Glucose-6-Phosphate Regulates Hepatic Bile Acid Synthesis in Mice.

It is well established that, besides facilitating lipid absorption, bile acids act as signaling molecules that modulate glucose and lipid metabolism. Bile acid metabolism, in turn, is controlled by several nutrient-sensitive transcription factors. Altered intrahepatic glucose signaling in type 2 diabetes associates with perturbed bile acid synthesis. We aimed to characterize the regulatory role of the primary intracellular metabolite of glucose, glucose-6-phosphate (G6P), on bile acid metabolism. Hepatic gene expression patterns and bile acid composition were analyzed in mice that accumulate G6P in the liver, that is, liver-specific glucose-6-phosphatase knockout (L-G6pc-/- ) mice, and mice treated with a pharmacological inhibitor of the G6P transporter. Hepatic G6P accumulation induces sterol 12α-hydroxylase (Cyp8b1) expression, which is mediated by the major glucose-sensitive transcription factor, carbohydrate response element-binding protein (ChREBP). Activation of the G6P-ChREBP-CYP8B1 axis increases the relative abundance of cholic-acid-derived bile acids and induces physiologically relevant shifts in bile composition. The G6P-ChREBP-dependent change in bile acid hydrophobicity associates with elevated plasma campesterol/cholesterol ratio and reduced fecal neutral sterol loss, compatible with enhanced intestinal cholesterol absorption. Conclusion: We report that G6P, the primary intracellular metabolite of glucose, controls hepatic bile acid synthesis. Our work identifies hepatic G6P-ChREBP-CYP8B1 signaling as a regulatory axis in control of bile acid and cholesterol metabolism.

Metabolic control of Ca2+/calmodulin-dependent protein kinase II (CaMKII)-mediated caspase-2 suppression by the B55ß/protein phosphatase 2A (PP2A).

High levels of metabolic activity confer resistance to apoptosis. Caspase-2, an apoptotic initiator, can be suppressed by high levels of nutrient flux through the pentose phosphate pathway. This metabolic control is exerted via inhibitory phosphorylation of the caspase-2 prodomain by activated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). We show here that this activation of CaMKII depends, in part, on dephosphorylation of CaMKII at novel sites (Thr(393)/Ser(395)) and that this is mediated by metabolic activation of protein phosphatase 2A in complex with the B55ß targeting subunit. This represents a novel locus of CaMKII control and also provides a mechanism contributing to metabolic control of apoptosis. These findings may have implications for metabolic control of the many CaMKII-controlled and protein phosphatase 2A-regulated physiological processes, because both enzymes appear to be responsive to alterations in glucose metabolized via the pentose phosphate pathway.

Structural basis for glucose-6-phosphate activation of glycogen synthase.

Regulation of the storage of glycogen, one of the major energy reserves, is of utmost metabolic importance. In eukaryotes, this regulation is accomplished through glucose-6-phosphate levels and protein phosphorylation. Glycogen synthase homologs in bacteria and archaea lack regulation, while the eukaryotic enzymes are inhibited by protein kinase mediated phosphorylation and activated by protein phosphatases and glucose-6-phosphate binding. We determined the crystal structures corresponding to the basal activity state and glucose-6-phosphate activated state of yeast glycogen synthase-2. The enzyme is assembled into an unusual tetramer by an insertion unique to the eukaryotic enzymes, and this subunit interface is rearranged by the binding of glucose-6-phosphate, which frees the active site cleft and facilitates catalysis. Using both mutagenesis and intein-mediated phospho-peptide ligation experiments, we demonstrate that the enzyme's response to glucose-6-phosphate is controlled by Arg583 and Arg587, while four additional arginine residues present within the same regulatory helix regulate the response to phosphorylation.

Deciphering the role of glucosamine-6-phosphate in the riboswitch action of glmS ribozyme.

