Characterization of the Polar Profile of Bacon and Fuerte Avocado Fruits by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry: Distribution of Non-structural Carbohydrates, Quinic Acid, and Chlorogenic Acid between Seed, Mesocarp, and Exocarp at Different Ripening Stages.

Avocado fruit growth and development, unlike that of other fruits, is characterized by the accumulation of oil and C7 sugars (in most fruits, the carbohydrates that prevail are C6). There are five essential carbohydrates which constitute 98% of the total content of soluble sugars in this fruit; these are fructose, glucose, sucrose, d-mannoheptulose, and perseitol, which together with quinic acid and chlorogenic acid have been the analytes under study in this work. After applying an efficient extraction procedure, a novel methodology based on hydrophilic interaction liquid chromatography coupled to mass spectrometry was applied to determine the levels of these seven substances in tissues─exocarp, seed, and mesocarp─from avocado fruits of two different varieties scarcely studied, Bacon and Fuerte, at three different ripening stages. Quantitative characterization of the selected tissues was performed, and the inter-tissue distribution of metabolites was described. For both varieties, d-mannoheptulose was the major component in the mesocarp and exocarp, whereas perseitol was predominant in the seed, followed by sucrose and d-mannoheptulose. Sucrose was found to be more abundant in seed tissues, with much lower concentrations in avocado mesocarp and exocarp. Quinic acid showed a predominance in the exocarp, and chlorogenic acid was exclusively determined in exocarp samples.

Characterization and Mechanisms of Action of Avocado Extract Enriched in Mannoheptulose as a Candidate Calorie Restriction Mimetic.

Increased consumer interest in the avocado (Persea americana or Persea gratissima) has been attributed to established health benefits of this fruit associated with a wide range of ingredients. In search of effective calorie restriction mimetics (CRM), we present herein a consideration of possible health benefits of the rare sugar, mannoheptulose (MH), which acts as an intracellular glycolytic inhibitor and presents the highest concentration of this inhibitor in unripe avocados. A method for producing an extract of unripe avocado (AvX) to enrich concentrations of MH is described. Experiments using myocyte cultures demonstrated a pattern of CRM-like responses when treated with AvX. In vivo experiments confirmed that orally consumed AvX is bioavailable in both mice and dogs, as observed in urine and blood samples. Additional experiments in both these species demonstrated CRM-like improvements in glucose and insulin responses. In sum, the MH-enriched AvX exhibits promise as a CRM.

Glucokinase intrinsically regulates glucose sensing and glucagon secretion in pancreatic alpha cells.

The secretion of glucagon by pancreatic alpha cells is regulated by a number of external and intrinsic factors. While the electrophysiological processes linking a lowering of glucose concentrations to an increased glucagon release are well characterized, the evidence for the identity and function of the glucose sensor is still incomplete. In the present study we aimed to address two unsolved problems: (1) do individual alpha cells have the intrinsic capability to regulate glucagon secretion by glucose, and (2) is glucokinase the alpha cell glucose sensor in this scenario. Single cell RT-PCR was used to confirm that glucokinase is the main glucose-phosphorylating enzyme expressed in rat pancreatic alpha cells. Modulation of glucokinase activity by pharmacological activators and inhibitors led to a lowering or an increase of the glucose threshold of glucagon release from single alpha cells, measured by TIRF microscopy, respectively. Knockdown of glucokinase expression resulted in a loss of glucose control of glucagon secretion. Taken together this study provides evidence for a crucial role of glucokinase in intrinsic glucose regulation of glucagon release in rat alpha cells.

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase and small chemical activators affect enzyme activity of activating glucokinase mutants by distinct mechanisms.

