Glucose Metabolites Research Articles

Grape pomace polyphenols improve insulin response to a standard meal in healthy individuals: A pilot study

Dietary polyphenols have beneficial effects on glucose/lipid metabolism in subjects at high risk to develop type 2 diabetes; however, the underlying mechanisms are not clear. We aimed to evaluate: 1) the acute effects of the consumption of a drink rich in polyphenols from red grape pomace (RGPD) on glucose/insulin and triglyceride responses to a standard meal in healthy individuals, and, 2) the relationship between plasma levels of phenolic metabolites and metabolic parameters. Twelve healthy men, aged 20-40 years participated in a randomized, controlled study according to a cross-over design. After a 3-day low-polyphenol diet, all participants consumed, on two different days and separated by a one week interval, after an overnight fast, a drink rich in polyphenols (1.562g gallic acid equivalents (GAE)) or a control drink (CD, no polyphenols), followed after 3h by a standard meal (960kcal, 18% protein, 30% fat, 52% CHO). Blood samples were taken at fasting, 3h after the drink, over 5h after the standard meal and at fasting on the next day to measure plasma concentrations of glucose, insulin, triglyceride and phenolic metabolites. Glycemic and triglyceride post-meal responses were similar after both the RGPD and the control drink. In contrast, postprandial insulin incremental area (iAUC0-5h) was 31% lower (p<0.05), insulin secretion index was 18% lower (p<0.016) and insulin sensitivity (SI) index was 36% higher (p=0.037) after the RGPD compared to CD. Among phenolic metabolites, gallic acid correlated inversely with the insulin response (r=-0.604; p=0.032) and positively with the SI index (r=0.588, p=0.037). RGPD consumption acutely reduced postprandial insulin levels and improved insulin sensitivity. This effect could be likely related to the increase in gallic acid levels. This drink, added to usual diet, could contribute to increase the daily intake of polyphenols, with potential health benefits. CLINICALTRIALS. NCT02865278.

Fructose acutely stimulates NKCC2 activity in rat thick ascending limbs by increasing surface NKCC2 expression.

The thick ascending limb (TAL) reabsorbs 25% of the filtered NaCl through the Na+-K+-2Cl- cotransporter (NKCC2). NKCC2 activity is directly related to surface NKCC2 expression and phosphorylation. Higher NaCl reabsorption by TALs is linked to salt-sensitive hypertension, which is linked to consumption of fructose in the diet. However, little is known about the effects of fructose on renal NaCl reabsorption. We hypothesized that fructose, but not glucose, acutely enhances TAL-dependent NaCl reabsorption by increasing NKCC2 activity via stimulation of surface NKCC2 levels and phosphorylation at Thr96/101. We found that fructose (5 mM) increased transport-related O2 consumption in TALs by 11.1 ± 3.2% ( P < 0.05). The effect of fructose on O2 consumption was blocked by furosemide. To study the effect of fructose on NKCC2 activity, we measured the initial rate of NKCC2-dependent thallium influx. We found that 20 min of treatment with fructose (5 mM) increased NKCC2 activity by 58.5 ± 16.9% ( P < 0.05). We then used surface biotinylation to measure surface NKCC2 levels in rat TALs. Fructose increased surface NKCC2 expression in a concentration-dependent manner (22 ± 5, 49 ± 10, and 101 ± 59% of baseline with 1, 5, and 10 mM fructose, respectively, P < 0.05), whereas glucose or a glucose metabolite did not. Fructose did not change NKCC2 phosphorylation at Thre96/101 or total NKCC2 expression. We concluded that acute fructose treatment increases NKCC2 activity by enhancing surface NKCC2 expression, rather than NKCC2 phosphorylation. Our data suggest that fructose consumption could contribute to salt-sensitive hypertension by stimulating NKCC2-dependent NaCl reabsorption in TALs.

The Metabolic Enzyme Hexokinase 2 Localizes to the Nucleus in AML and Normal Hematopoietic Stem/Progenitor Cells to Maintain Stemness

