2-Deoxy-glucose ameliorates the peritoneal mesothelial and endothelial barrier function perturbation occurring due to Peritoneal Dialysis fluids exposure.

Peritoneal dialysis (PD) and prolonged exposure to PD fluids (PDF) induce peritoneal membrane (PM) fibrosis and hypervascularity, leading to functional PM degeneration. 2-deoxy-glucose (2-DG) has shown potential as PM antifibrotic by inhibiting hyper-glycolysis induced mesothelial-to-mesenchymal transition (MMT). We investigated whether administration of 2-DG with several PDF affects the permeability of mesothelial and endothelial barrier of the PM. The antifibrotic effect of 2-DG was confirmed by the gel contraction assay with embedded mesothelial (MeT-5A) or endothelial (EA.hy926) cells cultured in Dianeal® 2.5 % (CPDF), BicaVera® 2.3 % (BPDF), Balance® 2.3 % (LPDF) with/without 2-DG addition (0.2 mM), and qPCR for αSMA, CDH2 genes. Moreover, 2-DG effect was tested on the permeability of monolayers of mesothelial and endothelial cells by monitoring the transmembrane resistance (RTM), FITC-dextran (10, 70 kDa) diffusion and mRNA expression levels of CLDN-1 to -5, ZO1, SGLT1, and SGLT2 genes. Contractility of MeT-5A cells in CPDF/2-DG was decreased, accompanied by αSMA (0.17 ± 0.03) and CDH2 (2.92 ± 0.29) gene expression fold changes. Changes in αSMA, CDH2 were found in EA.hy926 cells, though αSMA also decreased under LPDF/2-DG incubation (0.42 ± 0.02). Overall, 2-DG mitigated the PDF-induced alterations in mesothelial and endothelial barrier function as shown by RTM, dextran transport and expression levels of the CLDN-1 to -5, ZO1, and SGLT2. Thus, supplementation of PDF with 2-DG not only reduces MMT but also improves functional permeability characteristics of the PM mesothelial and endothelial barrier.

SLC15A4 mediates M1-prone metabolic shifts in macrophages and guards immune cells from metabolic stress.

The amino acid and oligopeptide transporter Solute carrier family 15 member A4 (SLC15A4), which resides in lysosomes and is preferentially expressed in immune cells, plays critical roles in the pathogenesis of lupus and colitis in murine models. Toll-like receptor (TLR)7/9- and nucleotide-binding oligomerization domain-containing protein 1 (NOD1)-mediated inflammatory responses require SLC15A4 function for regulating the mechanistic target of rapamycin complex 1 (mTORC1) or transporting L-Ala-γ-D-Glu-meso-diaminopimelic acid, IL-12: interleukin-12 (Tri-DAP), respectively. Here, we further investigated the mechanism of how SLC15A4 directs inflammatory responses. Proximity-dependent biotin identification revealed glycolysis as highly enriched gene ontology terms. Fluxome analyses in macrophages indicated that SLC15A4 loss causes insufficient biotransformation of pyruvate to the tricarboxylic acid cycle, while increasing glutaminolysis to the cycle. Furthermore, SLC15A4 was required for M1-prone metabolic change and inflammatory IL-12 cytokine productions after TLR9 stimulation. SLC15A4 could be in close proximity to AMP-activated protein kinase (AMPK) and mTOR, and SLC15A4 deficiency impaired TLR-mediated AMPK activation. Interestingly, SLC15A4-intact but not SLC15A4-deficient macrophages became resistant to fluctuations in environmental nutrient levels by limiting the use of the glutamine source; thus, SLC15A4 was critical for macrophage's respiratory homeostasis. Our findings reveal a mechanism of metabolic regulation in which an amino acid transporter acts as a gatekeeper that protects immune cells' ability to acquire an M1-prone metabolic phenotype in inflammatory tissues by mitigating metabolic stress.

2-Deoxy-D-glucose ameliorates inflammation and fibrosis in a silicosis mouse model by inhibiting hypoxia-inducible factor-1α in alveolar macrophages.

