Regulation of the tyrosine oxidizing system in fetal rat liver

The formation of glucose 6-arsenate and glucose 6-phosphate shows similar thermodynamic constants: both reactions are endothermic, endergonic, and occur with a decrease of entropy. However, the kinetic coefficients of the spontaneous formation of the arsenate esters are ca. 10⁵ times greater than those of their homologous phosphate esters. The activation energy of the spontaneous formation of glucose 6-arsenate (E = + 12 kcal mol^?1) is even smaller than that of the formation of glucose 6-phosphate by alkaline phosphate (E = + 13 kcal mol^?1). Similar to the case of the monoalkylphosphates, the monoanion species of glucose 6-arsenate is much more reactive than the dianion species. This is an important difference with respect to glucose 6-phosphate. The calculated half-lives at 25 °C and pH 7.0 of glucose 6-arsenate and 6-arsenogluconate are only ca. 6 and 30 min, respectively; they increase at lower temperatures and alkaline pH. At 0 °C and pH 9.0 the half-life of glucose 6-arsenate is ca. 20 h. Therefore, arsenate esters could probably be isolated for use as a tool in biochemical studies. Arsenate esters are good analogs of the phosphate esters for a variety of enzymes. Glucose-6-phosphate dehydrogenase shows nearly similar values of K_m and V for either glucose 6-phosphate or glucose 6-arsenate, and hexokinase is similarly inhibited by both compounds. 6-Phosphogluconate dehydrogenase has the same V with respect to 6-phosphogluconate and 6-arsenogluconate although the enzyme shows a much lower affinity for the latter substrate.     

Calcium sparks in the intact gerbil spiral modiolar artery

Background

We and others have demonstrated previously that ghrelin receptor (GhrR) knock out (KO) mice fed a high fat diet (HFD) have increased insulin sensitivity and metabolic flexibility relative to WT littermates. A striking feature of the HFD-fed GhrR KO mouse is the dramatic decrease in hepatic steatosis. To characterize further the underlying mechanisms of glucose homeostasis in GhrR KO mice, we conducted both hyperglycemic (HG) and hyperinsulinemic-euglycemic (HI-E) clamps. Additionally, we investigated tissue glucose uptake and specifically examined liver insulin sensitivity.

Results

Consistent with glucose tolerance-test data, in HG clamp experiments, GhrR KO mice showed a reduction in glucose-stimulated insulin release relative to WT littermates. Nevertheless, a robust 1^st phase insulin secretion was still achieved, indicating that a healthy β-cell response is maintained. Additionally, GhrR KO mice demonstrated both a significantly increased glucose infusion rate and significantly reduced insulin requirement for maintenance of the HG clamp, consistent with their relative insulin sensitivity. In HI-E clamps, both LFD-fed and HFD-fed GhrR KO mice showed higher peripheral insulin sensitivity relative to WT littermates as indicated by a significant increase in insulin-stimulated glucose disposal (Rd), and decreased hepatic glucose production (HGP). HFD-fed GhrR KO mice showed a marked increase in peripheral tissue glucose uptake in a variety of tissues, including skeletal muscle, brown adipose tissue and white adipose tissue. GhrR KO mice fed a HFD also showed a modest, but significant decrease in conversion of pyruvate to glucose, as would be anticipated if these mice displayed increased liver insulin sensitivity. Additionally, the levels of UCP2 and UCP1 were reduced in the liver and BAT, respectively, in GhrR KO mice relative to WT mice.

Conclusions

These results indicate that improved glucose homeostasis of GhrR KO mice is characterized by robust improvements of glucose disposal in both normal and metabolically challenged states, relative to WT controls. GhrR KO mice have an intact 1^st phase insulin response but require significantly less insulin for glucose disposal. Our experiments reveal that the insulin sensitivity of GhrR KO mice is due to both BW independent and dependent factors. We also provide several lines of evidence that a key feature of the GhrR KO mouse is maintenance of hepatic insulin sensitivity during metabolic challenge.     

Glucose Signaling in Yeast Is Partially Mimicked by Galactose and Does Not Require the Tps1 Protein

Glucose produces multiple effects inSaccharomyces cerevisiae,as it controls the expression of many genes and the activity of various enzymes. However, the elements involved in glucose signaling are not well characterized. In this work the capacity of galactose to bring about the same effects than glucose has been assessed. Galactose mimics glucose only partially; it is suggested that it does not interact with a “sensor” in the plasma membrane and that it produces a weaker intracellular signal than glucose. To examine whether trehalose-6P synthase (Tps1) is required to transduce the glucose signal, we have constructed atps1 hxk2/tps1 HXK2strain which, at difference of atps1strain, grows on glucose, and, at difference of atps1 hxk2strain, still possess the Hxk2 protein, possibly involved in glucose repression. From the response of this strain to glucose, we conclude that Tps1 does not play a prominent role in glucose signaling.     