Glucokinase (GK), a monomeric glucose-phosphorylating enzyme characterised by high structural flexibility, acts as a glucose sensor in pancreatic beta cells and liver. Pharmaceutical efforts to control the enzyme are hampered by an incomplete understanding of GK regulation. We investigated GK characteristics of wild-type and activating S64Y and G68V mutant proteins in the presence of various combinations of the synthetic activators RO-28-1675 and compound A, the endogenous activator fructose-2,6-bisphosphatase (FBPase-2), and the inhibitor mannoheptulose. S64Y impedes formation of a turn structure that is characteristic for the inactive enzyme conformation, and complex formation with compound A induces collision with the large domain. G68V evokes close contact of connecting region I and helix α13 with RO-28-1675 and compound A. Both mutants showed higher activity than the wild-type at low glucose and were susceptible to further activation by FBPase-2 and RO-28-1675, alone and additively. G68V was less active than S64Y, but was activatable by compound A. In contrast, compound A inhibited S64Y, and this effect was even more pronounced in combination with mannoheptulose. Mutant and wild-type GK showed comparable thermal stability and intracellular lifetimes. A GK-6-phosphofructo-2-kinase (PFK-2)/FBPase-2 complex predicted by in silico protein-protein docking demonstrated possible binding of the FBPase-2 domain near the active site of GK. In summary, activating mutations within the allosteric site of GK do not preclude binding of chemical activators (GKAs), but can alter their action into inhibition. Our postulated GK-PFK-2/FBPase-2 complex represents the endogenous principle of activation by substrate channelling which permits binding of other small molecules and proteins.

Reducing Glucokinase Activity Restores Endogenous Pulsatility and Enhances Insulin Secretion in Islets From db/db Mice.

An early sign of islet failure in type 2 diabetes (T2D) is the loss of normal patterns of pulsatile insulin release. Disruptions in pulsatility are associated with a left shift in glucose sensing that can cause excessive insulin release in low glucose (relative hyperinsulinemia, a hallmark of early T2D) and ß-cell exhaustion, leading to inadequate insulin release during hyperglycemia. Our hypothesis was that reducing excessive glucokinase activity in diabetic islets would improve their function. Isolated mouse islets were exposed to glucose and varying concentrations of the glucokinase inhibitor d-mannoheptulose (MH) to examine changes in intracellular calcium ([Ca2+]i) and insulin secretion. Acutely exposing islets from control CD-1 mice to MH in high glucose (20 mM) dose dependently reduced the size of [Ca2+]i oscillations detected by fura-2 acetoxymethyl. Glucokinase activation in low glucose (3 mM) had the opposite effect. We then treated islets from male and female db/db mice (age, 4 to 8 weeks) and heterozygous controls overnight with 0 to 10 mM MH to determine that 1 mM MH produced optimal oscillations. We then used 1 mM MH overnight to measure [Ca2+]i and insulin simultaneously in db/db islets. MH restored oscillations and increased insulin secretion. Insulin secretion rates correlated with MH-induced increases in amplitude of [Ca2+]i oscillations (R2 = 0.57, P < 0.01, n = 10) but not with mean [Ca2+]i levels in islets (R2 = 0.05, not significant). Our findings show that correcting glucose sensing can restore proper pulsatility to diabetic islets and improved pulsatility correlates with enhanced insulin secretion.

Dietary mannoheptulose does not alter glucose or lipid metabolism in adult Labrador Retrievers.

Mannoheptulose (MH), a glycolytic inhibitor, has been preliminarily investigated as a novel functional food ingredient for dogs. This study aimed to determine the effects of dietary MH, delivered as an extract of un-ripened avocados, on fatty acid and glucose kinetics in healthy adult Labrador Retriever dogs (n = 12 dogs). The study was a double-blindcrossover with each dog receiving both dietary treatments, control (CON) and MH (400 mg/kg of diet), in random order. Glucose and glycerol plasma turnover (Ra) and oxidation (Ox) were measured in fasting and in response to repeated meal feeding ("fed") with stable isotope tracers (U-13 C-glucose, 1,1,2,3,3-D5 -glycerol) and indirect calorimetry. Palmitate Ra and Ox were examined during repeated meal feeding only using an oral bolus of U-13 C-K2 -palmitate and indirect calorimetry. MH had no discernible effect on fasting glucose Ra (677, 722 SEM 36 µmol/min, CON, MH) or Ox (107, 109 µmol/min, CON, MH SEM 10 µmol/min) or fed glucose Ra (2913, 3626 SEM 644 µmol/min, CON, MH) or Ox (951, 936 SEM 174 µmol/min, CON, MH). Glycerol Ra, an index of the rate of lipolysis, was not different between dietary treatments (Fast 162, 113 SEM 35 µmol/min CON, MH; Fed 172, 135 SEM 21 µmol/min, CON, MH). Similarly, palmitate oxidation was not impacted by MH feeding (1966, 2276 SEM 79 µmol/min, CON, MH). Together, these findings do not support MH as a novel functional food ingredient at least at the dietary dose tested.