Inhaling silica causes the occupational illness silicosis, which mostly results in the gradual fibrosis of lung tissue. Previous research has demonstrated that hypoxia-inducible factor-1α (HIF-1α) and glycolysis-related genes are up-regulated in silicosis. The role of 2-deoxy-D-glucose (2-DG) as an inhibitor of glycolysis in silicosis mouse models and its molecular mechanisms remain unclear. Therefore, we used 2-DG to observe its effect on pulmonary inflammation and fibrosis in a silicosis mouse model. Furthermore, in vitro cell experiments were conducted to explore the specific mechanisms of HIF-1α. Our study found that 2-DG down-regulated HIF-1α levels in alveolar macrophages induced by silica exposure and reduced the interleukin-1ß (IL-1ß) level in pulmonary inflammation. Additionally, 2-DG reduced silica-induced pulmonary fibrosis. From these findings, we hypothesize that 2-DG reduced glucose transporter 1 (GLUT1) expression by inhibiting glycolysis, which inhibits the expression of HIF-1α and ultimately reduces transcription of the inflammatory cytokine, IL-1ß, thus alleviating lung damage. Therefore, we elucidated the important regulatory role of HIF-1α in an experimental silicosis model and the potential defense mechanisms of 2-DG. These results provide a possible effective strategy for 2-DG in the treatment of silicosis.

Convergent evolution of diverse Bacillus anthracis outbreak strains toward altered surface oligosaccharides that modulate anthrax pathogenesis.

Bacillus anthracis, a spore-forming gram-positive bacterium, causes anthrax. The external surface of the exosporium is coated with glycosylated proteins. The sugar additions are capped with the unique monosaccharide anthrose. The West African Group (WAG) B. anthracis have mutations rendering them anthrose deficient. Through genome sequencing, we identified 2 different large chromosomal deletions within the anthrose biosynthetic operon of B. anthracis strains from Chile and Poland. In silico analysis identified an anthrose-deficient strain in the anthrax outbreak among European heroin users. Anthrose-deficient strains are no longer restricted to West Africa so the role of anthrose in physiology and pathogenesis was investigated in B. anthracis Sterne. Loss of anthrose delayed spore germination and enhanced sporulation. Spores without anthrose were phagocytized at higher rates than spores with anthrose, indicating that anthrose may serve an antiphagocytic function on the spore surface. The anthrose mutant had half the LD50 and decreased time to death (TTD) of wild type and complement B. anthracis Sterne in the A/J mouse model. Following infection, anthrose mutant bacteria were more abundant in the spleen, indicating enhanced dissemination of Sterne anthrose mutant. At low sample sizes in the A/J mouse model, the mortality of ΔantC-infected mice challenged by intranasal or subcutaneous routes was 20% greater than wild type. Competitive index (CI) studies indicated that spores without anthrose disseminated to organs more extensively than a complemented mutant. Death process modeling using mouse mortality dynamics suggested that larger sample sizes would lead to significantly higher deaths in anthrose-negative infected animals. The model was tested by infecting Galleria mellonella with spores and confirmed the anthrose mutant was significantly more lethal. Vaccination studies in the A/J mouse model showed that the human vaccine protected against high-dose challenges of the nonencapsulated Sterne-based anthrose mutant. This work begins to identify the physiologic and pathogenic consequences of convergent anthrose mutations in B. anthracis.

Novel mutation in hexokinase 2 confers resistance to 2-deoxyglucose by altering protein dynamics.

Glucose is central to many biological processes, serving as an energy source and a building block for biosynthesis. After glucose enters the cell, hexokinases convert it to glucose-6-phosphate (Glc-6P) for use in anaerobic fermentation, aerobic oxidative phosphorylation, and the pentose-phosphate pathway. We here describe a genetic screen in Saccharomyces cerevisiae that generated a novel spontaneous mutation in hexokinase-2, hxk2G238V, that confers resistance to the toxic glucose analog 2-deoxyglucose (2DG). Wild-type hexokinases convert 2DG to 2-deoxyglucose-6-phosphate (2DG-6P), but 2DG-6P cannot support downstream glycolysis, resulting in a cellular starvation-like response. Curiously, though the hxk2G238V mutation encodes a loss-of-function allele, the affected amino acid does not interact directly with bound glucose, 2DG, or ATP. Molecular dynamics simulations suggest that Hxk2G238V impedes sugar binding by altering the protein dynamics of the glucose-binding cleft, as well as the large-scale domain-closure motions required for catalysis. These findings shed new light on Hxk2 dynamics and highlight how allosteric changes can influence catalysis, providing new structural insights into this critical regulator of carbohydrate metabolism. Given that hexokinases are upregulated in some cancers and that 2DG and its derivatives have been studied in anti-cancer trials, the present work also provides insights that may apply to cancer biology and drug resistance.

