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Acute Inhibition of Hepatic Glucose-6-phosphatase Does Not Affect Gluconeogenesis but Directs Gluconeogenic Flux toward Glycogen in Fasted Rats

2001; Elsevier BV; Volume: 276; Issue: 28 Linguagem: Inglês

10.1074/jbc.m101223200

1083-351X

van Theo Dijk, van der Sluijs, Coen Wiegman, Jfw Baller, L. A. Gustafson, Huibert Burger, AW Herling, Folkert Kuipers, A. J. Meijer, Dirk‐Jan Reijngoud,

Amino Acid Enzymes and Metabolism

Effects of acute inhibition of glucose-6-phosphatase activity by the chlorogenic acid derivative S4048 on hepatic carbohydrate fluxes were examined in isolated rat hepatocytes and in vivo in rats. Fluxes were calculated using tracer dilution techniques and mass isotopomer distribution analysis in plasma glucose and urinary paracetamol-glucuronide after infusion of [U-13C]glucose, [2-13C]glycerol, [1-2H]galactose, and paracetamol. In hepatocytes, glucose-6-phosphate (Glc-6-P) content, net glycogen synthesis, and lactate production from glucose and dihydroxyacetone increased strongly in the presence of S4048 (10 µm). In livers of S4048-treated rats (0.5 mg kg−1 min−1; 8 h) Glc-6-P content increased strongly (+440%), and massive glycogen accumulation (+1260%) was observed in periportal areas. Total glucose production was diminished by 50%. The gluconeogenic flux to Glc-6-P was unaffected (i.e. 33.3 ± 2.0 versus33.2 ± 2.9 µmol kg−1min−1 in control and S4048-treated rats, respectively). Newly synthesized Glc-6-P was redistributed from glucose production (62 ± 1 versus 38 ± 1%;p < 0.001) to glycogen synthesis (35 ± 5%versus 65 ± 5%; p < 0.005) by S4048. This was associated with a strong inhibition (−82%) of the flux through glucokinase and an increase (+83%) of the flux through glycogen synthase, while the flux through glycogen phosphorylase remained unaffected. In livers from S4048-treated rats, mRNA levels of genes encoding Glc-6-P hydrolase (∼9-fold), Glc-6-P translocase (∼4-fold), glycogen synthase (∼7-fold) and L-type pyruvate kinase (∼ 4-fold) were increased, whereas glucokinase expression was almost abolished. In accordance with unaltered gluconeogenic flux, expression of the gene encoding phosphoenolpyruvate carboxykinase was unaffected in the S4048-treated rats. Thus, acute inhibition of glucose-6-phosphatase activity by S4048 elicited 1) a repartitioning of newly synthesized Glc-6-P from glucose production into glycogen synthesis without affecting the gluconeogenic flux to Glc-6-P and 2) a cellular response aimed at maintaining cellular Glc-6-P homeostasis. Effects of acute inhibition of glucose-6-phosphatase activity by the chlorogenic acid derivative S4048 on hepatic carbohydrate fluxes were examined in isolated rat hepatocytes and in vivo in rats. Fluxes were calculated using tracer dilution techniques and mass isotopomer distribution analysis in plasma glucose and urinary paracetamol-glucuronide after infusion of [U-13C]glucose, [2-13C]glycerol, [1-2H]galactose, and paracetamol. In hepatocytes, glucose-6-phosphate (Glc-6-P) content, net glycogen synthesis, and lactate production from glucose and dihydroxyacetone increased strongly in the presence of S4048 (10 µm). In livers of S4048-treated rats (0.5 mg kg−1 min−1; 8 h) Glc-6-P content increased strongly (+440%), and massive glycogen accumulation (+1260%) was observed in periportal areas. Total glucose production was diminished by 50%. The gluconeogenic flux to Glc-6-P was unaffected (i.e. 33.3 ± 2.0 versus33.2 ± 2.9 µmol kg−1min−1 in control and S4048-treated rats, respectively). Newly synthesized Glc-6-P was redistributed from glucose production (62 ± 1 versus 38 ± 1%;p < 0.001) to glycogen synthesis (35 ± 