Dynamics of Insulin Secretion from EndoC-ßH1 ß-Cell Pseudoislets in Response to Glucose and Other Nutrient and Nonnutrient Secretagogues.

The dynamics of insulin secretion were characterized in response to a variety of physiological and pharmacological stimulators and other compounds in perifused pseudoislets generated from cells of the EndoC-ßH1 ß-cell line. Perifusion of EndoC-ßH1 pseudoislets with the physiological stimulus glucose (16.7 mM) induced sustained insulin secretion, which was inhibited by mannoheptulose. The adenylate cyclase activators IBMX and forskolin strongly potentiated this secretion. Glibenclamide, a Kir 6.2 potassium channel blocker, and Bay K 8644, an opener of the voltage-sensitive Ca2+ channel, also potentiated glucose-induced insulin secretion. The dynamics of insulin secretion from EndoC-ßH1 pseudoislets were characterized by an insulin secretory response to glucose starting within 1-2 min and passing over without interruption into a sustained phase of insulin release for the whole stimulation period. This lack of a transient decline between the first and the second phases of insulin release is an indication for a quick supply of insulin secretory granules from the reserve pool to the docking sites below the plasma membrane. Thereby, new secretory granules are directly made available for sustained exocytosis of insulin in EndoC-ßH1 ß-cells. The study shows that EndoC-ßH1 ß-cell pseudoislets are well suited for kinetic analyses of insulin secretion.

In vivo and ex vivo 19-fluorine magnetic resonance imaging and spectroscopy of beta-cells and pancreatic islets using GLUT-2 specific contrast agents.

The assessment of the ß-cell mass in experimental models of diabetes and ultimately in patients is a hallmark to understand the relationship between reduced ß-cell mass/function and the onset of diabetes. It has been shown before that the GLUT-2 transporter is highly expressed in both ß-cells and hepatocytes and that D-mannoheptulose (DMH) has high uptake specificity for the GLUT-2 transporter. As 19-fluorine MRI has emerged as a new alternative method for MRI cell tracking because it provides potential non-invasive localization and quantification of labeled cells, the purpose of this project is to validate ß-cell and pancreatic islet imaging by using fluorinated, GLUT-2 targeting mannoheptulose derivatives (19 FMH) both in vivo and ex vivo. In this study, we confirmed that, similar to DMH, 19 FMHs inhibit insulin secretion and increase the blood glucose level in mice temporarily (approximately two hours). We were able to assess the distribution of 19 FMHs in vivo with a temporal resolution of about 20 minutes, which showed a quick removal of 19 FMH from the circulation (within two hours). Ex vivo MR spectroscopy confirmed a preferential uptake of 19 FMH in tissue with high expression of the GLUT-2 transporter, such as liver, endocrine pancreas and kidney. No indication of further metabolism was found. In summary, 19 FMHs are potentially suitable for visualizing and tracking of GLUT-2 expressed cells. However, current bottlenecks of this technique related to the quick clearance of the compound and relative low sensitivity of 19 F MRI need to be overcome. Copyright © 2016 John Wiley & Sons, Ltd.

Dietary Mannoheptulose Does Not Significantly Alter Daily Energy Expenditure in Adult Labrador Retrievers.