Hematopoietic cells are arranged in a hierarchy where stem and progenitor cells differentiate into mature blood cells. Likewise, AML (Acute Myeloid Leukemia) is also hierarchical with leukemic stem and progenitor cells giving rise to more mature and differentiated blasts. Recent studies have shown that mitochondrial enzymes such as IDH2 can regulate AML stemness by altering metabolites that affect epigenetic marks. However, it is unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stemness in AML and normal hematopoietic cells. Here, we show that the mitochondrial enzyme, Hexokinase 2, localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stemness.We sought to identify mitochondrial metabolic enzymes that localize to the nucleus of stem cells, by evaluating the stem and bulk fractions from 8227 leukemia cells. 8227 leukemia cells are arranged in a hierarchy with functionally defined stem cells present in the CD34+CD38- fraction. We separated 8227 cells into CD34+CD38- and CD34-CD38+ populations by FACS sorting and prepared lysates of the nuclear and cytoplasmic fractions from each population. Using immunoblotting, we measured levels of mitochondrial enzymes in the subcellular fractions of each population. We discovered that the metabolic enzyme Hexokinase 2 (HK2) was increased in the nuclear fraction of 8227 stem cells compared to bulk cells. In contrast, other mitochondrial enzymes such as Aconitase 2 and Succinate Dehydrogenase B were not detected in the nuclear fractions. HK2 is an outer mitochondrial membrane protein that phosphorylates glucose to glucose-6-phosphate, thereby initiating glycolysis and the entry of glucose metabolites into the TCA cycle in the mitochondria. The nuclear localization of HK2 in mammalian cells has not been previous reported. We confirmed that 8227 cells have nuclear HK2 by confocal fluorescent microscopy and also demonstrated nuclear HK2 in AML cell lines (OCI-AML2, NB4, K562, and MV411) and primary AML samples.We also FACS sorted normal cord blood into populations of stem/progenitor (HSC, MPP, MLP, CMP, GMP and MEP) and differentiated (B cells, T cells, NK cells, Monocytes and Granulocytes) cells. The localization of HK2 in these cell fractions was measured by immunofluorescence and quantified by Metamorph and Imaris. Nuclear HK2 was detected in the stem/progenitor cells and progressively declined to minimal levels as the cells matured (Fig 1A).The mitochondrial localization of HK2 is dependent on AKT-mediated phosphorylation of Thr-473 and inhibited by dephosphorylation by the phosphatase PHLPP1. We asked whether phosphorylation of HK2 regulates the nuclear abundance of HK2. Using AML2 cells, we showed that knockdown of PHLPP1 decreased the abundance of nuclear HK2, while inhibition of AKT increased HK2 in the nucleus.Finally, we tested whether the nuclear localization of HK2 was functionally important to maintain stemness. We over-expressed HK2 tagged with nuclear localizing signals (PKKKRKV and PAAKRVKLD) in 8227 and NB4 leukemia cells. We confirmed the selective over-expression of HK2 in the nucleus of these cells by immunoblotting and immunofluorescence. Increasing nuclear HK2 did not alter the proliferation of the cells under basal conditions. However, increasing nuclear HK2 enhanced clonogenic growth and blocked retinoic acid-mediated cell differentiation.In summary, we discovered that the unphosphorylated form of the metabolic enzyme HK2 localizes to the nucleus in malignant and normal hematopoietic stem cells and is functionally important to maintain stem/progenitor state. Thus, we define a new role for mitochondrial enzymes in the regulation of stemness and differentiation. [Display omitted] DisclosuresSchimmer:Medivir AB: Research Funding; Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka Pharmaceuticals: Consultancy.

Extracellular glucose levels in cultures of undifferentiated mouse trophoblast stem cells affect gene expression during subsequent differentiation with replicable cell line-dependent variation.

Mouse trophoblast stem cells (TSCs) have been established and maintained using hyperglycemic conditions (11 mM glucose) for no apparent good reason. Because glucose metabolites are used as resources for cellular energy production, biosynthesis, and epigenetic modifications, differences in extracellular glucose levels may widely affect cellular function. Since the hyperglycemic culture conditions used for TSC culture have not been fully validated, the effect of extracellular glucose levels on the properties of TSCs remains unclear. To address this issue, we investigated the gene expression of stemness-related transcription factors in TSCs cultured in the undifferentiated state under various glucose concentrations. We also examined the expression of trophoblast subtype markers during differentiation, after returning the glucose concentration to the conventional culture concentration (11 mM). As a result, it appeared that the extracellular glucose conditions in the stem state not only affected the gene expression of stemness-related transcription factors before differentiation but also affected the expression of marker genes after differentiation, with some line-to-line variation. In the TS4 cell line, which showed the largest glucose concentration-dependent fluctuations in gene expression among all the lines examined, low glucose (1 mM glucose, LG) augmented H3K27me3 levels. An Ezh2 inhibitor prevented these LG-induced changes in gene expression, suggesting the possible involvement of H3K27me3 in the changes in gene expression seen in LG. These results collectively indicate that the response of the TSCs to the change in the extracellular glucose concentration is cell line-dependent and a part of which may be epigenetically memorized.