5%versus 65 ± 5%; p < 0.005) by S4048. This was associated with a strong inhibition (−82%) of the flux through glucokinase and an increase (+83%) of the flux through glycogen synthase, while the flux through glycogen phosphorylase remained unaffected. In livers from S4048-treated rats, mRNA levels of genes encoding Glc-6-P hydrolase (∼9-fold), Glc-6-P translocase (∼4-fold), glycogen synthase (∼7-fold) and L-type pyruvate kinase (∼ 4-fold) were increased, whereas glucokinase expression was almost abolished. In accordance with unaltered gluconeogenic flux, expression of the gene encoding phosphoenolpyruvate carboxykinase was unaffected in the S4048-treated rats. Thus, acute inhibition of glucose-6-phosphatase activity by S4048 elicited 1) a repartitioning of newly synthesized Glc-6-P from glucose production into glycogen synthesis without affecting the gluconeogenic flux to Glc-6-P and 2) a cellular response aimed at maintaining cellular Glc-6-P homeostasis. glucose 6-phosphate glucokinase (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.2), G6Pase, glucose-6-phosphatase glucose-6-phosphatase hydrolase (d-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) glucose transporter type 2, G6PT, glucose-6-phosphatase translocase glycogen phosphorylase (glycogen 1,4-α-d-glucan:orthophosphate α-d-glucosyltransferase, EC 2.4.1.1) glycogen synthase (UDP-glucose:glycogen 4-α-d-glucosyltransferase, EC 2.4.1.11) phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxylyase (transphosphorylating), EC 4.1.1.32) glucose-1-phosphate paracetamol-glucuronide 4-morpholinepropanesulfonic acid N,O-Bis(trimethylsilyl)trifluoroacetamide mass isotopomer distribution analysis magnetic resonance spectroscopy Glucose-6-phosphate (Glc-6-P)1 plays a pivotal role in hepatic carbohydrate metabolism both as a metabolite and as a signaling compound. Glc-6-P is the shared intermediate of gluconeogenesis (see Fig. 1, I + IV) and glycogenolysis (Fig. 1, II) and is formed by glucokinase (GK)-mediated glucose phosphorylation (Fig. 1, III). Glc-6-P provides the substrate for glucose production by the liver, via hydrolysis by glucose-6-phosphatase (G6Pase) (Fig. 1, IV). It serves as substrate for glycolysis (Fig. 1, V) and is the obligatory precursor for the synthesis of glycogen via UDP-glucose (Fig. 1, VI). Partitioning of newly synthesized Glc-6-P into glucose production, degradation via glycolysis, or storage as glycogen offers modes of autoregulating hepatic glucose production without affecting the rate of gluconeogenesis. Glc-6-P stimulates the activity of glycogen synthase (GS) b and of GS phosphatase (1Bollen M. Keppens S. Stalmans W. Biochem. J. 1998; 336: 19-31Crossref PubMed Scopus (322) Google Scholar). Glc-6-P and/or its pentose-phosphate derivative xylulose 5-phosphate act as signaling compound in the control of gene expression (see Ref. 2Girard J. Ferre P. Foufelle F. Annu. Rev. Nutr. 1997; 17: 325-352Crossref PubMed Scopus (302) Google Scholarfor a review). Recent data show that the effect of insulin on gene expression of hepatic enzymes involved in carbohydrate metabolism critically depends on concomitant intracellular metabolism of glucose (3Foretz M. Guichard C. Ferre P. Foufelle F. Proc. Natl. Acad. Sci. 2000; 96: 12737-12742Crossref Scopus (591) Google Scholar, 4Shimomura I. Bashkamov Y. Ikemoto S. Horton J.D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. 