Mannoheptulose (MH), a sugar found in avocados that inhibits glycolysis in vitro, has been preliminarily investigated as a novel food ingredient for dogs. This study aimed to determine the effects of dietary MH, delivered as an extract of un-ripened avocado, on energy expenditure (EE) in healthy adult Labrador Retriever dogs (total of 12 dogs, 26.99 ± 0.634 kg, 4.9 ± 0.2 y). The study was a double-blind, cross-over with each dog receiving both dietary treatments, control (CON) and MH (400 mg/kg of diet; 6 mg/kg BW), in random order. Resting and post-prandial (10 h) EE and respiratory quotient (RQ) were determined by indirect calorimetry (d 42). The following day, body composition was assessed using dual X-ray absorptiometry. Continuous activity monitoring was conducted using an Atical® accelerometer (d 43-47). A vastus lateralis muscle biopsy was obtained prior to the morning meal (d 49) and 4 h after consumption of their meal (d 56) to determine the protein content and phosphorylation of 5' adenosine monophosphate-activated protein kinase (AMPK). Diet did not affect body weight, resting EE or skeletal muscle AMPK phosphorylation. Dogs fed MH had significantly lower post-prandial RQ (p = 0.02) and ratio of fat to lean body mass (p = 0.02). Physical activity during light time periods (but not dark) was lower in dogs fed MH (p < 0.05) during weekends, but not on weekdays. These results suggest that MH affects energy balance of adult dogs, but that these effects are not dose dependent and not due to physical activity.

Physiological characterization of the human EndoC-ßH1 ß-cell line.

In the new human EndoC-ßH1 ß-cell line, a detailed analysis of the physiological characteristics was performed. This new human ß-cell line expressed all target structures on the gene and protein level, which are crucial for physiological function and insulin secretion induced by glucose and other secretagogues. Glucose influx measurements revealed an excellent uptake capacity of EndoC-ßH1 ß-cells by the Glut1 and Glut2 glucose transporters. A high expression level of glucokinase enabled efficient glucose phosphorylation, increasing the ATP/ADP ratio along with stimulation of insulin secretion in the physiological glucose concentration range. The EC50 value of glucose for insulin secretion was 10.3 mM. Mannoheptulose, a specific glucokinase inhibitor, blocked glucose-induced insulin secretion (GSIS). The nutrient insulin secretagogues l-leucine and 2-ketoisocaproate also stimulated insulin secretion, with a potentiating effect of l-glutamine. The Kir 6.2 potassium channel blocker glibenclamide and Bay K 8644, an opener of the voltage-sensitive Ca(2+) channel significantly potentiated GSIS. Potentiation of GSIS by IBMX and forskolin went along with a strong stimulation of cAMP generation. In conclusion, the new human EndoC-ßH1 ß-cell line fully mirrors the analogous physiological characteristics of primary mouse, rat and human ß-cells. Thus, this new human EndoC-ßH1 ß-cell line is very well suited for physiological ß-cell studies.

Imaging of the ß-cells of the islets of Langerhans.

The major aim of this paper is to review the present status of the techniques for the non-invasive imaging and quantification of insulin-producing pancreatic islet ß-cells. Emphasis is placed on both the expansion of prior work already considered in a prior review and novel achievements. Thus, the use of d-mannoheptulose analogs, hypoglycemic sulfonylureas and glinides, neural imaging agents, neuro-hormonal receptor ligands and nanoparticles is first dealt with. Thereafter, consideration is given on optical imaging technologies, the identification of new ß-cells specific binding and target proteins, the functional imaging of islets transplanted into the eye anterior chamber and in vivo manganese-enhanced magnetic resonance imaging.

Real-time analysis of intracellular glucose and calcium in pancreatic beta cells by fluorescence microscopy.