A dual-signal colorimetric and near-infrared fluorescence probe for the detection of exogenous and endogenous hydrogen peroxide in living cells

As a classic kind of reactive oxygen species (ROS), hydrogen peroxide (H2O2) plays a critical role in physiological and pathological processes. It is of significant importance to detect H2O2 with sufficient sensitivity, selectivity and universality in biological field. Herein a novel near-infrared probe (DCM-B) containing dicyanomethylene-4H-pyran as fluorophore and boronate ester group as the recognition unit was developed for the detection of H2O2. Experiments showed that the remarkable fluorescence enhancement and noticeable color change from yellow to purple were observed while H2O2 was added to DCM-B solution. It is proved that the boronate ester group of DCM-B cleavage and fluorescence off-on response based on intramolecular charge transfer (ICT) mechanism were obtained when the probe reacted with H2O2, and DCM-B showed excellent sensitivity, high selectivity to H2O2 detection in the presence of many competing analytes. Encouraged by the optical properties of near-infrared emission, it was successfully applied to the imaging of exogenous and endogenous hydrogen peroxide in living cells. More importantly, considering that the final metabolite of choline and glucose is H2O2 in living cells and organism, we expanded DCM-B to the indirect detection of choline and glucose with satisfactory performance.

Hyperglycaemia-induced methylglyoxal accumulation potentiates VEGF resistance of diabetic monocytes through the aberrant activation of tyrosine phosphatase SHP-2/SRC kinase signalling axis

Diabetes mellitus (DM) is a major cardiovascular risk factor contributing to cardiovascular complications by inducing vascular cell dysfunction. Monocyte dysfunction could contribute to impaired arteriogenesis response in DM patients. DM monocytes show blunted chemotactic responses to arteriogenic stimuli, a condition termed as vascular endothelial growth factor (VEGF) resistance. We hypothesize that methylglyoxal (MG), a glucose metabolite, induces monocyte dysfunction and aimed to elucidate the underlying molecular mechanisms. Human monocytes exposed to MG or monocytes from DM patients or mice (db/db) showed VEGF-resistance secondary to a pro-migratory phenotype. Mechanistically, DM conditions or MG exposure resulted in the upregulation of the expression of SHP-2 phosphatase. This led to the enhanced activity of SHP-2 and aided an interaction with SRC kinase. SHP-2 dephosphorylated the inhibitory phosphorylation site of SRC leading to its abnormal activation and phosphorylation of cytoskeletal protein, paxillin. We demonstrated that MG-induced molecular changes could be reversed by pharmacological inhibitors of SHP-2 and SRC and by genetic depletion of SHP-2. Finally, a SHP-2 inhibitor completely reversed the dysfunction of monocytes isolated from DM patients and db/db mice. In conclusion, we identified SHP-2 as a hitherto unknown target for improving monocyte function in diabetes. This opens novel perspectives for treating diabetic complications associated with impaired monocyte function.

Cytidine monophosphate N-acetylneuraminic acid synthetase enhances invasion of human triple-negative breast cancer cells.

BackgroundCancer cells have altered bioenergetics, which contributes to their ability to proliferate, survive in unusual microenvironments, and invade other tissues. Changes in glucose metabolism can have pleomorphic effects on tumor cells.MethodsTo investigate potential mechanisms responsible for the increased malignancy associated with altered glucose metabolism, we used an unbiased nuclear magnetic resonance spectroscopy screening method to identify glucose metabolites differentially produced in a highly malignant human triple-negative breast cancer (TNBC) cell line (BPLER) and a less malignant isogenic TNBC cell line (HMLER).ResultsN-acetylneuraminic acid (Neu5Ac), the predominant sialic acid derivative in mammalian cells, which forms the terminal sugar on mucinous cell surface glycoproteins, was the major glucose metabolite that differed. Neu5Ac was ~7-fold more abundant in BPLER than HMLER. Loss of Neu5Ac by enzymatic removal or siRNA knockdown of cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS), which activates cellular sialic acids for glycoprotein conjugation, had no significant effect on cell proliferation, but decreased the ability of BPLER to invade through a basement membrane. Conversely, overexpressing CMAS in HMLER increased invasivity. TNBCs in The Cancer Genome Atlas also had significantly more CMAS copy number variations and higher mRNA expression than non-TNBC, which have a better prognosis. CMAS knockdown in BPLER ex vivo blocked xenograft formation in mice.ConclusionNeu5Ac is selectively highly enriched in aggressive TNBC, and CMAS, the enzyme required for sialylation, may play an important role in TNBC tumor formation and invasivity.