Directed evolution of Saccharomyces cerevisiae for low volatile acidity during winemaking under aerobic conditions.

The use of yeast respiratory metabolism has been proposed as a promising approach to solve the problem of increasing ethanol content in wine, which is largely due to climate change. The use of S. cerevisiae for this purpose is mostly hampered by acetic acid overproduction generated under the necessary aerobic conditions. However, it was previously shown that a reg1 mutant, alleviated for carbon catabolite repression (CCR), showed low acetic acid production under aerobic conditions. In this work directed evolution of three wine yeast strains was performed to recover CCR-alleviated strains, expecting they will also be improved concerning volatile acidity. This was done by subculturing strains on galactose, in the presence of 2-deoxyglucose for around 140 generations. As expected, all evolved yeast populations released less acetic acid than their parental strains in grape juice, under aerobic conditions. Single clones were isolated from the evolved populations, either directly or after one cycle of aerobic fermentation. Only some clones from one of three original strains showed lower acetic acid production than their parental strain. Most clones isolated from EC1118 showed slower growth. However, even the most promising clones failed to reduce acetic acid production under aerobic conditions in bioreactors. Therefore, despite the concept of selecting low acetic acid producers by using 2-deoxyglucose as selective agent was found to be correct, especially at the population level, the recovery of strains with potential industrial utility by this experimental approach remains a challenge.

Enteral glucose, absorbed and metabolized, potently enhances mesenteric lymph flow in chow- and high-fat-fed rats.

A portion of absorbed dietary triglycerides (TG) is retained in the intestine after the postprandial period, within intracellular and extracellular compartments. This pool of TG can be mobilized in response to several stimuli, including oral glucose. The objective of this study was to determine whether oral glucose must be absorbed and metabolized to mobilize TG in rats and whether high-fat feeding, a model of insulin resistance, alters the lipid mobilization response to glucose. Lymph flow, TG concentration, TG output, and apolipoprotein B48 (apoB48) concentration and output were assessed after an intraduodenal lipid bolus in rats exposed to the following intraduodenal administrations 5 h later: saline (placebo), glucose, 2-deoxyglucose (2-DG, absorbed but not metabolized), or glucose + phlorizin (intestinal glucose absorption inhibitor). Glucose alone, but not 2-DG or glucose + phlorizin treatments, stimulated lymph flow, TG output, and apoB48 output compared with placebo. The effects of glucose in high-fat-fed rats were similar to those in chow-fed rats. In conclusion, glucose must be both absorbed and metabolized to enhance lymph flow and intestinal lipid mobilization. This effect is qualitatively and quantitatively similar in high-fat- and chow-fed rats. The precise signaling mechanism whereby enteral glucose enhances lymph flow and mobilizes enteral lipid remains to be determined.NEW & NOTEWORTHY Glucose potently enhances mesenteric lymph flow in chow- and high-fat-fed rats. The magnitude of glucose effect on lymph flow is no different in chow- and high-fat-fed rats. Glucose must be absorbed and metabolized to enhance lymph flow and mobilize intestinal lipid.

Lewy Body Disease Primate Model with α-Synuclein Propagation from the Olfactory Bulb.