Glucose is the physiological stimulus for insulin secretion in pancreatic beta cells. The uptake and phosphorylation of glucose initiate and control downstream pathways, resulting in insulin secretion. However, the temporal coordination of these events in beta cells is not fully understood. The recent development of the FLII(12)Pglu-700µ-δ6 glucose nanosensor facilitates real-time analysis of intracellular glucose within a broad concentration range. Using this fluorescence-based technique, we show the shift in intracellular glucose concentration upon external supply and removal in primary mouse beta cells with high resolution. Glucose influx, efflux, and metabolism rates were calculated from the time-dependent plots. Comparison of insulin-producing cells with different expression levels of glucose transporters and phosphorylating enzymes showed that a high glucose influx rate correlated with GLUT2 expression, but was largely also sustainable by high GLUT1 expression. In contrast, in cells not expressing the glucose sensor enzyme glucokinase glucose metabolism was slow. We found no evidence of oscillations of the intracellular glucose concentration in beta cells. Concomitant real-time analysis of glucose and calcium dynamics using FLII(12)Pglu-700µ-δ6 and fura-2-acetoxymethyl-ester determined a glucose threshold of 4mM for the [Ca(2+)](i) increase in beta cells. Indeed, a glucose concentration of 7mM had to be reached to evoke large amplitude [Ca(2+)](i) oscillations. The K(ATP) channel closing agent glibenclamide was not able to induce large amplitude [Ca(2+)](i) oscillations in the absence of glucose. Our findings suggest that glucose has to reach a threshold to evoke the [Ca(2+)](i) increase and subsequently initiate [Ca(2+)](i) oscillations in a K(ATP) channel independent manner.

Additive activation of glucokinase by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and the chemical activator LY2121260.

The glucose phosphorylating enzyme glucokinase plays a crucial role in stimulus-secretion coupling in pancreatic beta cells and in glucose metabolism in liver. Glucose mediates a shift of the enzyme's conformational equilibrium towards the closed conformation with high glucokinase activity. Further activation of glucokinase is endogenously mediated by interaction with the bisphosphatase domain (FBPase-2) of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) and can be achieved also by a new class of glucokinase activators (GKA), chemical compounds that might be suited for type 2 diabetes therapy. While FBPase-2 increased only the phosphorylating capacity of glucokinase, the GKA LY2121260 augmented in addition the affinity of glucokinase for glucose. PFK-2/FBPase-2 but not LY2121260 antagonized glucokinase inhibition by the competitive glucokinase inhibitor mannoheptulose at increasing glucose concentrations. Interestingly, an additive activation of glucokinase was observed by use of recombinant FBPase-2 together with LY2121260. This new crucial observation could be confirmed with cellular extracts containing the glucokinase and PFK-2/FBPase-2 proteins. Addition of LY2121260 resulted in a further significant increase in glucokinase activity. Because the glucokinase-PFK-2/FBPase-2 complex was conserved under LY2121260 treatment as shown by size exclusion chromatography a concerted action of both activators towards the closed active glucokinase conformation can be anticipated. Thus, as a result of the additive effect of both activators on glucokinase activity, the largest increase of glucose-induced insulin secretion was observed in the combined presence of PFK-2/FBPase-2 and LY2121260.

Brain glucose sensors play a significant role in the regulation of pancreatic glucose-stimulated insulin secretion.

As patients decline from health to type 2 diabetes, glucose-stimulated insulin secretion (GSIS) typically becomes impaired. Although GSIS is driven predominantly by direct sensing of a rise in blood glucose by pancreatic ß-cells, there is growing evidence that hypothalamic neurons control other aspects of peripheral glucose metabolism. Here we investigated the role of the brain in the modulation of GSIS. To examine the effects of increasing or decreasing hypothalamic glucose sensing on glucose tolerance and insulin secretion, glucose or inhibitors of glucokinase, respectively, were infused into the third ventricle during intravenous glucose tolerance tests (IVGTTs). Glucose-infused rats displayed improved glucose handling, particularly within the first few minutes of the IVGTT, with a significantly lower area under the excursion curve within the first 10 min (AUC0-10). This was explained by increased insulin secretion. In contrast, infusion of the glucokinase inhibitors glucosamine or mannoheptulose worsened glucose tolerance and decreased GSIS in the first few minutes of IVGTT. Our data suggest a role for brain glucose sensors in the regulation of GSIS, particularly during the early phase. We propose that pharmacological agents targeting hypothalamic glucose-sensing pathways may represent novel therapeutic strategies for enhancing early phase insulin secretion in type 2 diabetes.