Steroid Receptor Coactivator-2 Controls the Pentose Phosphate Pathway through RPIA in Human Endometrial Cancer Cells

Steroid receptor coactivator-2 (SRC-2) is a transcriptional coregulator that modulates the activity of many transcription factors. Levels of SRC-2 are elevated in endometrial biopsies from polycystic ovary syndrome patients, a population predisposed to endometrial cancer (EC). Increased expression of SRC-2 is also detected in neoplastic endometrium suggesting a causal link between elevated SRC-2 expression and the emergence of endometrial disorders that can lead to cancer. Here, we reveal that SRC-2 knockdown reduces EC cell proliferation and anchorage-independence. Additionally, SRC-2 is required to maintain cellular glycolytic capacity and oxidative phosphorylation, processes essential for EC cell proliferation. Importantly, SRC-2 is critical for the normal performance of the pentose phosphate pathway (PPP). Perturbation of the PPP due to loss of SRC-2 expression may result from the depletion of ribose-5-P isomerase (RPIA), a key enzyme of the PPP. As with SRC-2, RPIA knockdown reduces EC cell proliferation, which is accompanied by a decrease in glycolytic capacity and oxidative phosphorylation. Glucose metabolite tracking experiments confirmed that knockdown of SRC-2 and RPIA downregulates the metabolic rate of both glycolysis and the PPP, highlighting a novel regulatory cross-talk between glycolysis and the PPP modulated by SRC-2.

Effect of sex on glucose handling by adipocytes isolated from rat subcutaneous, mesenteric and perigonadal adipose tissue.

BackgroundAdult rat epididymal adipocytes are able to convert large amounts of glucose to lactate and glycerol. However, fatty acid efflux is much lower than that expected from glycerol levels if they were the product of lipolysis. Use of glucose for lipogenesis is limited, in contrast with the active glycolysis-derived lactate (and other 3-carbon substrates). In this study, we analyzed whether white adipose tissue (WAT) site and sex affect these processes.MethodsMature adipocytes from perigonadal, mesenteric and subcutaneous WAT of female and male rats were isolated, and incubated with 7 or 14 mM glucose during 1 or 2 days. Glucose consumption, metabolite efflux and gene expression of glycolytic and lipogenesis-related genes were measured.ResultsThe effects of medium initial glucose concentration were minimal on most parameters studied. Sex-induced differences that were more extensive; however, the most marked, distinct, effects between WAT sites, were dependent on the time of incubation. In general, the production of lactate was maintained during the incubation, but glycerol release rates increased with time, shifting from a largely glycolytic origin to its triacylglycerol (TAG) lipolytic release. Glycerol incorporation was concurrent with increased TAG turnover: lipolytic glycerol was selectively secreted, while most fatty acids were recycled again into TAG. Fatty acid efflux increased with incubation, but was, nevertheless, minimal compared with that of glycerol. Production of lactate and glycerol from glucose were maximal in mesenteric WAT.DiscussionFemale rats showed a higher adipocyte metabolic activity than males. In mesenteric WAT, gene expression (and substrate efflux) data suggested that adipocyte oxidation of pyruvate to acetyl-CoA was higher in females than in males, with enhanced return of oxaloacetate to the cytoplasm for its final conversion to lactate. WAT site differences showed marked tissue specialization-related differences. Use of glucose for lipogenesis was seriously hampered over time, when TAG turnover-related lipolysis was activated. We postulate that these mechanisms may help decrease glycaemia and fat storage, producing, instead, a higher availability of less-regulated 3-carbon substrates, used for energy elsewhere.

CT quantitative analysis study for angiogenesis, and degree of ischemic necrosis and glucose metabolite in non-small cell lung cancer.

We aimed at exploring the feasibility of noninvasive late arterial phase enhanced CT imaging in evaluating tumor angiogenesis, ischemic necrosis, and glucose metabolism, thereby providing pathological information for the comprehensive treatment plan in non-small cell lung cancer (NSCLC). 52 cases of NSCLC were enrolled in this study. The mean ischemia necrosis CT quantitative value (INCTQ) and CT enhanced value (CTe) of the tumor were determined, and the immunohistochemical staining of factors relating to tumor angiogenesis, ischemic necrosis and glucose metabolism, including VEGF, VEGFR-2, HIF-1α, CAIX, GLUT1, and GLUT3, were conducted. The mean INCTQ values of different expression grades of VEGF, VEGFR-2, HIF-1α, and CAIX have no significant difference, but the mean INCTQ values of different expression grades of GLUT1 or GLUT3 have significant differences (p < 0.001), respectively. However, INCTQ value has a positive correlation with CAIX expression. In addition, CTe value was positively correlated with VEGF. To sum up, late arterial phase CT enhanced images of NSCLC not only can assess the tumor angiogenesis, but also can reflect the degree of ischemic necrosis, effectively reflecting the level of glucose metabolism in tumor and tumor angiogenesis, for the comprehensive treatment program.