BACKGROUND: Lewy body diseases (LBDs), which are pathologically defined as the presence of intraneuronal α-synuclein (α-Syn) inclusions called Lewy bodies, encompass Parkinson's disease, Parkinson's disease with dementia, and dementia with Lewy bodies. Autopsy studies have shown that the olfactory bulb (OB) is one of the regions where Lewy pathology develops and initiates its spread in the brain. OBJECTIVE: This study aims to clarify how Lewy pathology spreads from the OB and affects brain functions using nonhuman primates. METHODS: We inoculated α-Syn preformed fibrils into the unilateral OBs of common marmosets (Callithrix jacchus) and performed pathological analyses, manganese-enhanced magnetic resonance imaging, and 18 F-fluoro-2-deoxy-d-glucose positron emission tomography up to 6 months postinoculation. RESULTS: Severe α-Syn pathology was observed within the olfactory pathway and limbic system, while mild α-Syn pathology was seen in a wide range of brain regions, including the substantia nigra pars compacta, locus coeruleus, and even dorsal motor nucleus of the vagus nerve. The brain imaging analyses showed reduction in volume of the OB and progressive glucose hypometabolism in widespread brain regions, including the occipital lobe, and extended beyond the pathologically affected regions. CONCLUSIONS: We generated a novel nonhuman primate LBD model with α-Syn propagation from the OB. This model suggests that α-Syn propagation from the OB is related to OB atrophy and cerebral glucose hypometabolism in LBDs. © 2022 International Parkinson and Movement Disorder Society.

An Integrated Proteomic and Glycoproteomic Investigation Reveals Alterations in the N-Glycoproteomic Network Induced by 2-Deoxy-D-Glucose in Colorectal Cancer Cells.

As a well-known glycolysis inhibitor for anticancer treatment, 2-Deoxy-D-glucose (2DG) inhibits the growth and survival of cancer cells by interfering with the ATP produced by the metabolism of D-glucose. In addition, 2DG inhibits protein glycosylation in vivo by competing with D-mannose, leading to endoplasmic reticulum (ER) stress and unfolded protein responses in cancer cells. However, the molecular details underlying the impact of 2DG on protein glycosylation remain largely elusive. With an integrated approach to glycoproteomics and proteomics, we characterized the 2DG-induced alterations in N-glycosylation, as well as the cascading impacts on the whole proteome using the HT29 colorectal cancer cell line as a model system. More than 1700 site-specific glycoforms, represented by unique intact glycopeptides (IGPs), were identified. The treatment of 2DG had a broad effect on the N-glycoproteome, especially the high-mannose types. The glycosite occupancy of the high-mannose N-glycans decreased the most compared with the sialic acid and fucose-containing N-glycans. Many of the proteins with down-regulated high-mannose were implicated in functional networks related to response to topologically incorrect protein, integrin-mediated signaling, lysosomal transport, protein hydroxylation, vacuole, and protein N-glycosylation. The treatment of 2DG also functionally disrupted the global cellular proteome, evidenced by significant up-regulation of the proteins implicated in protein folding, endoplasmic reticulum, mitochondrial function, cellular respiration, oxidative phosphorylation, and translational termination. Taken together, these findings reveal the complex changes in protein glycosylation and expression underlying the various effects of 2DG on cancer cells, and may provide insightful clues to inform therapeutic development targeting protein glycosylation.

Colchicine treatment impairs skeletal muscle mitochondrial function and insulin sensitivity in an age-specific manner.

The aim of the study was to investigate the impact of autophagy inhibition on skeletal muscle mitochondrial function and glucose homeostasis in young and aged mice. The transcriptional co-activator PGC-1α regulates muscle oxidative phenotype which has been shown to be linked with basal autophagic capacity. Therefore, young and aged inducible muscle-specific PGC-1α knockout (iMKO) mice and littermate lox/lox controls were used in three separate experiments performed after either saline or colchicine injections on two consecutive days: (1) Euthanization in the basal state obtaining skeletal muscle for mitochondrial respirometry, (2) whole body glucose tolerance test, and (3) in vivo insulin-stimulated 2-deoxyglucose (2-DG) uptake into skeletal muscle. Muscle PGC-1α was not required for maintaining basal autophagy flux, regardless of age. Colchicine-induced inhibition of autophagy was associated with impairments of skeletal muscle mitochondrial function, including reduced ADP sensitivity and altered mitochondrial redox balance in both young and aged mice. Colchicine treatment reduced the glucose tolerance in aged, but not young mice, and similarly in iMKO and lox/lox mice. Colchicine reduced insulin-stimulated 2-DG uptake in soleus muscle in aged mice, independently of PGC-1α, and without affecting insulin-regulated phosphorylation of proximal or distal mediators of insulin signaling. In conclusion, the results indicate that autophagy regulates the mitochondrial ADP sensitivity and redox balance as well as whole body glucose tolerance and skeletal muscle insulin sensitivity in aged mice, with no additional effects of inducible PGC-1α deletion.