(19)F-heptuloses as tools for the non-invasive imaging of GLUT2-expressing cells.

Suitable analogs of d-mannoheptulose are currently considered as possible tools for the non-invasive imaging of pancreatic islet insulin-producing cells. Here, we examined whether (19)F-heptuloses could be used for non-invasive imaging of GLUT2-expressing cells. After 20 min incubation, the uptake of (19)F-heptuloses (25 mM) by rat hepatocytes, as assessed by (19)F NMR spectroscopy, ranged from 0.50 (1-deoxy-1-fluoro-d-mannoheptulose and 3-deoxy-3-fluoro-d-mannoheptulose) to 0.25 (1,3-dideoxy-1,3-difluoro-d-mannoheptulose) and 0.13 (1-deoxy-1-fluoro-d-glucoheptulose, 3-deoxy-3-fluoro-d-glucoheptulose and 1,3-dideoxy-1,3-difluoro-d-glucoheptulose) µmol per 3×10(6)cells. (19)F MRI experiments also allowed the detection of 1-deoxy-1-fluoro-d-mannoheptulose in rat hepatocytes. All three (19)F-mannoheptuloses cited above, as well as 7-deoxy-7-fluoro-d-mannoheptulose and 1-deoxy-1-fluoro-d-glucoheptulose inhibited insulin release evoked in rat isolated pancreatic islets by 10mM d-glucose to the same extent as that observed with an equivalent concentration (10mM) of d-mannoheptulose, while 3-deoxy-3-fluoro-d-glucoheptulose and 1,3-dideoxy-1,3-difluoro-d-glucoheptulose (also 10mM) were less potent than d-mannoheptulose in inhibiting insulin release. The 1-deoxy-1-fluoro-d-mannoheptulose and 3-deoxy-3-fluoro-d-mannoheptulose only marginally affected INS-1 cell viability. These findings are compatible with the view that selected (19)F-heptuloses may represent suitable tools for the non-invasive imaging of hepatocytes and insulin-producing cells by (19)F MRI.

Brain glucosamine boosts protective glucoprivic feeding.

The risk of iatrogenic hypoglycemia is increased in diabetic patients who lose defensive glucoregulatory responses, including the important warning symptom of hunger. Protective hunger symptoms during hypoglycemia may be triggered by hypothalamic glucose-sensing neurons by monitoring changes downstream of glucose phosphorylation by the specialized glucose-sensing hexokinase, glucokinase (GK), during metabolism. Here we investigated the effects of intracerebroventricular (ICV) infusion of glucosamine (GSN), a GK inhibitor, on food intake at normoglycemia and protective feeding responses during glucoprivation and hypoglycemia in chronically catheterized rats. ICV infusion of either GSN or mannoheptulose, a structurally different GK inhibitor, dose-dependently stimulated feeding at normoglycemia. Consistent with an effect of GSN to inhibit competitively glucose metabolism, ICV coinfusion of d-glucose but not l-glucose abrogated the orexigenic effect of ICV GSN at normoglycemia. Importantly, ICV infusion of a low GSN dose (15 nmol/min) that was nonorexigenic at normoglycemia boosted feeding responses to glucoprivation in rats with impaired glucose counterregulation. ICV infusion of 15 nmol/min GSN also boosted feeding responses to threatened hypoglycemia in rats with defective glucose counterregulation. Altogether our findings suggest that GSN may be a potential therapeutic candidate for enhancing defensive hunger symptoms during hypoglycemia.

An efficient synthesis of d-mannoheptulose via oxidation of an olefinated sugar with potassium permanganate in aqueous acetone.

An efficient three-step synthesis of d-mannoheptulose was successfully accomplished from 2,3,4,5,6-penta-O-benzyl-d-mannose. First, an olefinated sugar was prepared from 2,3,4,5,6-penta-O-benzyl-d-mannose via a Wittig reaction. Second, the key step, a 2-hydroxyoxirane product was unexpectedly obtained by oxidation of the olefinated sugar with potassium permanganate in aqueous acetone. Finally d-mannoheptulose was synthesized through debenzylation and hydrolysis in an overall yield of 39%.