The Complement-1q-Like-3 Inhibits Insulin Secretion by an Adhesion G-Protein Coupled Receptor, BAI3 in Pancreatic ß Cells

Secreted proteins are important metabolic regulators and our objective was to identify proteins that are important regulators of β-cell function. Microarray data from multiple tissues including liver, adipose, gastrocnemius, islets from lean and obese mice were analyzed to identify candidate regulators. For this, we developed a data filtering approach to identify candidate secreted protein regulators. Next, cross-tissue correlation network and gene enrichment based analyses were performed to determine cellular pathways affected by each candidate secreted protein in the target tissue. By using this methodology, complement-1q-like-3 (C1ql3) was identified to affect ’secretory processes’ in islets. Preliminary data showed that the expression of C1ql3 was increased by 32-fold in islets of obese mice. Incubating islets with C1ql3 containing culture media inhibited high glucose, GLP-1 receptor agonist (exendin-4), cAMP, and potassium-induced insulin secretion in mouse islets. Interestingly, C1ql3 did not affect insulin secretion in response to low and submaximal concentrations of glucose, fatty acids, amino acids, and mitochondrial metabolites. We further observed that the expression of BAI3, a receptor for C1ql3, is elevated in islets of obese compared to lean mice. Knockdown of BAI3 in β-cells increased glucose-stimulated insulin secretion by 2-fold and soluble C1ql3-binding fragment of BAI3 completely blocked the inhibitory effects of C1ql3 on cAMP-stimulated insulin secretion. These findings report the identification of a novel pathway in C1ql3/BAI3 that antagonizes GLP-1/cAMP signaling to inhibit insulin secretion from β-cells. We propose that the activation of C1ql3/BAI3 signaling contributes to type 2 diabetes and may offer therapeutic alternatives to treat type 2 diabetes. Disclosure R. Gupta: None. M. Schaid: None. M.E. Kimple: None. J.E. Koltes: None. S. Bhatnagar: None.

Apabetalone (RVX-208) Lowers Major Adverse Cardiovascular Events (MACE) in Diabetes Mellitus Patients with CVD by Attenuating Monocyte Adhesion to Endothelial Cells

Apabetalone (RVX-208) leads to a 57% relative risk reduction of MACE in patients with diabetes mellitus (DM) and CVD. To test whether RVX-208, an inhibitor (BETi) of epigenetic readers bromodomain extra-terminal (BET) proteins lowers CVD by affecting genes mediating monocyte adhesion to endothelial cells in response to high glucose(HG) and dietary metabolite trimethyl-amine oxide (TMAO). Cultured THP-1 monocytes, HUVEC endothelial cells and primary human hepatocytes (PHH) were exposed to varying amts of glucose and TMAO. Results showed that HG (25.6 mM) induced Very Late Antigen-4 (VLA-4) mRNA, a gene mediating THP-1 adhesion by 1.3-fold and RVX-2suppressed it &amp;gt;50%. BETi blocked TMAO induction of VLA-4 mRNA by &amp;gt;50% in THP-1. In HUVECs RVX-2abrogated HG induction of E-selectin and MYD88 mRNA by 2- and 1.3-fold, respectively and lowered TMAO induction of both mRNAs by &amp;gt;50%. Microbiome action on dietary phospholipids followed by hepatic flavin mono-oxygenase-3 (FMO3) processing yields TMAO. In PHH exposed for 24 hours to RVX-208, FMO3 mRNA was lower by 40% but it also suppressed a transcriptional regulator of FMO3, farnesoid X receptor (FXR). BETi for 6 hours suppressed both FXR mRNA and protein within 6 hours by &amp;gt;80% suggesting a direct effect of BETi on the FXR gene. Furthermore, in ChiP assays BRD4, a BET protein, dissociated immediately from FXR DNA upon exposure to RVX-208. BRD4 guides a complex containing RNA pol II along active genes, its dissociation would halt transcription of the gene. In summary, Apabetalone inhibits HG and TMAO enhanced adhesion of THP-1 to HUVECs, this process mimics a step in CVD pathogenesis. RVX-2suppresses cellular adhesion genes; VLA-4 in THP-1 and both E-selectin plus MYD88 in HUVECs. BETi blocks TMAO activity and its production by inhibiting FXR expression, a regulator of FMO3 gene transcription. Rapid actions of BETi in dissociating BRD4 from FXR DNA suggests a direct effect of RVX-2on transcription of this gene. Disclosure L. Tsujikawa: None. E. Kulikowski: Employee; Self; Resverlogix Corp.. C. Calosing: None. S. Wasiak: Employee; Self; Resverlogix Corp. D. Gilham: Employee; Self; Resverlogix Corp.. Stock/Shareholder; Self; Resverlogix Corp.. C. Halliday: None. J.O. Johansson: Employee; Self; Resverlogix Corp., Artery Therapeutics, Inc. M. Sweeney: Employee; Self; Resverlogix Corp. N.C. Wong: Stock/Shareholder; Self; Resverlogix Corp..

