 corresponded to that of the WT. A167E mutation, selected in analogy to the XylE, introduced an additional protonatable site and perturbed the protonation state of Asp22, with the latter now exhibiting a pKa of 6.4. These studies confirm that Asp22 is the proton-binding residue in GlcPSe and show that charged residues in its vicinity affect the pKa of glucose/H1 symport. [ABSTRACT FROM AUTHOR]

Adipose tissue is essential for metabolic homeostasis, balancing lipid storage and mobilization based on nutritional status. This is coordinated by insulin, which triggers kinase signaling cascades to modulate numerous metabolic proteins, leading to increased glucose uptake and anabolic processes like lipogenesis. Given recent evidence that glucose is dispensable for adipocyte respiration, we sought to test whether glucose is necessary for insulin-stimulated anabolism. Examining lipogenesis in cultured adipocytes, glucose was essential for insulin to stimulate the synthesis of fatty acids and glyceride-glycerol. Importantly, glucose was dispensable for lipogenesis in the absence of insulin, suggesting that distinct carbon sources are used with or without insulin. Metabolic tracing studies revealed that glucose was required for insulin to stimulate pathways providing carbon substrate, NADPH, and glycerol 3-phosphate for lipid synthesis and storage. Glucose also displaced leucine as a lipogenic substrate and was necessary to suppress fatty acid oxidation. Together, glucose provided substrates and metabolic control for insulin to promote lipogenesis in adipocytes. This contrasted with the suppression of lipolysis by insulin signaling, which occurred independently of glucose. Given previous observations that signal transduction acts primarily before glucose uptake in adipocytes, these data are consistent with a model whereby insulin initially utilizes protein phosphorylation to stimulate lipid anabolism, which is sustained by subsequent glucose metabolism. Consequently, lipid abundance was sensitive to glucose availability, both during adipogenesis and in Drosophila flies in vivo. Together, these data highlight the importance of glucosemetabolismto support insulin action, providing a complementary regulatory mechanismto signal transduction to stimulate adipose anabolism. [ABSTRACT FROM AUTHOR]

The cystine/glutamate transporter system xc consists of the light-chain subunit xCT (SLC7A11) and the heavy-chain subunit CD98 (4F2hc or SLC3A2) and exchanges extracellular cystine for intracellular glutamate at the plasma membrane. The imported cystine is reduced to cysteine and used for synthesis of GSH, one of the most important antioxidants in cancer cells. Because cancer cells have increased levels of reactive oxygen species, xCT, responsible for cystine-glutamate exchange, is overexpressed in many cancers, including glioblastoma. However, under glucose-limited conditions, xCT overexpression induces reactive oxygen species accumulation and cell death. Here we report that cell survival under glucose deprivation depends on cell density. We found that high cell density (HD) down-regulates xCT levels and increases cell viability under glucose deprivation. We also found that growth of glioblastoma cells at HD inactivates mTOR and that treatment of cells grown at low density with the mTOR inhibitor Torin 1 down-regulates xCT and inhibits glucose deprivation-induced cell death. The lysosome inhibitor bafilomycin A1 suppressed xCT down-regulation in HD-cultured glioblastoma cells and in Torin 1-treated cells grown at low density. Additionally, bafilomycin A1 exposure or ectopic xCT expression restored glucose deprivation- induced cell death at HD. These results suggest that HD inactivates mTOR and promotes lysosomal degradation of xCT, leading to improved glioblastoma cell viability under glucoselimited conditions. Our findings provide evidence that control of xCT protein expression via lysosomal degradation is an important mechanism for metabolic adaptation in glioblastoma cells. [ABSTRACT FROM AUTHOR]