Experimental and Computational Studies on Structure and Energetic Properties of Halogen Derivatives of 2-Deoxy-D-Glucose.

The results of structural studies on a series of halogen-substituted derivatives of 2-deoxy-D-glucose (2-DG) are reported. 2-DG is an inhibitor of glycolysis, a metabolic pathway crucial for cancer cell proliferation and viral replication in host cells, and interferes with D-glucose and D-mannose metabolism. Thus, 2-DG and its derivatives are considered as potential anticancer and antiviral drugs. X-ray crystallography shows that a halogen atom present at the C2 position in the pyranose ring does not significantly affect its conformation. However, it has a noticeable effect on the crystal structure. Fluorine derivatives exist as a dense 3D framework isostructural with the parent compound, while Cl- and I-derivatives form layered structures. Analysis of the Hirshfeld surface shows formation of hydrogen bonds involving the halogen, yet no indication for the existence of halogen bonds. Density functional theory (DFT) periodic calculations of cohesive and interaction energies (at the B3LYP level of theory) have supported these findings. NMR studies in the solution show that most of the compounds do not display significant differences in their anomeric equilibria, and that pyranose ring puckering is similar to the crystalline state. For 2-deoxy-2-fluoro-D-glucose (2-FG), electrostatic interaction energies between the ligand and protein for several existing structures of pyranose 2-oxidase were also computed. These interactions mostly involve acidic residues of the protein; single amino-acid substitutions have only a minor impact on binding. These studies provide a better understanding of the structural chemistry of halogen-substituted carbohydrates as well as their intermolecular interactions with proteins determining their distinct biological activity.

Neuregulin 4 Downregulation Induces Insulin Resistance in 3T3-L1 Adipocytes through Inflammation and Autophagic Degradation of GLUT4 Vesicles.

The adipokine Neuregulin 4 (Nrg4) protects against obesity-induced insulin resistance. Here, we analyze how the downregulation of Nrg4 influences insulin action and the underlying mechanisms in adipocytes. Validated shRNA lentiviral vectors were used to generate scramble (Scr) and Nrg4 knockdown (KD) 3T3-L1 adipocytes. Adipogenesis was unaffected in Nrg4 KD adipocytes, but there was a complete impairment of the insulin-induced 2-deoxyglucose uptake, which was likely the result of reduced insulin receptor and Glut4 protein. Downregulation of Nrg4 enhanced the expression of proinflammatory cytokines. Anti-inflammatory agents recovered the insulin receptor, but not Glut4, content. Proteins enriched in Glut4 storage vesicles such as the insulin-responsive aminopeptidase (IRAP) and Syntaxin-6 as well as TBC1D4, a protein involved in the intracellular retention of Glut4 vesicles, also decreased by Nrg4 KD. Insulin failed to reduce autophagy in Nrg4 KD adipocytes, observed by a minor effect on mTOR phosphorylation, at the time that proteins involved in autophagy such as LC3-II, Rab11, and Clathrin were markedly upregulated. The lysosomal activity inhibitor bafilomycin A1 restored Glut4, IRAP, Syntaxin-6, and TBC1D4 content to those found in control adipocytes. Our study reveals that Nrg4 preserves the insulin responsiveness by preventing inflammation and, in turn, benefits the insulin regulation of autophagy.

Reduction of 3-Deoxyglucosone by Epigallocatechin Gallate Results Partially from an Addition Reaction: The Possible Mechanism of Decreased 5-Hydroxymethylfurfural in Epigallocatechin Gallate-Treated Black Garlic.