Mutation of the ptsG Gene Results in Increased Production of Succinate in Fermentation of Glucose by Escherichia coli

Escherichia coli NZN111 is blocked in the ability to grow fermentatively on glucose but gave rise spontaneously to a mutant that had this ability. The mutant carries out a balanced fermentation of glucose to give approximately 1 mol of succinate, 0.5 mol of acetate, and 0.5 mol of ethanol per mol of glucose. The causative mutation was mapped to the ptsG gene, which encodes the membrane-bound, glucose-specific permease of the phosphotransferase system, protein EIICB^glc. Replacement of the chromosomal ptsG gene with an insertionally inactivated form also restored growth on glucose and resulted in the same distribution of fermentation products. The physiological characteristics of the spontaneous and null mutants were consistent with loss of function of the ptsG gene product; the mutants possessed greatly reduced glucose phosphotransferase activity and lacked normal glucose repression. Introduction of the null mutant into strains not blocked in the ability to ferment glucose also increased succinate production in those strains. This phenomenon was widespread, occurring in different lineages of E. coli, including E. coli B.     

Intracellular Glucose Concentration in Derepressed Yeast Cells Consuming Glucose Is High Enough To Reduce the Glucose Transport Rate by 50%

In Saccharomyces cerevisiae cells exhibiting high-affinity glucose transport, the glucose consumption rate at extracellular concentrations above 10 mM was only half of the zero trans-influx rate. To determine if this regulation of glucose transport might be a consequence of intracellular free glucose we developed a new method to measure intracellular glucose concentrations in cells metabolizing glucose, which compares glucose stereoisomers to correct for adhering glucose. The intracellular glucose concentration was 1.5 mM, much higher than in most earlier reports. We show that for the simplest model of a glucose carrier, this concentration is sufficient to reduce the glucose influx by 50%. We conclude that intracellular glucose is the most likely candidate for the observed regulation of glucose import and hence glycolysis. We discuss the possibility that intracellular glucose functions as a primary signal molecule in these cells.     

An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells

In several organisms solute transport is mediated by the simultaneous operation of saturable and non-saturable (diffusion-like) uptake, but often the nature of the diffusive component remains elusive. The present work investigates the nature of the diffusive glucose transport in Olea europaea cell cultures. In this system, glucose uptake is mediated by a glucose-repressible, H⁺-dependent active saturable transport system that is superimposed on a diffusional component. The latter represents the major mode of uptake when high external glucose concentrations are provided. In glucose-sufficient cells, initial velocities of d- and l-[U-¹⁴C]glucose uptake were equal and obeyed linear concentration dependence up to 100 mM sugar. In sugar starved cells, where glucose transport is mediated by the saturable system, countertransport of the sugar pairs 3-O-methyl-d-glucose/d-[U-¹⁴C]glucose and 3-O-methyl-d-glucose/3-O-methyl-d-[U-¹⁴C]glucose was demonstrated. This countertransport was completely absent in glucose-sufficient cells, indicating that linear glucose uptake is not mediated by a typical sugar permease. The endocytic inhibitors wortmannin-A and NH₄Cl inhibited neither the linear component of d- and l-glucose uptake nor the absorption of the nonmetabolizable glucose analog 3-O-methyl-d-[U-¹⁴C]glucose, thus excluding the involvement of endocytic mediated glucose uptake. Furthermore, the formation of endocytic vesicles assessed with the marker FM1-43 proceeded at a very slow rate. Activation energies for glucose transport in glucose sufficient cells and plasma membrane vesicles were 7 and 4 kcal mol^− 1, respectively, lower than the value estimated for diffusion of glucose through the lipid bilayer of phosphatidylethanolamine liposomes (12 kcal mol^− 1). Mercury chloride inhibited both the linear component of sugar uptake in sugar sufficient cells and plasma membrane vesicles, and the incorporation of the fluorescent glucose analog 2-NBDG, suggesting protein-mediated transport. Diffusive uptake of glucose was inhibited by a drop in cytosolic pH and stimulated by the protein kinase inhibitor staurosporine. The data demonstrate that the low-affinity, high-capacity, diffusional component of glucose uptake occurs through a channel-like structure whose transport capacity may be regulated by intracellular protonation and phosphorylation/dephosphorylation.     