2000; 96: 13656-13661Crossref Scopus (625) Google Scholar), supporting a sequence of events starting with the direct induction of GK expression by insulin. Enhanced activity of GK results in increased intracellular concentrations of Glc-6-P and/or xylulose 5-phosphate. This appears to be essential in the action of insulin on the stimulation of expression of genes involved in glucose production, glycolysis, and lipogenesis (e.g. the hydrolytic subunit of glucose-6-phosphatase (G6PH), glucose transporter type 2 (GLUT2), liver-type pyruvate kinase ATP-citrate lyase, acetyl-CoA carboxylase, and fatty acid synthase (see Ref. 2Girard J. Ferre P. Foufelle F. Annu. Rev. Nutr. 1997; 17: 325-352Crossref PubMed Scopus (302) Google Scholar for a review). Since Glc-6-P participates in so many reactions in hepatic glucose metabolism, the relationship between hepatic glucose production and gluconeogenesis in vivo is very complex. A major problem in studying Glc-6-P partitioning in vivo resides in the choice of precursor, label, and isotopic model. In earlier studies, substrates labeled with 14C or 13C have been applied followed by determination of positional isotopomer distribution in either plasma glucose (5Consoli A. Kennedy F. Miles J. Gerich J. J. Clin. Invest. 1987; 80: 1303-1310Crossref PubMed Scopus (125) Google Scholar) or in urinaryN-phenylacetylglutamine (6Landau B.R. Schumann W.C. Chandramouli V. Magnusson I. Kumaran K. Wahren J. Am. J. Physiol. 1993; 265: E636-E647PubMed Google Scholar). Relative gluconeogenic fractions obtained in this way were converted into absolute rates of gluconeogenesis by multiplying with the plasma glucose turnover rate. With this method, the contribution of a particular substrate to the gluconeogenic flux directed into plasma glucose can be calculated. More recent methods estimate gluconeogenic flux from precursors directed to plasma glucose; these methods comprise 2H incorporation into specific positions in plasma glucose from2H2O (7Landau B.R. Wahren J. Chandramouli V. Schumann W.C. Ekberg K. Kalhan S.C. J. Clin. Invest. 1995; 95: 172-178Crossref PubMed Scopus (180) Google Scholar) or incorporation of [2-13C]glycerol into mass isotopomers of plasma glucose (8Neese R.A. Schwarz J.-M. Faix D. Turner S. Letscher A. Vu D. Hellerstein M.K. J. Biol. Chem. 1995; 270: 14452-14463Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 9Peroni O. Large V. Beylot M. Am. J. Physiol. 1995; 269: E516-E523PubMed Google Scholar). The development of an improved isotopic model based on the last method allows for the calculation of flux rates of newly synthesized Glc-6-P into plasma glucose as well as into glycogen (10Hellerstein M.K. Neese R.A. Linfoot P. Christiansen M. Turner S. Letscher A. J. Clin. Invest. 1997; 100: 1305-1319Crossref PubMed Scopus (169) Google Scholar). In the latter model, incorporation of [2-13C]glycerol is measured in plasma glucose and urinary paracetamol-glucuronide (p-GlcUA), as markers of two major metabolic routes of Glc-6-P (e.g. hepatic glucose production and glycogen synthesis via UDP-glucose, respectively (Fig. 1, I + IV andI + VI, respectively). The obtained fractional contributions for plasma glucose and UDP-glucose (via p-GlcUA), respectively, are subsequently converted in absolute rates of gluconeogenic flux, directed to each of the compounds, by multiplying with the rates of appearance of plasma glucose and UDP-glucose (via p-GlcUA), respectively. After correction for exchange of newly synthesized Glc-6-P between plasma glucose and glycogen, via UDP-glucose, the total gluconeogenic flux into Glc-6-P is obtained. It should be realized, however, that the gluconeogenic flux into Glc-6-P thus obtained represents a minimal estimate, since the flux of Glc-6-P into glycolysis (Fig. 1, V) is not considered in this isotopic model. Using this isotopic model, we have studied the effects of acute pharmacological inhibition of G6Pase in vitro and in vivo on the rate of gluconeogenesis and on the partitioning of Glc-6-P. Recently, a novel class of chlorogenic acid derivatives has been developed that inhibit G6Pase activity by blocking glucose-6-phosphate translocase (G6PT) (11Hemmerle H. Burger H.