The cellular energy sensor AMP-activated protein kinase (AMPK) is a metabolic regulator that mediates adaptation to nutritional variations to maintain a proper energy balance in cells. We show here that suckling-weaning and fasting-refeeding transitions in rodents are associated with changes in AMPK activation and the cellular energy state in the liver. These nutritional transitions were characterized by a metabolic switch from lipid to glucose utilization, orchestrated by modifications in glucose levels and the glucagon/insulin ratio in the bloodstream. We therefore investigated the respective roles of glucose and pancreatic hormones on AMPK activation in mouse primary hepatocytes. We found that glucose starvation transiently activatesAMPK, whereas changes in glucagon and insulin levels had no impact on AMPK. Challenge of hepatocytes with metformininduced metabolic stress strengthened both AMPK activation and cellular energy depletion under limited-glucose conditions, whereas neither glucagon nor insulin altered AMPK activation. Although both insulin and glucagon induced AMPKα phosphorylation at its Ser485/491 residue, they did not affect its activity. Finally, the decrease in cellular ATP levels in response to an energy stress was additionally exacerbated under fasting conditions and by AMPK deficiency in hepatocytes, revealing metabolic inflexibility and emphasizing the importance ofAMPKfor maintaining hepatic energy charge. Our results suggest that nutritional changes (i.e. glucose availability), rather than the related hormonal changes (i.e. the glucagon/insulin ratio), sensitize AMPK activation to the energetic stress induced by the dietary transition during fasting. This effect is critical for preserving the cellular energy state in the liver. [ABSTRACT FROM AUTHOR]

Hypersecretion of glucagon from pancreatic α-cells strongly contributes to diabetic hyperglycemia. Moreover, failure of α-cells to increase glucagon secretion in response to falling blood glucose concentrations compromises the defense against hypoglycemia, a common complication in diabetes therapy. However, the mechanisms underlying glucose regulation of glucagon secretion are poorly understood and likely involve both α-cell-intrinsic and intraislet paracrine signaling. Among paracrine factors, glucose-stimulated release of the GABA metabolite γ-hydroxybutyric acid (GHB) from pancreatic-cells might mediate glucose suppression of glucagon release viaGHBreceptors on β-cells. However, the direct effects of GHB on β-cell signaling and glucagon release have not been investigated. Here, we found that GHB (4-10M) lacked effects on the cytoplasmic concentrations of the secretion-regulating messengers Ca2+ and cAMP in mouseβ-cells. Glucagon secretion from perifused mouse islets was also unaffected by GHB at both 1 and 7 mM glucose. The GHB receptor agonist 3-chloropropanoic acid and the antagonist NCS-382 had no effects on glucagon secretion and did not affect stimulation of secretion induced by a drop in glucose from 7 to 1mM. Inhibition of endogenous GHB formation with the GABA transaminase inhibitor vigabatrin also failed to influence glucagon secretion at 1 mM glucose and did not prevent the suppressive effect of 7 mM glucose. In human islets, GHB tended to stimulate glucagon secretion at 1 mM glucose, an effect mimicked by 3-chloropropanoic acid. We conclude thatGHBdoes not mediate the inhibitory effect of glucose on glucagon secretion. [ABSTRACT FROM AUTHOR]

The chronic effects of metformin on liver gluconeogenesis involve repression of the G6pc gene, which is regulated by the carbohydrate-response element-binding protein through raised cellular intermediates of glucose metabolism. In this study we determined the candidate mechanisms by which metformin lowers glucose 6-phosphate (G6P) in mouse and rat hepatocytes challenged with high glucose or gluconeogenic precursors. Cell metformin loads in the therapeutic range lowered cell G6P but not ATP and decreased G6pc mRNA at high glucose. The G6P lowering by metformin was mimicked by a complex 1 inhibitor (rotenone) and an uncoupler (dinitrophenol) and by overexpression of mGPDH, which lowers glycerol 3-phosphate and G6P and also mimics the G6pc repression by metformin. In contrast, direct allosteric activators of AMPK (A-769662, 991, and C-13) had opposite effects from metformin on glycolysis, gluconeogenesis, and cell G6P. The G6P lowering by metformin, which also occurs in hepatocytes from AMPK knockout mice, is best explained by allosteric regulation of phosphofructokinase-1 and/or fructose bisphosphatase-1, as supported by increased metabolism of [3-3H]glucose relative to [2-3H]glucose; by an increase in the lactate m2/m1 isotopolog ratio from [1,2-13C2]glucose; by lowering of glycerol 3-phosphate an allosteric inhibitor of phosphofructokinase-1; and by marked G6P elevation by selective inhibition of phosphofructokinase-1; but not by a more reduced cytoplasmic NADH/NAD redox state. We conclude that therapeutically relevant doses of metformin lower G6P in hepatocytes challenged with high glucose by stimulation of glycolysis by an AMP-activated protein kinase-independent mechanism through changes in allosteric effectors of phosphofructokinase-1 and fructose bisphosphatase-1, including AMP, Pi, and glycerol 3-phosphate. [ABSTRACT FROM AUTHOR]