Influence of reduced glucose levels on the in vitro hatching,attachment, and trophoblast outgrowth of the mouse blastocyst

The influence of glucose on the in vitro hatching, attachment, and trophoblast outgrowth of 856 mouse blastocysts has been examined. Results indicate that glucose is a necessary factor for the in vitro hatching of the mouse blastocyst and that a delay in hatching can be accomplished by culturing blastocysts in culture media devoid of glucose.     

Influence of Familial Renal Glycosuria Due to Mutations in the SLC5A2 Gene on Changes in Glucose Tolerance over Time

Familial renal glycosuria is an inherited disorder resulting in glucose excretion in the urine despite normal blood glucose concentrations. It is most commonly due to mutations in the SLC5A2 gene coding for the glucose transporter SGLT2 in the proximal tubule. Several drugs have been introduced as means to lower glucose in patients with type 2 diabetes targeting SGLT2 resulting in renal glycosuria, but no studies have addressed the potential effects of decreased renal glucose reabsorption and chronic glycosuria on the prevention of glucose intolerance. Here we present data on a large pedigree with renal glycosuria due to two mutations (c.300-303+2del and p.A343V) in the SLC5A2 gene. The mutations, which in vitro affected glucose transport in a cell line model, and the ensuing glycosuria were not associated with better glycemic control during a follow-up period of more than 10 years. One individual, who was compound heterozygous for mutations in the SLC5A2 gene suffered from severe urogenital candida infections and postprandial hypoglycemia. In conclusion, in this family with familial glycosuria we did not find any evidence that chronic loss of glucose in the urine would protect from deterioration of the glucose tolerance over time.     

Glycolysis Controls Plasma Membrane Glucose Sensors To Promote Glucose Signaling in Yeasts

Sensing of extracellular glucose is necessary for cells to adapt to glucose variation in their environment. In the respiratory yeast Kluyveromyces lactis, extracellular glucose controls the expression of major glucose permease gene RAG1 through a cascade similar to the Saccharomyces cerevisiae Snf3/Rgt2/Rgt1 glucose signaling pathway. This regulation depends also on intracellular glucose metabolism since we previously showed that glucose induction of the RAG1 gene is abolished in glycolytic mutants. Here we show that glycolysis regulates RAG1 expression through the K. lactis Rgt1 (KlRgt1) glucose signaling pathway by targeting the localization and probably the stability of Rag4, the single Snf3/Rgt2-type glucose sensor of K. lactis. Additionally, the control exerted by glycolysis on glucose signaling seems to be conserved in S. cerevisiae. This retrocontrol might prevent yeasts from unnecessary glucose transport and intracellular glucose accumulation.     

Involvement of the Clock Gene Rev-erb alpha in the Regulation of Glucagon Secretion in Pancreatic Alpha-Cells

Disruption of pancreatic clock genes impairs pancreatic beta-cell function, leading to the onset of diabetes. Despite the importance of pancreatic alpha-cells in the regulation of glucose homeostasis and in diabetes pathophysiology, nothing is known about the role of clock genes in these cells. Here, we identify the clock gene Rev-erb alpha as a new intracellular regulator of glucagon secretion. Rev-erb alpha down-regulation by siRNA (60–70% inhibition) in alphaTC1-9 cells inhibited low-glucose induced glucagon secretion (p<0.05) and led to a decrease in key genes of the exocytotic machinery. The Rev-erb alpha agonist GSK4112 increased glucagon secretion (1.6 fold) and intracellular calcium signals in alphaTC1-9 cells and mouse primary alpha-cells, whereas the Rev-erb alpha antagonist SR8278 produced the opposite effect. At 0.5 mM glucose, alphaTC1-9 cells exhibited intrinsic circadian Rev-erb alpha expression oscillations that were inhibited by 11 mM glucose. In mouse primary alpha-cells, glucose induced similar effects (p<0.001). High glucose inhibited key genes controlled by AMPK such as Nampt, Sirt1 and PGC-1 alpha in alphaTC1-9 cells (p<0.05). AMPK activation by metformin completely reversed the inhibitory effect of glucose on Nampt-Sirt1-PGC-1 alpha and Rev-erb alpha. Nampt inhibition decreased Sirt1, PGC-1 alpha and Rev-erb alpha mRNA expression (p<0.01) and glucagon release (p<0.05). These findings identify Rev-erb alpha as a new intracellular regulator of glucagon secretion via AMPK/Nampt/Sirt1 pathway.     