-J. Below P. Schubert G. Rippel R. Schindler P.W. Paulus E. Herling A.W. J. Med. Chem. 1997; 40: 137-145Crossref PubMed Scopus (259) Google Scholar). In experiments in anesthetized rats and perfused rat livers, it was demonstrated that these compounds inhibit hepatic glucose production and lower blood glucose concentration in a dose-dependent way (12Herling A.W. Burger H.-J. Schwab D. Hemmerle H. Below P. Schubert G. Am. J. Physiol. 1998; 274: G1087-G1093PubMed Google Scholar, 13Herling A.W. Burger H.-J. Schubert G. Hemmerle H. Schaefer H.-L. Kramer W. Eur. J. Pharmacol. 1999; 386: 75-82Crossref PubMed Scopus (57) Google Scholar). We addressed the following questions. 1) Does inhibition of hepatic glucose production by G6PT blockade result in an inhibition of gluconeogenic flux into Glc-6-P and/or a change in the partitioning of Glc-6-P? 2) Does inhibition of G6PT acutely influence gene expression of enzymes involved in Glc-6-P metabolism? [1-2H]Galactose (99.6% 2H APE) was purchased from Isotec, Inc. (Miamisburg, OH), and [2-13C]glycerol (99.9% 13C APE) and [U-13C]glucose 99.9% 13C APE) were purchased from CIL, Inc. (Andover, MA). All chemicals were pro analysis grade. Infusates were freshly made and sterilized by the Hospital Pharmacy the day before an experiment. Hepatocytes were isolated from 20–24-h-starved male Wistar rats (250 g) by ex situ liver perfusion with collagenase (14Meijer A.J. Lof C. Ramos I.C. Verhoeven A.J. Eur. J. Biochem. 1985; 148: 189-196Crossref PubMed Scopus (91) Google Scholar). Incubations of freshly isolated hepatocytes (5–10 mg dry mass/ml) were carried out at 37 °C in closed 25-ml plastic scintillation vials containing 2 ml in Krebs-Henseleit bicarbonate medium plus 10 mm sodium HEPES (pH 7.4) and, where indicated, either 10 mm dihydroxyacetone or 20 mm glucose as substrate; the gas phase was 95% O2 and 5% CO2 (v/v). Male Wistar rats (275 ± 14 g) were bred at the Central Animal Laboratory, University of Groningen (The Netherlands). The animals were housed in Plexiglas cages (25 × 25 × 30 cm), with a controlled light-dark regime (12 h dark and 12 h light) and had free access to water and food (RMH-B, Hope Farms BV, Woerden, The Netherlands). One week before the experiment the animals were equipped with two permanent heart catheters, one for infusion and one to draw blood samples, as described by Kuipers et al.(15Kuipers F. Havinga R. Bosschieter H. Toorop G.P. Hindriks F.R. Vonk R.J. Gastroenterology. 1985; 88: 403-411Abstract Full Text PDF PubMed Google Scholar). Twenty-four hours before the start of the experiments, food was removed, but the animals had still free access to water. On the day of the experiment, the animals were placed in metabolic cages that allowed continuous collection of urine. The animals were infused with [U-13C]glucose (1.0 ± 0.1 µmol kg−1 min−1), [2-13C]glycerol (9.2 ± 0.5 µmol kg−1 min−1), [1-2H]galactose (4.7 ± 0.2 µmol kg−1 min−1), paracetamol (total dose: 212 ± 10 mg kg−1), and, where indicated, S4048 (total dose: 265 ± 13 mg kg−1) in a sterile isotonic solution consisting of phosphate-buffered saline (pH 7.2) with Me2SO (6.1% v/v). Blood samples (200 µl) were drawn before the start of the infusion and 3, 6, 7, and 8 h thereafter. Timed urine samples were collected at hourly intervals. The blood samples were collected in heparin-containing tubes and centrifuged immediately. Plasma and urine samples were stored at −20 °C until analysis. At the end of the experiment, the animals were anesthetized with pentobarbital; a large blood sample was taken by heart puncture; and the liver was excised and weighed, and parts were frozen immediately in liquid N2. Glucose and lactate in hepatocyte incubations were determined in HClO4-extracted, KOH-neutralized samples with ATP, NADP+, hexokinase, and Glc-6-P dehydrogenase (glucose) and with NAD+ and lactate dehydrogenase (16Bergmeyer H.U. Methods of Enzymatic Analysis. 