The GlmS ribozyme is believed to exploit a general acid-base catalytic mechanism in the presence of glucosamine-6-phosphate (GlcN6P) to accelerate self-cleavage by approximately six orders of magnitude. The general acid and general base are not known, and the role of the GlcN6P cofactor is even less well understood. The amine group of GlcN6P has the ability to either accept or donate a proton and could therefore potentially act as an acid or a base. In order to decipher the role of GlcN6P in the self-cleavage of glmS, we have determined the preferred protonation state of the amine group in the wild-type and an inactive G40A mutant using molecular dynamics simulations and free energy calculations. Here we show that, upon binding of GlcN6P to wild-type glmS, the pK(a) of the amine moiety is altered by the active site environment, decreasing by about 2.2 from a solution pK(a) of about 8.2. On the other hand, we show that the pK(a) of the amine group slightly increases to about 8.4 upon binding to the G40A inactive mutant of glmS. These results suggest that GlcN6P acts as a general acid in the self-cleavage of glmS. Upon binding to glmS, GlcN6P can easily release a proton to the 5'-oxygen of G1 during self-cleavage of the backbone phosphodiester bond. However, in the G40A inactive mutant of glmS, the results suggest that the ability of GlcN6P to easily release its proton is diminished, in addition to the possible lack of G40 as an effective base.

Glucokinase and molecular aspects of liver glycogen metabolism.

Conversion of glucose into glycogen is a major pathway that contributes to the removal of glucose from the portal vein by the liver in the postprandial state. It is regulated in part by the increase in blood-glucose concentration in the portal vein, which activates glucokinase, the first enzyme in the pathway, causing an increase in the concentration of glucose 6-P (glucose 6-phosphate), which modulates the phosphorylation state of downstream enzymes by acting synergistically with other allosteric effectors. Glucokinase is regulated by a hierarchy of transcriptional and post-transcriptional mechanisms that are only partially understood. In the fasted state, glucokinase is in part sequestered in the nucleus in an inactive state, complexed to a specific regulatory protein, GKRP (glucokinase regulatory protein). This reserve pool is rapidly mobilized to the cytoplasm in the postprandial state in response to an elevated concentration of glucose. The translocation of glucokinase between the nucleus and cytoplasm is modulated by various metabolic and hormonal conditions. The elevated glucose 6-P concentration, consequent to glucokinase activation, has a synergistic effect with glucose in promoting dephosphorylation (inactivation) of glycogen phosphorylase and inducing dephosphorylation (activation) of glycogen synthase. The latter involves both a direct ligand-induced conformational change and depletion of the phosphorylated form of glycogen phosphorylase, which is a potent allosteric inhibitor of glycogen synthase phosphatase activity associated with the glycogen-targeting protein, GL [hepatic glycogen-targeting subunit of PP-1 (protein phosphatase-1) encoded by PPP1R3B]. Defects in both the activation of glucokinase and in the dephosphorylation of glycogen phosphorylase are potential contributing factors to the dysregulation of hepatic glucose metabolism in Type 2 diabetes.

'Last In-First Out': seasonal variations of non-structural carbohydrates, glucose-6-phosphate and ATP in tubers of two Arum species.

Knowledge on the metabolism of polysaccharide reserves in wild species is still scarce. In natural sites we collected tubers of Arum italicum Mill. and A. maculatum L. - two geophytes with different apparent phenological timing, ecology and chorology - during five stages of the annual cycle in order to understand patterns of reserve accumulation and degradation. Both the entire tuber and its proximal and distal to shoot portion were utilised. Pools of non-structural carbohydrates (glucose, sucrose and starch), glucose-6-phosphate and ATP were analysed as important markers of carbohydrate metabolism. In both species, starch and glucose content of the whole tuber significantly increased from sprouting to the maturation/senescence stages, whereas sucrose showed an opposite trend; ATP and glucose-6-phosphate were almost stable and dropped only at the end of the annual cycle. Considering the two different portions of the tuber, both ATP and glucose-6-phosphate concentrations were higher in proximity to the shoot in all seasonal stages, except the flowering stage. Our findings suggest that seasonal carbon partitioning in the underground organ is driven by phenology and occurs independently of seasonal climate conditions. Moreover, our results show that starch degradation, sustained by elevated ATP and glucose-6-phosphate pools, starts in the peripheral, proximal-to-shoot portion of the tuber, consuming starch accumulated in the previous season, as a 'Last In-First Out' mechanism of carbohydrate storage.