Glucose transport in membrane vesicles from Azotobacter vinelandii,: Properties of the glucose carrierin situ

The active transport of d-glucose by membrane vesicles prepared from Azotobactervinelandii strain O is coupled to the oxidation of l-malate. The glucose carrier, but not the energy coupling system of the vesicles, is induced by growth of the cells on d-glucose medium. Vesicles isolated from A. vinelandii grown in the presence of sucrose or acetate accumulate glucose at less than 7% of the rate observed for vesicles from glucose-grown cells. Nevertheless, vesicles from sucrose- or acetate-grown cells transport sucrose or calcium, respectively, in the presence of malate.The transport system expressed in vesicles from glucose-cultured cells is highly specific for d-glucose. Studies of glucose analog uptake and of the competitive effect of analogs reveal that: (i) The glucose carrier is stereospecific. (ii) The affinity of hexoses for the transport system is inversely related to the bulk of substituents on the pyranose ring, especially at the C-1 and C-2 positions, (iii) The most effective competitors, 6-deoxyglucose and 2-deoxyglucose, exhibit affinities only 10–20% that of d-glucose for the transport system, (iv) Phloretin, but not phlorizin, is a competitive inhibitor of glucose transport, having an apparent K_i of 9 μm at pH 7.0. These latter findings suggest a similarity of the glucose transport system of fxA. vinelandii and those of eukaryotes with regard to the glucose carrier.     

Assimilation of Polysaccharides and Glucose by Major Bacterial Groups in the Delaware Estuary

The contribution of major bacterial groups to the assimilation of extracellular polymeric substances (EPS) and glucose in the Delaware Estuary was assessed using microautoradiography and fluorescence in situ hybridization. Bacterial groups contributed to EPS and glucose assimilation in part according to their distribution in the estuary. Abundance of the phylogenetic groups explained 35% and 55% of the variation in EPS and glucose assimilation, respectively. Actinobacteria contributed 70% to glucose assimilation in freshwater, while Alphaproteobacteria assimilated 60% of this compound in saline water. In contrast, various bacterial groups dominated the assimilation of EPS. Actinobacteria and Betaproteobacteria contributed the most in the freshwater section, whereas Cytophaga-like bacteria and Alpha- and Gammaproteobacteria participated in EPS assimilation in the lower part of the estuary. In addition, we examined the fraction of bacteria in each group that assimilated glucose or EPS. Overall, the fraction of bacteria in all groups that assimilated glucose was higher than the fraction that assimilated EPS (15 to 30% versus 5 to 20%, respectively). We found no correlation between the relative abundance of a group in the estuary and the fraction of bacteria actively assimilating glucose or EPS; the more active groups were often less abundant. Our results imply that the bacterial community in the Delaware Estuary is not controlled solely by “bottom-up” factors such as dissolved organic matter.     

Partial purification and characterization of the glucosidases involved in the processing of asparagine-linked oligosaccharides

A glucosidase preparation with activity toward certain glucose-containing oligosaccharides was partially purified from calf liver membranes by Triton X-100 solubilization and DEAE-cellulose and hydroxylapatite chromatography. The enzyme preparation hydrolyzed the glucose residues from (glucose)₁,(mannose)₉(N-acetylglucosamine)₁, and (glucose)₂(mannose) ₉(N-acetylglucosamine)₁ but was totally inactive toward (glucose)₃(mannose)₉(N-acetylglucosamine) ₁. In contrast, crude membrane preparations of the calf liver were active toward all three substrates. The partially purified enzyme had a pH optimum of 6.7 and was very unstable in the absence of added 20% glycerol. The rate of glucose release from the one-and two-glucose-containing oligosaccharides was significantly decreased when four or five of the mannose residues were first removed from the substrate. The release of glucose from (glucose)₁(mannose)₉(N-acetylglucosamine)₁ was inhibited by p-nitrophenyl-α-d-glucoside much more effectively than by p-nitrophenyl-β-d-glucoside, suggesting that this glucose residue may be linked α to the mannose residue. We conclude that during oligosaccharide processing at least two different glucosidases are involved in glucose removal.     