Effect of long-term nitrite administration on browning of white adipose tissue in type 2 diabetic rats: A stereological study

IntroductionNitric oxide (NO) deficiency is associated with obesity and type 2 diabetes. Nitrite, a NO donor, is considered as a new therapeutic agent in diabetes. This study aims at determining effects of long-term nitrite administration on browning of white adipose tissue (WAT) in type 2 diabetic rats. MethodsMale rats were divided into 4 groups: Control, control + nitrite, diabetes, and diabetes + nitrite. Sodium nitrite (50 mg/L in drinking water) was administered for 3 months. Body weight was measured weekly. Fasting serum levels of glucose and nitric oxide metabolites (NOx) were measured monthly. Histological evaluations and measurement of cyclic guanosine monophosphate (cGMP) and NOx levels in adipose tissue were done at the end of the study. ResultsNitrite decreased serum glucose concentration and body weight gain in diabetic rats by 27.6% and 37.9%, respectively. In diabetic rats, nitrite increased NOx and cGMP levels in inguinal WAT by 95.7% and 33.1%, respectively. Numerical density in WAT of nitrite-treated diabetic rats was higher than in diabetic ones (995 ± 83 vs. 2513 ± 256 cell/mm3, P < 0.001); in addition, total surface area (4.84 ± 0.32 vs. 44.26 ± 9.7, mm2, P < 0.001) and volume of inguinal beige adipose tissue (7.2 ± 0.49 vs. 66.4 ± 14.51 mm3, <0.001) were higher in nitrite-treated diabetic rats compared to diabetic ones. ConclusionsFavorable effects of long-term nitrite administration in obese type 2 diabetic rats is, at least in part, due to browning of WAT and also associated with increased NOx and cGMP level in adipose tissue. These findings may have potential applications for management of diabesity.

Effects of Bacillus amyloliquefaciens and different phosphorus sources on Maize plants as revealed by NMR and GC-MS based metabolomics

Plant growth-promoting bacteria of the genus Bacillus are known to solubilize phosphates and enhance plant growth in many plant species. We explored the effects of the inoculation with a commercial isolate Bacillus amyloliquefaciens on the growth and metabolic processes of maize plants in pot soils treated with triple superphosphate, rock phosphate, and either cow- or horse-manure composts, as P-fertilizers. The metabolic profiles of maize leaves in the different treatments were determined by both Gas Chromatography–Mass Spectrometry and Nuclear Magnetic Resonance spectroscopy. Principle Components Analysis (PCA) based on data matrix from both techniques revealed a relationship between treatments and specific plant metabolites. Inoculated plants showed larger P and N contents and a more differentiated metabolome when treated with the two composts than with inorganic fertilizers. B. amyloliquefaciens in combination with composts significantly increased glucose, fructose, alanine and GABA metabolites in maize leaves, thus suggesting an improved photosynthetic activity due to enhanced P and N uptake. Both composts sustained plant growth and the phosphate solubilizing activity of B. amyloliquefaciens, while differences in P and N contents in plant leaves were attributed to the different content in compost of lignin residues and alkyl moieties, and consequent impact on microbial growth. The combination of B. amyloliquefaciens inoculation with composted organic P-fertilizers rich in available metabolic carbon appears as an efficient alternative to mineral fertilizers to enhance nutrients uptake and foster growth mechanisms in maize plants.

Safety of dietary supplementation with arginine in adult humans.