The role of glucose 6-phosphate in mediating the effects of glucokinase overexpression on hepatic glucose metabolism.

Pharmacological activation or overexpression of glucokinase in hepatocytes stimulates glucose phosphorylation, glycolysis and glycogen synthesis. We used an inhibitor of glucose 6-phosphate (Glc6P) hydrolysis, namely the chlorogenic derivative, 1-[2-(4-chloro-phenyl)-cyclopropylmethoxy]-3, 4-dihydroxy-5-(3-imidazo[4,5-b]pyridin-1-yl-3-phenyl-acryloyloxy)-cyclohexanecarboxylic acid (also known as S4048), to determine the contribution of Glc6P concentration, as distinct from glucokinase protein or activity, to the control of glycolysis and glycogen synthesis by glucokinase overexpression. The validity of S4048 for testing the role of Glc6P was supported by its lack of effect on glucokinase binding and its nuclear/cytoplasmic distribution. The stimulation of glycolysis by glucokinase overexpression correlated strongly with glucose phosphorylation, whereas glycogen synthesis correlated strongly with Glc6P concentration. Metabolic control analysis was used to determine the sensitivity of glycogenic flux to glucokinase or Glc6P at varying glucose concentrations (5-20 mm). The concentration control coefficient of glucokinase on Glc6P (1.4-1.7) was relatively independent of glucose concentration, whereas the flux control coefficients of Glc6P (2.4-1.0) and glucokinase (3.7-1.8) on glycogen synthesis decreased with glucose concentration. The high sensitivity of glycogenic flux to Glc6P at low glucose concentration is consistent with covalent modification by Glc6P of both phosphorylase and glycogen synthase. The high control strength of glucokinase on glycogenic flux is explained by its concentration control coefficient on Glc6P and the high control strength of Glc6P on glycogen synthesis. It is suggested that the regulatory strength of pharmacological glucokinase activators on glycogen metabolism can be predicted from their effect on the Glc6P content.

Glucose 6-phosphate causes translocation of phosphorylase in hepatocytes and inactivates the enzyme synergistically with glucose.

The role of glucose 6-P (glucose 6-phosphate) in regulating the activation state of glycogen synthase and its translocation is well documented. In the present study, we investigated the effects of glucose 6-P on the activation state and compartmentation of phosphorylase in hepatocytes. Glucose 6-P levels were modulated in hepatocytes by glucokinase overexpression or inhibition with 5-thioglucose and the effects of AMP were tested using AICAR (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside), which is metabolized to an AMP analogue. Inhibition of glucokinase partially counteracted the effect of glucose both on the inactivation of phosphorylase and on the translocation of phosphorylase a from a soluble to a particulate fraction. The increase in glucose 6-P caused by glucokinase overexpression caused translocation of phosphorylase a to the pellet and had additive effects with glucose on inactivation of phosphorylase. It decreased the glucose concentration that caused half-maximal inactivation from 20 to 11 mM, indicating that it acts synergistically with glucose. AICAR activated phosphorylase and counteracted the effect of glucose 6-P on phosphorylase inactivation. However, it did not counteract translocation of phosphorylase by glucose 6-P. Glucose 6-P and AICAR had opposite effects on the activation state of glycogen synthase, but they had additive effects on translocation of the enzyme to the pellet. There was a direct correlation between the translocation of phosphorylase a and of glycogen synthase to the pellet, suggesting that these enzymes translocate in tandem. In conclusion, glucose 6-P causes both translocation of phosphorylase and inactivation, indicating a more complex role in the regulation of glycogen metabolism than can be explained from regulation of glycogen synthase alone.

ADP-glucose drives starch synthesis in isolated maize endosperm amyloplasts: characterization of starch synthesis and transport properties across the amyloplast envelope.