The hexosamine biosynthesis pathway (HBP) branches from glycolysis and forms UDP-GlcNAc, the moiety for O-linked β-GlcNAc (O-GlcNAc) post-translational modifications. An inability to directly measure HBP flux has hindered our understanding of the factors regulating protein O-GlcNAcylation. Our goals in this study were to (i) validate a LC-MS method that assesses HBP flux as UDP-GlcNAc (13C)-molar percent enrichment (MPE) and concentration and (ii) determine whether glucose availability or workload regulate cardiac HBP flux. For (i), we perfused isolated murine working hearts with [U-13C6]glucosamine (1, 10, 50, or 100 µM), which bypasses the rate-limiting HBP enzyme. We observed a concentration-dependent increase in UDP-GlcNAc levels and MPE, with the latter reaching a plateau of 56.3 ± 2.9%. For (ii), we perfused isolated working hearts with [U-13C6]glucose (5.5 or 25 mM). Glycolytic efflux doubled with 25 mM [U-13C6]glucose; however, the calculated HBP flux was similar among the glucose concentrations at ~2.5 nmol/g of heart protein/min, representing ~0.003-0.006% of glycolysis. Reducing cardiac workload in beating and nonbeating Langendorff perfusions had no effect on the calculated HBP flux at ~2.3 and 2.5 nmol/g of heart protein/min, respectively. To the best of our knowledge, this is the first direct measurement of glucose flux through the HBP in any organ. We anticipate that these methods will enable foundational analyses of the regulation of HBP flux and protein O-GlcNAcylation. Our results suggest that in the healthy ex vivo perfused heart, HBP flux does not respond to acute changes in glucose availability or cardiac workload. [ABSTRACT FROM AUTHOR]

Metabolic reprogramming in cancer cells can increase their dependence on metabolic substrates such as glucose. As such, the vulnerability of cancer cells to glucose deprivation creates an attractive opportunity for therapeutic intervention. Because it is not possible to starve tumors of glucose in vivo, here we sought to identify the mechanisms in glucose deprivation-induced cancer cell death and then designed inhibitor combinations to mimic glucose deprivation-induced cell death. Using metabolomic profiling, we found that cells undergoing glucose deprivation-induced cell death exhibited dramatic accumulation of intracellular L-cysteine and its oxidized dimer, L-cystine, and depletion of the antioxidant GSH. Building on this observation, we show that glucose deprivation-induced cell death is driven not by the lack of glucose, but rather by L-cystine import. Following glucose deprivation, the import of L-cystine and its subsequent reduction to L-cysteine depleted both NADPH and GSHpools, thereby allowing toxic accumulation of reactive oxygen species. Consistent with this model, we found that the glutamate/cystine antiporter (xCT) is required for increased sensitivity to glucose deprivation. We searched for glycolytic enzymes whose expression is essential for the survival of cancer cells with high xCT expression and identified glucose transporter type 1 (GLUT1). Testing a drug combination that co-targeted GLUT1 and GSH synthesis, we found that this combination induces synthetic lethal cell death in high xCT-expressing cell lines susceptible to glucose deprivation. These results indicate that co-targeting GLUT1 and GSH synthesis may offer a potential therapeutic approach for targeting tumors dependent on glucose for survival. [ABSTRACT FROM AUTHOR]

MicroRNA 199 (miR-199) negatively impacts pancreatic β-cell function and its expression is highly increased in islets from diabetic mice as well as in plasma of diabetic patients. Here we investigatedhowmiR- 199 expression is regulated inβ-cells by assessing expression of miR-199 precursors (primiR-199a1, primiR-199a2, and primiR-199b) and mature miR-199 (miR-199-3p and miR-199-5p) and promoter transcriptional activity assays in mouse islets and mouse insulinoma cells (MIN6) under different stimuli. We found that mouse islets equally express miR-199-3p and miR- 199-5p. However, the primiRNA expression levels differed; although primiR-199a1 expression was about 30% greater than that of primiR-199a2, primiR-199b is barely detected in islets. We observed a 2-fold increase in primiR-199a1 and primiR-199a2 mRNA levels in mouse islets cultured in 10mM glucose compared with 5.5mMglucose. Similar responses to glucose were observed in MIN6 cells. Exposure to 30mM KCl to induce membrane depolarization and calcium influx increased expression of primiR-199a2 but not of primiR-199a1 in MIN6 cells, indicating that calcium influx was involved. Transcriptional activity studies in MIN6 cells also revealed that primiR-199a2 promoter activity was enhanced by glucose and reduced by 2-deoxy-D-glucose-induced starvation. KCl and the potassium channel blocker tolbutamide also stimulated primiR-199a2 promoter activity. Calcium channel blockade by nifedipine reduced primiR-199a2 promoter activity in MIN6 cells, and diazoxide-mediated calcium influx inhibition blunted glucose up-regulation of miR-199-3p in islets. In conclusion, we uncover that glucose acutely up-regulates miR-199 family expression in β-cells. Glucose metabolism and calcium influx are involved in primiR-199a2 expression but not primiR-199a1 expression. [ABSTRACT FROM AUTHOR]