5-Hydroxymethylfurfural (5-HMF) is a harmful substance generated during the processing of black garlic. Our previous research demonstrated that impregnation of black garlic with epigallocatechin gallate (EGCG) could reduce the formation of 5-HMF. However, there is still a lack of relevant research on the mechanism and structural identification of EGCG inhibiting the production of 5-HMF. In this study, an intermediate product of 5-HMF, 3-deoxyglucosone (3-DG), was found to be decreased in black garlic during the aging process, and impregnation with EGCG for 24 h further reduced the formation of 3-DG by approximately 60% in black garlic compared with that in the untreated control. The aging-mimicking reaction system of 3-DG + EGCG was employed to determine whether the reduction of 3-DG was the underlying mechanism of decreased 5-HMF formation in EGCG-treated black garlic. The results showed that EGCG accelerated the decrease of 3-DG and further attenuated 5-HMF formation, which may be caused by an additional reaction with 3-DG, as evidenced by LC-MS/MS analysis. In conclusion, this study provides new insights regarding the role of EGCG in blocking 5-HMF formation.

Novel Facet of an Old Dietary Molecule? Direct Influence of Caffeine on Glucose and Biogenic Amine Handling by Human Adipocytes.

Caffeine is a plant alkaloid present in food and beverages consumed worldwide. It has high lipid solubility with recognized actions in the central nervous system and in peripheral tissues, notably the adipose depots. However, the literature is scant regarding caffeine's influence on adipocyte functions other than lipolysis, such as glucose incorporation into lipids (lipogenesis) and amine oxidation. The objective of this study was to explore the direct effects of caffeine and of isobutylmethylxanthine (IBMX) on these adipocyte functions. Glucose transport into fat cells freshly isolated from mice, rats, or humans was monitored by determining [3H]-2-deoxyglucose (2-DG) uptake, while the incorporation of radiolabeled glucose into cell lipids was used as an index of lipogenic activity. Oxidation of benzylamine by primary amine oxidase (PrAO) was inhibited by increasing doses of caffeine in human adipose tissue preparations with an inhibition constant (Ki) in the millimolar range. Caffeine inhibited basal and insulin-stimulated glucose transport as well as lipogenesis in rodent adipose cells. The antilipogenic action of caffeine was also observed in adipocytes from mice genetically invalidated for PrAO activity, indicating that PrAO activity was not required for lipogenesis inhibition. These caffeine inhibitory properties were extended to human adipocytes: relative to basal 2-DG uptake, set at 1.0 ± 0.2 for 6 individuals, 0.1 mM caffeine tended to reduce uptake to 0.83 ± 0.08. Insulin increased uptake by 3.86 ± 1.11 fold when tested alone at 100 nM, and by 3.21 ± 0.80 when combined with caffeine. Our results reinforce the recommendation of caffeine's potential in the treatment or prevention of obesity complications.

KD025 Shifts Pulmonary Endothelial Cell Bioenergetics and Decreases Baseline Lung Permeability.

KD025 is a ROCK2 inhibitor currently being tested in clinical trials for the treatment of fibrotic lung diseases. The therapeutic effects of KD025 are partly due to its inhibition of profibrotic pathways and fat metabolism. However, whether KD025 affects pulmonary microvascular endothelial cell (PMVEC) function is unknown, despite evidence that alveolar-capillary membrane disruption constitutes major causes of death in fibrotic lung diseases. We hypothesized that KD025 regulates PMVEC metabolism, pH, migration, and survival, a series of interrelated functional characteristics that determine pulmonary barrier integrity. We used PMVECs isolated from Sprague Dawley rats. KD025 dose-dependently decreased lactate production and glucose consumption. The inhibitory effect of KD025 was more potent compared with other metabolic modifiers, including 2-deoxy-glucose, extracellular acidosis, dichloroacetate, and remogliflozin. Interestingly, KD025 increased oxidative phosphorylation, whereas 2-deoxy-glucose did not. KD025 also decreased intracellular pH and induced a compensatory increase in anion exchanger 2. KD025 inhibited PMVEC migration, but fasudil (nonspecific ROCK inhibitor) did not. We tested endothelial permeability in vivo using Evans Blue dye in the bleomycin pulmonary fibrosis model. Baseline permeability was decreased in KD025-treated animals independent of bleomycin treatment. Under hypoxia, KD025 increased PMVEC necrosis as indicated by increased lactate dehydrogenase release and propidium iodide uptake and decreased ATP; it did not affect Annexin V binding. ROCK2 knockdown had no effect on PMVEC metabolism, pH, and migration, but it increased nonapoptotic caspase-3 activity. Together, we report that KD025 promotes oxidative phosphorylation; decreases glycolysis, intracellular pH, and migration; and strengthens pulmonary barrier integrity in a ROCK2-independent manner.