Impaired Glucose Metabolism in Mice Lacking the Tas1r3 Taste Receptor Gene

The G-protein-coupled sweet taste receptor dimer T1R2/T1R3 is expressed in taste bud cells in the oral cavity. In recent years, its involvement in membrane glucose sensing was discovered in endocrine cells regulating glucose homeostasis. We investigated importance of extraorally expressed T1R3 taste receptor protein in age-dependent control of blood glucose homeostasis in vivo, using nonfasted mice with a targeted mutation of the Tas1r3 gene that encodes the T1R3 protein. Glucose and insulin tolerance tests, as well as behavioral tests measuring taste responses to sucrose solutions, were performed with C57BL/6ByJ (Tas1r3+/+) inbred mice bearing the wild-type allele and C57BL/6J-Tas1r3^tm1Rfm mice lacking the entire Tas1r3 coding region and devoid of the T1R3 protein (Tas1r3-/-). Compared with Tas1r3+/+ mice, Tas1r3-/- mice lacked attraction to sucrose in brief-access licking tests, had diminished taste preferences for sucrose solutions in the two-bottle tests, and had reduced insulin sensitivity and tolerance to glucose administered intraperitoneally or intragastrically, which suggests that these effects are due to absence of T1R3. Impairment of glucose clearance in Tas1r3-/- mice was exacerbated with age after intraperitoneal but not intragastric administration of glucose, pointing to a compensatory role of extraoral T1R3-dependent mechanisms in offsetting age-dependent decline in regulation of glucose homeostasis. Incretin effects were similar in Tas1r3+/+ and Tas1r3-/- mice, which suggests that control of blood glucose clearance is associated with effects of extraoral T1R3 in tissues other than the gastrointestinal tract. Collectively, the obtained data demonstrate that the T1R3 receptor protein plays an important role in control of glucose homeostasis not only by regulating sugar intake but also via its extraoral function, probably in the pancreas and brain.     

Characterization of Xylose Uptake in the Yeasts Pichia heedii and Pichia stipitis

The kinetics of xylose uptake were investigated in the efficient xylose fermenter Pichia stipitis and in the more readily genetically manipulated, strictly respiratory yeast Pichia heedii. Both yeasts demonstrated more than one xylose uptake system, differing in substrate affinity. The K_m of high-affinity xylose uptake in both organisms was similar to that of the efficient high-affinity glucose uptake system of Saccharomyces cerevisiae. In P. heedii, low-affinity xylose uptake was enhanced with growth on 2% but not 0.05% xylose and high-affinity uptake was reduced. In contrast to glucose uptake, xylose uptake in P. heedii was inhibited by dinitrophenol. Dinitrophenol inhibited both glucose and xylose uptake by P. stipitis. Glucose uptake was not inhibited by a 100-fold molar excess of xylose in P. heedii. It is suggested that xylose uptake in P. heedii is via a carrier system(s) distinct from those for glucose uptake.     

The hexokinase gene family in the zebrafish: Structure,expression, functional and phylogenetic analysis

Hexokinase-catalyzed glucose phosphorylation is the first and crucial step for glucose utilization. Although there are reported studies on glucose metabolism in commercial species, knowledge on it is almost nil in zebrafish (Danio rerio), an important model organism for biological research. We have searched these fish hexokinase genes by BLAST analysis; determined their expression in liver, muscle, brain and heart; measured their response to fasting and glucose administration; and performed homology sequences studies to glimpse their evolutionary history. We have confirmed by RT-qPCR studies that the six DNA sequences annotated as possible hexokinases in the NCBI GenBank are transcribed. The organ distribution of the HXK genes is similar in zebrafish as in mammals, to which they are distantly related. Of these, DrGLK and DrSHXK1 are expressed in the fish liver, DrHXK1 in brain and heart, and DrHXK2 in muscle. The only gene responsive to glucose was liver DrGLK. Its expression is induced approximately 1 h after glucose intraperitoneal injection, but not after saline solution injection. The comparison of the fish sequences and the corresponding mammalian ones imply that in both taxa the main muscle and brain isoforms are fusion products of the ancestral gene, their amino halves having separated before than their carboxy ones, followed by the fusion event, whereas fish and mammalian glucokinase genes remained unduplicated.     

Contributions of XylR,CcpA and cre to diauxic growth of Bacillus megaterium and to xylose isomerase expression in the presence of glucose and xylose

Bacillus megaterium shows diauxic growth in minimal medium containing glucose and xylose. We have examined the influence of three elements that regulate xyl operon expression on diauxic growth and expression of a xylA-lacZ fusion. xylA is 13-fold repressed during growth on glucose. Induction occurs at the onset of the lag phase after glucose is consumed. Inactivation of xylR yields a two-fold increase in expression of xylA on glucose. Deletion of the catabolite responsive element (cre) has a more pronounced effect, reducing glucose repression from 13-fold in the wild type to about 2.5-fold. When xylR and cre are inactivated together a residual two-fold repression of xylA is found. Inactivation of xylR affects diauxic growth by shortening the lag phase from 70 to 40?min. In-frame deletion of ccpA results in the loss of diauxic growth, an increase in doubling time and simultaneous use of both sugars. In contrast, a strain with an inactivated cre site in xylA exhibits diauxic growth without an apparent lag phase on glucose and xylose, whereas fructose and xylose are consumed simultaneously.