2nd Ed. Academic Press, Inc., New York1974Google Scholar). The glycogen content of hepatocytes was measured as follows. Aliquots of cells were diluted with 4 volumes of ice-cold 0.9% NaCl with 10 mmMOPS (pH 7.4) and centrifuged. After removal of the clear supernatant, the pellets were dissolved in 0.1 m KOH and heated for 40 min at 85 °C. The solution was acidified to pH 4.5 with acetic acid (3 m) and centrifuged to remove the protein. To 100 µl of the supernatant 0.14 units of amyloglucosidase was added, and the mixture was incubated for 2 h at 40 °C. The glucose formed was measured fluorometrically as described (16Bergmeyer H.U. Methods of Enzymatic Analysis. 2nd Ed. Academic Press, Inc., New York1974Google Scholar). Background glucose was measured in identically treated samples, without addition of amyloglucosidase (16Bergmeyer H.U. Methods of Enzymatic Analysis. 2nd Ed. Academic Press, Inc., New York1974Google Scholar). For measurement of intracellular Glc-6-P, an aliquot of the cell suspension was diluted with 4 volumes of ice-cold 0.9% NaCl plus 10 mm MOPS (pH 7.4) and centrifuged for 1 s in a microcentrifuge. The cell pellet was immediately extracted with HClO4 (4%, w/v), and the precipitate was neutralized with a mixture of 2 m KOH and 0.5 mMOPS. Glc-6-P was determined fluorometrically with NADP+and Glc-6-P dehydrogenase (16Bergmeyer H.U. Methods of Enzymatic Analysis. 2nd Ed. Academic Press, Inc., New York1974Google Scholar). Samples for measurement of glycogen and Glc-6-P of liver tissue were prepared by extracting liquid N2-cooled liver powder (about 100 mg wet weight) with either 1 ml of 0.1 m KOH (glycogen) or HClO4(4%, w/v; Glc-6-P); this was then followed by the same procedure as described above for hepatocytes. Plasma insulin was determined by a radioimmunoassay RI-13K (Linco Research, Inc., St Charles, MO). Plasma glucose concentration was determined enzymatically by use of the Beckman glucose analyzer II (Beckman Instruments, Palo Alto, CA). To visualize glycogen deposition in the liver, staining with PAS was performed on 4-µm-thick slices from frozen livers excised from the studied rats according to standard procedures. Total RNA was isolated from ∼30 mg of liver tissue using the Trizol method (Life Technologies, Inc.) followed by the SV Total RNA Isolation System (Promega, Madison, WI) according to the protocols provided by the manufacturer. Isolated total RNA was converted to single-stranded cDNA by a reverse transcription procedure with M-Mulv-RT (Roche Molecular Biochemicals) according to the manufacturer's protocol. For polymerase chain reaction amplification studies, amounts of cDNA corresponding to ∼30 ng of RNA were amplified with Taq DNA polymerase (Roche Molecular Biochemicals) and the appropriate forward and reverse primers (Life Technologies), essentially according to the manufacturer's protocols and optimized for the particular amplification cycler used. In the same experiments, calibration curves were run on serial dilutions of a 4× concentrated cDNA solution, resulting in a series containing 4×, 2×, 1×, 0.5×, 0.125×, 0.062×, and 0.031× of the cDNA present in the assay incubation. Gel electrophoresis of both assay and calibration incubations were done simultaneously. All gels were photographed with an