Cooperative effects of galanin and leptin on alleviation of insulin resistance in adipose tissue of diabetic rats.

It was reported that either orexigenic neuropeptide galanin or anorexigenic hormone leptin caught benefit insulin sensitivity through increasing the translocation of glucose transporter 4 (GLUT4) in patients with diabetes. To date, it is unknown whether galanin can potentiate the effect of leptin on alleviation of insulin resistance. Therefore, in the current study we sought to assess the combined effect of central leptin and galanin on insulin resistance in the adipose tissues of type 2 diabetic rats. Galanin and leptin were injected into the intracerebroventricle of the diabetic rats, respectively, or cooperatively once a day for 2 weeks. Then, several indexes of insulin resistance were examined. The results showed that glucose infusion rates in the hyperinsulinaemic-euglycaemic clamp test, plasma adiponectin content and GLUT4 translocation, as well as Akt phosphorylation in fat cells, were higher, not GLUT4 protein and GLUT4 mRNA expression, but HOMA index was lower in the galanin + leptin group than either one of them. Furthermore, treatment with MK-2206, an Akt inhibitor, blocked the combined effects of galanin + leptin on alleviation of insulin resistance. These results suggest that galanin can improve the leptin-induced mitigative effects on insulin resistance in the fat cells, and those provided new insights into the potential tactics for prevention and remedy of insulin resistance.

Impacts of rat hindlimb Fndc5/irisin overexpression on muscle and adipose tissue metabolism.

Myokines, such as irisin, have been purported to exert physiological effects on skeletal muscle in an autocrine/paracrine fashion. In this study, we aimed to investigate the mechanistic role of in vivo fibronectin type III domain-containing 5 (Fndc5)/irisin upregulation in muscle. Overexpression (OE) of Fndc5 in rat hindlimb muscle was achieved by in vivo electrotransfer, i.e., bilateral injections of Fndc5 harboring vectors for OE rats (n = 8) and empty vector for control rats (n = 8). Seven days later, a bolus of D2O (7.2 mL/kg) was administered via oral gavage to quantify muscle protein synthesis. After an overnight fast, on day 9, 2-deoxy-d-glucose-6-phosphate (2-DG6P; 6 mg/kg) was provided during an intraperitoneal glucose tolerance test (2 g/kg) to assess glucose handling. Animals were euthanized, musculus tibialis cranialis muscles and subcutaneous fat (inguinal) were harvested, and metabolic and molecular effects were evaluated. Muscle Fndc5 mRNA increased with OE (~2-fold; P = 0.014), leading to increased circulating irisin (1.5 ± 0.9 to 3.5 ± 1.2 ng/mL; P = 0.049). OE had no effect on protein anabolism or mitochondrial biogenesis; however, muscle glycogen was increased, along with glycogen synthase 1 gene expression (P = 0.04 and 0.02, respectively). In addition to an increase in glycogen synthase activation in OE (P = 0.03), there was a tendency toward increased glucose transporter 4 protein (P = 0.09). However, glucose uptake (accumulation of 2-DG6P) was identical. Irisin elicited no endocrine effect on mitochondrial biogenesis or uncoupling proteins in white adipose tissue. Hindlimb overexpression led to physiological increases in Fndc5/irisin. However, our data indicate limited short-term impacts of irisin in relation to muscle anabolism, mitochondrial biogenesis, glucose uptake, or adipose remodeling.

Nucleotide polymorphism assay for the identification of west African group Bacillus anthracis: a lineage lacking anthrose.