Insulin action in adipose tissue is crucial for whole-body glucose homeostasis, with insulin resistance being a major risk factor for metabolic diseases such as type 2 diabetes. Recent studies have proposed mitochondrial oxidants as a unifying driver of adipose insulin resistance, serving as a signal of nutrient excess. However, neither the substrates for nor sites of oxidant production are known. Because insulin stimulates glucose utilization, we hypothesized that glucose oxidation would fuel respiration, in turn generating mitochondrial oxidants. This would impair insulin action, limiting further glucose uptake in a negative feedback loop of "glucose-dependent" insulin resistance. Using primary rat adipocytes and cultured 3T3-L1 adipocytes, we observed that insulin increased respiration, but notably this occurred independently of glucose supply. In contrast, glucose was required for insulin to increase mitochondrial oxidants. Despite rising to similar levels as when treated with other agents that cause insulin resistance, glucose-dependent mitochondrial oxidants failed to cause insulin resistance. Subsequent studies revealed a temporal relationship whereby mitochondrial oxidants needed to increase before the insulin stimulus to induce insulin resistance. Together, these data reveal that (a) adipocyte respiration is principally fueled from nonglucose sources; (b) there is a disconnect between respiration and oxidative stress, whereby mitochondrial oxidant levels do not rise with increased respiration unless glucose is present; and (c) mitochondrial oxidative stress must precede the insulin stimulus to cause insulin resistance, explaining why short-term, insulin-dependent glucose utilization does not promote insulin resistance. These data provide additional clues to mechanistically link nutrient excess to adipose insulin resistance. [ABSTRACT FROM AUTHOR]

Increased carbohydrate consumption increases hepatic de novo lipogenesis, which has been linked to the development of chronic metabolic diseases, including obesity, hepatic steatosis, and insulin resistance. Stearoyl CoA desaturase 1 (SCD1) is a critical lipogenic enzyme that catalyzes the synthesis of two monounsaturated fatty acids, oleate and palmitoleate, from the saturated fatty acids stearate and palmitate, respectively. SCD1- deficient mouse models are protected against diet-induced adiposity, hepatic steatosis, and hyperglycemia. However, the mechanism of this protection by SCD1 deficiency is unclear. Using liver-specific SCD1 knockout (LKO) mice fed a high-carbohydrate, low-fat diet, we show that hepatic SCD1 deficiency increases systemic glucose uptake. Hepatic SCD1 deficiency enhanced glucose transporter type 1 (GLUT1) expression in the liver and also up-regulated GLUT4 and adiponectin expression in adipose tissue. The enhanced glucose uptake correlated with increased liver expression and elevated plasma levels of fibroblast growth factor 21 (FGF21), a hepatokine known to increase systemic insulin sensitivity and regulate whole-body lipid metabolism. Feeding LKO mice a triolein-supplemented but not tristearin-supplemented high-carbohydrate, low-fat diet reduced FGF21 expression and plasma levels. Consistently, SCD1 inhibition in primary hepatocytes induced FGF21 expression, which was repressed by treatment with oleate but not palmitoleate. Moreover, deletion of the transcriptional coactivator PPARγ coactivator 1α (PGC-1α) reduced hepatic and plasma FGF21 and white adipocyte tissue-specific GLUT4 expression and raised plasma glucose levels in LKO mice. These results suggest that hepatic oleate regulates glucose uptake in adipose tissue either directly or partially by modulating the hepatic PGC-1α-FGF21 axis. [ABSTRACT FROM AUTHOR]