Previous studies with animals and humans have shown beneficial effects of dietary supplementation with L-arginine (Arg) on reducing white fat and improving health. At present, a long-term safe level of Arg administration to adult humans is unknown. The objective of this study was to conduct a randomized, placebo-controlled, clinical trial to evaluate the safety and tolerability of oral Arg in overweight or obese but otherwise healthy adults with a body mass index of ≥ 25kg/m2. A total of 142 subjects completed a 7-day wash-in period using a 12g Arg/day dose. All the remaining eligible 101 subjects who tolerated the wash-in dose (45 men and 56 women) were assigned randomly to ingest 0, 15 or 30g Arg (as pharmaceutical-grade Arg-HCl) per day for 90days. Arg was taken daily in at least two divided doses by mixing with a flavored beverage. At Days 0 and 90, blood pressures of study subjects were recorded,their physical examinations were performed, andtheirblood and 24-h urine samples were obtained to measure: (1) serum concentrations of amino acids, glucose, fatty acids, and related metabolites; and (2) renal, hepatic, endocrine and metabolic parameters. Our results indicate that the serum concentration of Arg in men or women increased (P < 0.05) progressively with increasing oral Arg doses from 0 to 30g/day. Dietary supplementation with 30g Arg/day reduced (P < 0.05) systolic blood pressure and serum glucose concentration in females, as well as serum concentrations of free fatty acids in both males and females. Based on physiological and biochemical variables, study subjects tolerated oral administration of 15 and 30g Arg/day without adverse events. We conclude that a long-term safe level of dietary Arg supplementation is at least 30g/day in adult humans.

Site-specific glycations of apolipoprotein A-I lead to differentiated functional effects on lipid-binding and on glucose metabolism

Prolonged hyperglycemia in poorly controlled diabetes leads to an increase in reactive glucose metabolites that covalently modify proteins by non-enzymatic glycation reactions. Apolipoprotein A-I (apoA-I) of high-density lipoprotein (HDL) is one of the proteins that becomes glycated in hyperglycemia. The impact of glycation on apoA-I protein structure and function in lipid and glucose metabolism were investigated.ApoA-I was chemically glycated by two different glucose metabolites (methylglyoxal and glycolaldehyde). Synchrotron radiation and conventional circular dichroism spectroscopy were used to study apoA-I structure and stability. The ability to bind lipids was measured by lipid-clearance assay and native gel analysis, and cholesterol efflux was measured by using lipid-laden J774 macrophages. Diet induced obese mice with established insulin resistance, L6 rat and C2C12 mouse myocytes, as well as INS-1E rat insulinoma cells, were used to determine in vivo and in vitro glucose uptake and insulin secretion.Site-specific, covalent modifications of apoA-I (lysines or arginines) led to altered protein structure, reduced lipid binding capability and a reduced ability to catalyze cholesterol efflux from macrophages, partly in a modification-specific manner. The stimulatory effects of apoA-I on the in vivo glucose clearance were negatively affected when apoA-I was modified with methylglyoxal, but not with glycolaldehyde. The in vitro data showed that both glucose uptake in muscle cells and insulin secretion from beta cells were affected. Taken together, glycation modifications impair the apoA-I protein functionality in lipid and glucose metabolism, which is expected to have implications for diabetes patients with poorly controlled blood glucose.

Metabolomic analysis and biochemical changes in the urine and serum of streptozotocin-induced normal- and obese-diabetic rats.

Diabetes mellitus (DM) is a chronic disease that can affect metabolism of glucose and other metabolites. In this study, the normal- and obese-diabetic rats were compared to understand the diabetes disorders of type 1 and 2 diabetes mellitus. This was done by evaluating their urine metabolites using proton nuclear magnetic resonance (1H NMR)-based metabolomics and comparing with controls at different time points, considering the induction periods of obesity and diabetes. The biochemical parameters of the serum were also investigated. The obese-diabetic model was developed by feeding the rats a high-fat diet and inducing diabetic conditions with a low dose of streptozotocin (STZ) (25mg/kg bw). However, the normal rats were induced by a high dose of STZ (55mg/kg bw). A partial least squares discriminant analysis (PLS-DA) model showed the biomarkers of both DM types compared to control. The synthesis and degradation of ketone bodies, tricarboxylic (TCA) cycles, and amino acid pathways were the ones most involved in the variation with the highest impact. The diabetic groups also exhibited a noticeable increase in the plasma glucose level and lipid profile disorders compared to the control. There was also an increase in the plasma cholesterol and low-density lipoprotein (LDL) levels and a decline in the high-density lipoprotein (HDL) of diabetic rats. The normal-diabetic rats exhibited the highest effect of all parameters compared to the obese-diabetic rats in the advancement of the DM period. This finding can build a platform to understand the metabolic and biochemical complications of both types of DM and can generate ideas for finding targeted drugs.