BACKGROUND: The exosporium of the anthrax-causing Bacillus anthracis endospores display a tetrasaccharide composed of three rhamnose residues and an unusual sugar termed anthrose. Anthrose is a proposed potential target for immunotherapy and for specific detection of B. anthracis. Although originally thought to be ubiquitous in B. anthracis, previous work identified an anthrose negative strain from a West African lineage isolated from cattle that could represent a vaccine escape mutant. These strains carry genes required for expression of the anthrose operon but premature stop codons resulting from an 8-bp insertion in BAS3320 (an amino-transferase) and a C/T substitution at position 892 of the BAS3321 (a glycosyltransferase) gene prevent anthrose expression. Various other single nucleotide polymorphisms (SNPs) have been identified throughout the operon and could be the basis for detection of anthrose-deficient strains. RESULTS: In this study, we evaluated rhAmp genotypic assays based on SNPs at positions 892 and 1352 of BAS3321 for detection and differentiation of anthrose negative (Ant-) West African strains. Discrimination of anthrose negative West African isolates was achieved with as low as 100 fg of DNA, whereas consistent genotyping of Sterne necessitated at least 1 pg of DNA. CONCLUSIONS: Screening of a global panel of B. anthracis isolates showed anthrose-expressing alleles are prevalent worldwide whereas the anthrose-deficient phenotype is to date limited to West Africa. Our work also revealed a third, previously unreported anthrose genotype in which the operon is altogether missing from a Polish B. anthracis isolate.

Leucine and mTORc1 act independently to regulate 2-deoxyglucose uptake in L6 myotubes.

Chronic mTORc1 hyperactivation via obesity-induced hyperleucinaemia has been implicated in the development of insulin resistance, yet the direct impact of leucine on insulin-stimulated glucose uptake in muscle cells remains unclear. To address this, differentiated L6 myotubes were subjected to various compounds designed to either inhibit mTORc1 activity (rapamycin), blunt leucine intracellular import (BCH), or activate mTORc1 signalling (3BDO), prior to the determination of the uptake of the glucose analogue, 2-deoxyglucose (2-DG), in response to 1 mM insulin. In separate experiments, L6 myotubes were subject to various media concentrations of leucine (0-0.8 mM) for 24 h before 2-DG uptake in response to insulin was assessed. Both rapamycin and BCH blunted 2-DG uptake, irrespective of insulin administration, and this occurred in parallel with a decline in mTOR, 4E-BP1, and p70S6K phosphorylation status, but little effect on AKT phosphorylation. In contrast, reducing leucine media concentrations suppressed 2-DG uptake, both under insulin- and non-insulin-stimulated conditions, but did not alter the phosphorylation state of AKT-mTORc1 components examined. Unexpectedly, 3BDO failed to stimulate mTORc1 signalling, but, nonetheless, caused a significant increase in 2-DG uptake under non-insulin-stimulated conditions. Both leucine and mTORc1 influence glucose uptake in muscle cells independent of insulin administration, and this likely occurs via distinct but overlapping mechanisms.

Hypothesis: A Novel Neuroprotective Role for Glucose-6-phosphatase (G6PC3) in Brain-To Maintain Energy-Dependent Functions Including Cognitive Processes.

The isoform of glucose-6-phosphatase in liver, G6PC1, has a major role in whole-body glucose homeostasis, whereas G6PC3 is widely distributed among organs but has poorly-understood functions. A recent, elegant analysis of neutrophil dysfunction in G6PC3-deficient patients revealed G6PC3 is a neutrophil metabolite repair enzyme that hydrolyzes 1,5-anhydroglucitol-6-phosphate, a toxic metabolite derived from a glucose analog present in food. These patients exhibit a spectrum of phenotypic characteristics and some have learning disabilities, revealing a potential linkage between cognitive processes and G6PC3 activity. Previously-debated and discounted functions for brain G6PC3 include causing an ATP-consuming futile cycle that interferes with metabolic brain imaging assays and a nutritional role involving astrocyte-neuron glucose-lactate trafficking. Detailed analysis of the anhydroglucitol literature reveals that it competes with glucose for transport into brain, is present in human cerebrospinal fluid, and is phosphorylated by hexokinase. Anhydroglucitol-6-phosphate is present in rodent brain and other organs where its accumulation can inhibit hexokinase by competition with ATP. Calculated hexokinase inhibition indicates that energetics of brain and erythrocytes would be more adversely affected by anhydroglucitol-6-phosphate accumulation than heart. These findings strongly support the paradigm-shifting hypothesis that brain G6PC3 removes a toxic metabolite, thereby maintaining brain glucose metabolism- and ATP-dependent functions, including cognitive processes.