Enzymes that degrade lignocellulose to simple sugars are of great interest in research and for biotechnology because of their role in converting plant biomass to fuels and chemicals. The synthesis of cellulolytic enzymes in filamentous fungi is tightly regulated at the transcriptional level, with the transcriptional activator ClrB/CLR-2 playing a critical role in many species. In Penicillium oxalicum, clrB overexpression could not relieve the dependence of cellulase expression on cellulose as an inducer, suggesting that clrB is controlled post-transcriptionally. In this study, using a reporter gene system in yeast, we identified the C-terminal region of ClrB/CLR-2 as a transcriptional activation domain. Expression of clrBID, encoding a ClrB derivative in which the DNA-binding and transcriptional activation domains are fused together to remove the middle region, led to cellulase production in the absence of cellulose in P. oxalicum. Strikingly, the clrBID-expressing strain produced cellulase on carbon sources that normally repress cellulase expression, including glucose and glycerol. Results from deletion of the carbon catabolite repressor gene creA in the clrBID-expressing strain suggested that the effect of clrBID is independent of CreA's repressive function. A similar modification of clrB in Aspergillus niger resulted in the production of a mannanase in glucose medium. Taken together, these results indicate that ClrB suppression under noninducing conditions involves its middle region, suggesting a potential strategy to engineer fungal strains for improved cellulase production on commonly used carbon sources. [ABSTRACT FROM AUTHOR]

Mitochondria undergo morphological and dynamic changes in response to environmental stresses. Few studies have focused on addressing mitochondrial remodeling under stress. Using the fission yeast Schizosaccharomyces pombe as a model organism, here we investigated mitochondrial remodeling under glucose starvation. We employed live-cell microscopy to monitor mitochondrial morphology and dynamics of cells in profusion chambers under glucose starvation. Our results revealed that mitochondria fragment within minutes after glucose starvation and that the dynamin GTPase Dnm1 is required for promoting mitochondrial fragmentation. Moreover, we found that glucose starvation enhancesDnm1localization to mitochondria and increases the frequency of mitochondrial fission but decreases PKA activity. We further demonstrate that low PKA activity enhances glucose starvation-induced mitochondrial fragmentation, whereas high PKA activity confers resistance to glucose starvation-induced mitochondrial fragmentation. Moreover, we observed that AMP-activated protein kinase is not involved in regulating mitochondrial fragmentation under glucose starvation. Of note, glucose starvation-induced mitochondrial fragmentation was associated with enhanced reactive oxygen species production. Our work provides detailed mechanistic insights into mitochondrial remodeling in response to glucose starvation. [ABSTRACT FROM AUTHOR]

Cells use multiple mechanisms to regulate their metabolic states in response to changes in their nutrient environment. One example is the response of cells to glucose. In Saccharomyces cerevisiae growing in glucose-depleted medium, the re-availability of glucose leads to the down-regulation of gluconeogenesis and the activation of glycolysis, leading to “glucose repression.” However, our knowledge of the mechanisms mediating the glucose-dependent down-regulation of the gluconeogenic transcription factors is limited. Using the major gluconeogenic transcription factor Rds2 as a candidate, we identify here a novel role for the E3 ubiquitin ligase Pib1 in regulating the stability and degradation of Rds2. Glucose addition to cells growing under glucose limitation results in a rapid ubiquitination of Rds2, followed by its proteasomal degradation. Through in vivo and in vitro experiments, we establish Pib1 as the ubiquitin E3 ligase that regulates Rds2 ubiquitination and stability. Notably, this Pib1-mediated Rds2 ubiquitination, followed by proteasomal degradation, is specific to the presence of glucose. This Pib1-mediated ubiquitination of Rds2 depends on the phosphorylation state of Rds2, suggesting a cross-talk between ubiquitination and phosphorylation to achieve a metabolic state change. Using stable isotope-based metabolic flux experiments, we find that the loss of Pib1 results in an imbalanced gluconeogenic state, regardless of glucose availability. Pib1 is required for complete glucose repression and enables cells to optimally grow in competitive environments when glucose again becomes available. Our results reveal the existence of a Pib1-mediated regulatory program that mediates glucose repression when glucose availability is restored. [ABSTRACT FROM AUTHOR]