Glucosamine causes overproduction of reactive oxygen species, leading to repression of hypocotyl elongation through a hexokinase-mediated mechanism in Arabidopsis.

Glucosamine (GlcN) is a naturally occurring amino-sugar that is synthesized by amidation of fructose-6-phosphate. Although a number of reports have examined the biological effects of GlcN on insulin resistance in mammalian systems, little is known about its effects on plant growth. In this study, we have shown that exogenous GlcN inhibits hypocotyl elongation in Arabidopsis, whereas glucose and its analogs alleviate this inhibitory effect. The hexokinase (HXK)-specific inhibitor mannoheptulose also restored hypocotyl elongation. The gin2-1 mutants with an alteration in AtHXK1 exhibited higher tolerance to GlcN. We also found that GlcN induces a significant increase in the production of reactive oxygen species (ROS). In addition, the GlcN-mediated inhibition of hypocotyl elongation was relieved by reducing agents such as ascorbic acid and glutathione. GlcN treatment resulted in significant induction of expression of GST1, GST2 and GST6, which are marker genes for ROS production. The gin2 mutation also represses the ROS production and the GST2 induction by GlcN treatment. Taken together, these results provide evidence that GlcN induces HXK-mediated induction of oxidative stress, leading to growth repression in Arabidopsis thaliana.

Flow cytometric quantification of glucose-stimulated beta-cell metabolic flux can reveal impaired islet functional potency.

The objective of this study was to develop a multiparametric flow cytometry assay to simultaneously quantify isolated pancreatic islet cell viability, apoptosis, and glucose-induced metabolic flux. INS-1 and rat islet beta-cells were stained with fluorescent probes for cell viability (ToPro3), apoptosis (Annexin V and VADFMK), and intracellular calcium (Ca2+(i)) (Fura Red), stimulated with glucose, and analyzed on a FACS Vantage flow cytometer. Glucose-induced metabolic activity was indicated by changes in Fura Red fluorescence and the autofluorescence of the pyridine [NAD(P)H] and flavin (FAD/FMN) nucleotides. Rat islets cultured under conditions of proinflammatory cytokine-induced oxidative stress were evaluated by flow cytometry and transplantation into diabetic mice. INS-1 and rat islet beta-cell health and metabolic activity were quantified in response to elevated glucose dose and inhibitors of glycolysis and mitochondrial function. Changes in metabolite fluorescence were converted to an area under the curve (AUC) value. Rat islets cultured under oxidative stress conditions showed decreased viability, increased apoptosis, and decreased glucose-induced metabolic activity indicated by reduced AUC for pyridine and flavin nucleotides and Ca2+(i). Reduced metabolite AUC measured by flow cytometry correlated with the inability to reverse diabetes in mice. Single cell flow cytometry can simultaneously quantify both overall islet cell health and beta-cell glucose responsiveness as indicators of functional potency.

Detection of transaldolase deficiency by quantification of novel seven-carbon chain carbohydrate biomarkers in urine.

Transaldolase deficiency, a recently discovered disorder of carbohydrate metabolism with multisystem involvement, has been diagnosed in 6 patients. Affected patients have abnormal concentrations of polyols in body fluids and in all patients we have previously found increased amounts of a seven-carbon chain carbohydrate which we suspected of being sedoheptulose. We report development of a liquid chromatography-tandem mass spectrometry method for quantitation of the seven-carbon carbohydrates sedoheptulose and mannoheptulose in urine. Additionally, other seven-carbon chain carbohydrates were characterized in urine, including sedoheptitol, perseitol and sedoheptulose 7-phosphate. Transaldolase-deficient patients had significantly increased urinary sedoheptulose and sedoheptulose 7-phosphate, associated with subtle elevations of mannoheptulose, sedoheptitol and perseitol. Our findings reveal novel urinary biomarkers for identification of transaldolase deficiency.