Changes in energy metabolism, and levels of stress-related hormones and electrolytes in horses after intravenous administration of romifidine and the peripheral \u03b1-2 adrenoceptor antagonist vatinoxan

BackgroundRomifidine, an α-2 adrenoceptor agonist, is a widely-used sedative in equine medicine. Besides the desired sedative and analgesic actions, α-2 adrenoceptor agonists have side effects like alterations of plasma concentrations of glucose and certain stress-related hormones and metabolites in various species. Vatinoxan (previously known as MK-467), in turn, is an antagonist of α-2 adrenoceptors. Because vatinoxan does not cross the blood brain barrier in significant amounts, it has only minor effect on sedation induced by α-2 adrenoceptor agonists. Previously, vatinoxan is shown to prevent the hyperglycaemia, increase of plasma lactate concentration and the decrease of insulin and non-esterified free fatty acids (FFAs) caused by α-2 adrenoceptor agonists in different species. The aim of our study was to investigate the effects of intravenous romifidine and vatinoxan, alone and combined, on plasma concentrations of glucose and some stress-related hormones and metabolites in horses.ResultsPlasma glucose concentration differed between all intravenous treatments: romifidine (80 μg/kg; ROM), vatinoxan (200 μg/kg; V) and the combination of these (ROM + V). Glucose concentration was the highest after ROM and the lowest after V. Serum FFA concentration was higher after V than after ROM or ROM + V. The baseline serum concentration of insulin varied widely between the individual horses. No differences were detected in serum insulin, cortisol or plasma adrenocorticotropic hormone (ACTH) concentrations between the treatments. Plasma lactate, serum triglyceride or blood sodium and chloride concentrations did not differ from baseline or between the treatments. Compared with baseline, plasma glucose concentration increased after ROM and ROM + V, serum cortisol, FFA and base excess increased after all treatments and plasma ACTH concentration increased after V. Serum insulin concentration decreased after V and blood potassium decreased after all treatments.ConclusionsRomifidine induced hyperglycaemia, which vatinoxan partially prevented despite of the variations in baseline levels of serum insulin. The effects of romifidine and vatinoxan on the insulin concentration in horses need further investigation.

P-51 - Free radical transformations of glucose metabolites in aqueous solutions

It is well known that ROS, generated via various enzymatic and non-enzymatic pathways, can cause the damage of biomolecules. Using γ-irradiation and/or Fe2+/t-BuOOH reagent system for •OH and •OR generation we have shown that glucose metabolites and their analogs (fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, glycerol-1-phosphate etc.) undergo free radical destruction in aqueous solutions. As a result of such reactions inorganic phosphate and carbonyl products were formed. Among free radical transformation products of starting compounds glyoxal and methylglyoxal (MG) have also been found. The mechanisms for such transformations were proposed. It is known that dicarbonyls possess high toxicity, MG is a potent protein glycating agent, MG-derived hydroimidazolone (MG-H1) is often the major advanced glycation end-product in proteins of tissues and body fluids, increasing in diabetes and associated vascular complications, renal failure, cirrhosis, Alzheimer's disease, arthritis, Parkinson's disease and ageing. The obtained data are recommended to consider during estimation of ROS-induced pathophysiological processes involving glucose metabolites.

Regional differences in brain glucose metabolism determined by imaging mass spectrometry

ObjectiveGlucose is the major energy substrate of the brain and crucial for normal brain function. In diabetes, the brain is subject to episodes of hypo- and hyperglycemia resulting in acute outcomes ranging from confusion to seizures, while chronic metabolic dysregulation puts patients at increased risk for depression and Alzheimer's disease. In the present study, we aimed to determine how glucose is metabolized in different regions of the brain using imaging mass spectrometry (IMS). MethodsTo examine the relative abundance of glucose and other metabolites in the brain, mouse brain sections were subjected to imaging mass spectrometry at a resolution of 100 μm. This was correlated with immunohistochemistry, qPCR, western blotting and enzyme assays of dissected brain regions to determine the relative contributions of the glycolytic and pentose phosphate pathways to regional glucose metabolism. ResultsIn brain, there are significant regional differences in glucose metabolism, with low levels of hexose bisphosphate (a glycolytic intermediate) and high levels of the pentose phosphate pathway (PPP) enzyme glucose-6-phosphate dehydrogenase (G6PD) and PPP metabolite hexose phosphate in thalamus compared to cortex. The ratio of ATP to ADP is significantly higher in white matter tracts, such as corpus callosum, compared to less myelinated areas. While the brain is able to maintain normal ratios of hexose phosphate, hexose bisphosphate, ATP, and ADP during fasting, fasting causes a large increase in cortical and hippocampal lactate. ConclusionThese data demonstrate the importance of direct measurement of metabolic intermediates to determine regional differences in brain glucose metabolism and illustrate the strength of imaging mass spectrometry for investigating the impact of changing metabolic states on brain function at a regional level with high resolution.