Hypoglycemic drugs such as metformin increase glucose uptake and utilization by peripheral tissues to maintain glucose homeostasis, and the AMP-activated protein kinase (AMPK) signaling pathway is an important component of this pharmacological activity. Liver kinase B1 (LKB1) acts as a kinase upstream of AMPK and plays an important regulatory role in glucose metabolism. In recent years, as a tumor suppressor, LKB1's antitumor activity has been widely studied, yet its hypoglycemic activity is not clear. Here, using biochemical and cell viability assays, site-directed mutagenesis, immunoblotting, and immunofluorescence staining, we found that a natural product, antroalbol H isolated from the basidiomycete mushroom Antrodiella albocinnamomea, increases cellular glucose uptake in murine L6 myotubes and 3T3-L1 adipocytes. Of note, our results indicated that this effect is related to LKB1-mediated Thr-172 phosphorylation of AMPKα. Furthermore, we observed that antroalbol H induces the phosphorylation of LKB1 specifically at Thr-189 and changes subcellular localization of LKB1. Finally, antroalbol H treatment strikingly promoted glucose transporter type 4 (GLUT4) translocation to the plasma membrane. We conclude that antroalbol H promotes Thr-189 phosphorylation of LKB1, leading to AMPK activation, revealing this residue as a potential target for increasing glucose uptake, and that antroalbol H therefore has potential for managing hyperglycemia. [ABSTRACT FROM AUTHOR]

Whether growing cancer cells prefer lactate as a fuel over glucose or vice versa is an important yet controversial issue. Labeling of tricarboxylic acid (TCA) cycle intermediates with glucose or lactate isotope tracers is often used to report the relative contributions of these two metabolites to the TCA cycle. However, this approach may not yield accurate results, as isotopic labeling may not accurately reflect net contributions of each metabolite. This may be due to isotopic exchange occurring during the conversion between pyruvate and lactate. To evaluate this quantitatively, we used an equation (CG - CG' = CL' - CL) assessing the relationship between isotopic labeling and net consumption measurements in vitro. CG and CL refer to the contributions of glucose and lactate to the TCA cycle as measured by their net consumption, whereas CG' and CL' refer to glucose and lactate's contributions determined with isotopic labeling. We found that the isotopic labeling data overestimate the net contribution of lactate to the TCA cycle and underestimate that of glucose. The overestimated amount is equal to the isotopic exchange amount between pyruvate and lactate. After excluding the interference of isotopic exchange, the major carbon contribution (i.e. acetyl-CoA) to the TCA cycle comes from glucose rather than lactate in vitro. We propose that these relative contributions of glucose and lactate may also be present in cancer cells in vivo. [ABSTRACT FROM AUTHOR]

Amides of fatty acids with ethanolamine (FAE) are biologically active lipids that participate in a variety of biological functions, including the regulation of feeding. The polyunsaturated FAE anandamide (arachidonoylethanolamide) increases food intake by activating G protein-coupled cannabinoid receptors. On the other hand, the monounsaturated FAE oleoylethanolamide (OEA) reduces feeding and body weight gain by activating the nuclear receptor PPAR-alpha (peroxisome proliferator-activated receptor alpha). In the present report, we examined whether OEA can also influence energy utilization. OEA (1-20 microm) stimulated glycerol and fatty acid release from freshly dissociated rat adipocytes in a concentration-dependent and structurally selective manner. Under the same conditions, OEA had no effect on glucose uptake or oxidation. OEA enhanced fatty acid oxidation in skeletal muscle strips, dissociated hepatocytes, and primary cardiomyocyte cultures. Administration of OEA in vivo (5 mg kg(-1), intraperitoneally) produced lipolysis in both rats and wild-type mice, but not in mice in which PPAR-alpha had been deleted by homologous recombination (PPAR-alpha(-/-)). Likewise, OEA was unable to enhance lipolysis in adipocytes or stimulate fatty acid oxidation in skeletal muscle strips isolated from PPAR-alpha mice. The synthetic PPAR-alpha agonist Wy-14643 produced similar effects, which also were dependent on the presence of PPAR-alpha. Subchronic treatment with OEA reduced body weight gain and triacylglycerol content in liver and adipose tissue of diet-induced obese rats and wild-type mice, but not in obese PPAR-alpha(-/-) mice. The results suggest that OEA stimulates fat utilization through activation of PPAR-alpha and that this effect may contribute to its anti-obesity actions.