We recently developed a method of purifying amyloplasts from developing maize (Zea mays L.) endosperm tissue [Neuhaus, Thom, Batz and Scheibe (1993) Biochem. J. 296, 395-401]. In the present paper we analyse how glucose 6-phosphate (Glc6P) and other phosphorylated compounds enter the plastid compartment. Using a proteoliposome system in which the plastid envelope membrane proteins are functionally reconstituted, we demonstrate that this type of plastid is able to transport [14C]Glc6P or [32P]Pi in counter exchange with Pi, Glc6P, dihydroxyacetone phosphate and phosphoenolpyruvate. Glucose 1-phosphate, fructose 6-phosphate and ribose 5-phosphate do not act as substrates for counter exchange. Besides hexose phosphates, ADP-glucose (ADPGlc) also acts as a substrate for starch synthesis in isolated maize endosperm amyloplasts. This process exhibits saturation kinetics with increasing concentrations of exogenously supplied [14C]ADPGlc, reaching a maximum at 2mM. Ultrasonication of isolated amyloplasts greatly reduces the rate of ADPGlc-dependent starch synthesis, indicating that the process is dependent on the intactness of the organelles. The plastid ATP/ADP transporter is not responsible for ADPGlc uptake. Data are presented that indicate that ADPGlc is transported by another translocator in counter exchange with AMP. To analyse the physiology of starch synthesis in more detail, we examined how Glc6P- and ADPGlc-dependent starch synthesis in isolated maize endosperm amyloplasts interact. Glc6P-dependent starch synthesis is not inhibited by increasing concentrations of ADPGlc. In contrast, the rate of ADPGlc-dependent starch synthesis is reduced by increasing concentrations of ATP necessary for Glc6P-dependent starch synthesis. The possible modes of inhibition of ADPGlc-dependent starch synthesis by ATP are discussed with respect to the stromal generation of AMP required for ADPGlc uptake.

Glucose 6-phosphate produced by glucokinase, but not hexokinase I, promotes the activation of hepatic glycogen synthase.

In a previous study (O'Doherty, R. M., Lehman, D. L., Seoane, J., Gómez-Foix, A. M., Guinovart, J. J., and Newgard, C.B. (1996) J. Biol. Chem. 271, 20524-20530), we demonstrated that adenovirus-mediated overexpression of glucokinase but not hexokinase I has a potent enhancing effect on glycogen synthesis in primary hepatocytes. In an effort to understand the underlying mechanism of this differential effect of the two hexokinase isoforms, we have investigated changes in key intracellular metabolites and the activation state of glycogen synthase in cells treated with recombinant adenoviruses expressing the liver isoform of glucokinase (AdCMV-GKL) or hexokinase I (AdCMV-HKI). Glucose 6-phosphate (Glu-6-P) levels are elevated from approximately 1.5 nmol/mg protein to 8-10 nmol/mg protein in both AdCMV-GKL- and AdCMV-HKI-treated hepatocytes as glucose is raised from 1 to 5 mM, levels four times higher than those in untreated cells. In AdCMV-GKL-treated cells, Glu-6-P continues to accumulate at glucose levels greater than 5 mM, reaching a maximum of 120 nmol/mg protein in cells incubated at 25 mM glucose, a value 10 and 50 times greater than the maximal levels achieved in AdCMV-HKI-treated and untreated cells, respectively. In parallel with the changes observed in Glu-6-P levels, increases in UDP-Glc in AdCMV-HKI- and AdCMV-GKL-treated cells were most pronounced at low (1-5 mM) and high (25 mM) glucose levels, respectively. Despite the significant increases in Glu-6-P and UDP-Glc achieved in AdCMV-HKI-treated cells, only AdCMV-GKL-treated cells exhibited increases in glycogen synthase activity ratio and translocation of the enzyme from a soluble to a particulate form relative to untreated control cells. We conclude that Glu-6-P produced by overexpressed glucokinase is glycogenic because it effectively promotes activation of glycogen synthase. Glu-6-P produced by overexpressed hexokinase, in contrast, appears to be unable to exert the same regulatory effects, probably due to the different subcellular distribution of the two glucose-phosphorylating enzymes.