Acylation stimulating protein (ASP) is a potent stimulator of triglyceride synthesis in adipocytes. In the present study, we have examined the effect of an ASP functional knockout (ASP(-/-)) on lipid metabolism in male mice. In both young (14 weeks) and older (26 weeks) mice there were marked delays in postprandial triglyceride clearance (80% increase at 14 weeks and 120% increase at 26 weeks versus wild type (+/+)). Postprandial nonesterified fatty acids were also increased in ASP(-/-) mice versus ASP(+/+) mice by 37% (low fat 10% Kcal) and by 73% (high fat 40% Kcal) diets, although there were no differences in fasting lipid levels. The ASP(-/-) mice had moderately increased energy intake (16% +/- 2% p < 0.0001) and reduced feed efficiency (33% increase in calories/g of body weight gained on low fat diet) versus wild type. The ASP(-/-) mice also had modest changes in insulin/glucose metabolism (30% to 40% decrease in insulin.glucose product), implying increased insulin sensitivity. As well, there were decreases in leptin (29% shift in leptin to body weight ratio) and up to a 26% decrease in specific adipose tissue depots versus the wild type mice on both low fat and high fat diets. These results demonstrate that ASP plays an important role in adipose tissue metabolism and fat partitioning.

Mesangial expansion underlies diabetic nephropathy, leading to sclerosis and renal failure. The glycosaminoglycan heparin inhibits mesangial cell growth, but the molecular mechanism is unclear. Here, rat mesangial cells (RMCs) were growth-arrested in the G0/G1 phase of cell division, stimulated to divide in normal glucose (5.6 mM) or high glucose (25.6 mM) with or without heparin, and analyzed for glucose uptake. We observed that RMCs entering the G1 phase in normal glucose with or without heparin rapidly cease glucose uptake. RMCs entering G1 in high glucose sustained glucose uptake for the first 3 h, and high-glucose exposure of RMCs only in the first 8 h of G1 induced the formation of an extracellular monocyte-adhesive hyaluronan matrix after cell division was completed. Moreover, a low heparin concentration under high-glucose conditions blocked glucose uptake by 1 h intoG1. Of note, glucose transporter 4 (glut4) localized on theRMCsurface atG0/G1 and was internalized into G1 cells under normal glucose conditions with or without heparin within 30 min. We also noted that, under high-glucose conditions, glut4 remained on the RMC surface for at least 2 h into G1 and was internalized by 4 h without heparin and within 1 h with heparin. These results provide evidence that the influx of glucose in hyperglycemic dividing RMCs initiates intermediate glucose metabolism, leading to increased cytosolic UDP sugars, and induces abnormal intracellular hyaluronan synthesis during the S phase of cell division. [ABSTRACT FROM AUTHOR]

Levoglucosan is the 1,6-anhydrosugar of D-glucose formed by pyrolysis of glucans and is found in the environment and industrial waste.Twotypes of microbial levoglucosan metabolic pathways are known. Although the eukaryotic pathway involving levoglucosan kinase has been well-studied, the bacterial pathway involving levoglucosan dehydrogenase (LGDH) has not been well-investigated. Here, we identified and cloned the lgdh gene from the bacterium Pseudarthrobacter phenanthrenivorans and characterized the recombinant protein. The enzyme exhibited high substrate specificity toward levoglucosan and NAD+ for the oxidative reaction and was confirmed to be LGDH. LGDH also showed weak activities (~4%) toward L-sorbose and 1,5-anhydro-D-glucitol. The reverse (reductive) reaction using 3-keto-levoglucosan and NADH exhibited significantly lower Km and higher kcat values than those of the forward reaction. The crystal structures of LGDH in the apo and complex forms with NADH, NADH + levoglucosan, and NADH + L-sorbose revealed that LGDH has a typical fold of Gfo/Idh/ MocA family proteins, similar to those of scyllo-inositol dehydrogenase, aldose--aldose oxidoreductase, 1,5-anhydro-D-fructose reductase, and glucose--fructose oxidoreductase. The crystal structures also disclosed that the active site of LGDH is distinct from those of these enzymes. The LGDH active site extensively recognized the levoglucosan molecule with six hydrogen bonds, and the C3 atom of levoglucosan was closely located to the C4 atom of NADH nicotinamide. Our study is the first molecular characterization of LGDH, providing evidence for C3-specific oxidation and representing a starting point for future biotechnological use of LGDH and levoglucosan-metabolizing bacteria. [ABSTRACT FROM AUTHOR]