Image Master VDS system (Amersham Pharmacia Biotech), and intensities were quantified by video-scanning densitometry, using the software program Image Master 1D Elite 3.0 (Amersham Pharmacia Biotech). All quantified intensities of experimental samples were within the linear part of the calibration curves. The following primer sequences were used: G6PH forward primer (ACT TTG GGA TCC AGT CGA CT) and reverse primer (ACA GCA ATG CCT GAC AAG AC); G6PT forward primer (ATG AGA TCG CTC TGG ACA AG) and reverse primer (TTC GGA GTC CAA CAT CAG CA); GK forward primer (GTG GGC TTC ACC TTC TCC TT) and reverse primer (TCA CCA TTG CCA CCA CAT CC); GLUT2 forward primer (GGA TCT GCT CAC ATA GTC AC) and reverse primer (TCT GGA CAG AAG AGC AGT AG); GS forward primer (CCA ATT CCA TGA ATG GCA GG) and reverse primer (GCC TGG ATA AGG ATT CTA GG); GP forward primer (GAG ACT ACA TTC AGG CTG TG) and reverse primer (CTA GCT CAC TGA AGT CCT TG); liver-type pyruvate kinase forward primer (TAC ATT GAC GAC GGG CTC AT) and reverse primer (ATG CTC TCC AGC ATC TGT GT); PEP-CK forward primer (GCC AGG ATC GAA AGC AAG AC) and reverse primer (CCA GTT GTT GAC CAA AGG CT); and β-actin forward primer (AAC ACC CCA GCC ATG TAC G) and reverse primer (ATG TCA CGC ACG ATT TCC C). Fifty microliters of plasma was deproteinized by adding 500 µl of ice-cold ethanol. The mixture was placed on ice for 30 min and then centrifuged. The supernatant was divided into two equal portions. Each portion was transferred to a reaction vial with a Teflon-faced cap and dried by evaporation at 60 °C under N2. After cooling down, the first portion was derivatized to glucose pentaacetate by adding 150 µl of pyridine/acetic anhydride (1:2) (v/v) to the dry residue and incubating for 30 min at 60 °C, followed by drying at 60 °C under N2. The dry residue was dissolved in 500 µl of ethyl acetate and transferred to an injection vial. The second portion was derivatized to glucose-aldonitrile-pentaacetate by adding 50 µl of pyridine containing hydroxylamine (2%; v/v) to the dry residue and incubating for 45 min at 100 °C. After cooling, 100 µl of acetic anhydride was added, and the mixture was incubated for another 30 min at 60 °C, followed by drying at 60 °C under N2. The dry residue was dissolved in 500 µl of ethyl acetate and transferred to an injection vial. For isolation of p-GlcUA, urine samples (0.5 ml) were centrifuged to remove any debris, and the supernatant was injected onto a Nucleosil 7C18 SP250/10 column. The high pressure liquid chromatography system consisted of a Milton Roy CM4000 pump and a Milton Roy SM4000 variable wavelength ultraviolet detector (Interscience, Breda, The Netherlands). Millennium software (Waters, Etten Leur, The Netherlands) was used for peak integration. To achieve base-line separation of the p-GlcUA peak, a two-buffer gradient program was applied consisting of buffer A containing 2% (w/v) ammonium formate in water (pH 4.8) and buffer B containing 40% CH3CN in water. The program started with 100% A and 0% B at 3.3 ml/min. At 10.7 min, the composition was changed to 92.5% A and 7.5% B within 0.1 min, and at 20 min buffer B was increased to 100% within 2 min. Under these conditions, the p-GlcUA peak eluted at 18.7 min, in a volume of 1.2 ml. The collected fraction was divided into two portions of 0.6 ml each. Each fraction was transferred to a Teflon-capped reaction vial, and both fractions were dried at 115 °C under N2. After cooling, p-GlcUA was derivatized to its tetratrimethylsilyl-ethyl ester by adding 400 µl of ethanol/acetylchloride, 10:1 (v/v), to the dry residue and incubating for 45 min at room temperature, followed by drying at 60 °C under N2. To the dry residue, 200 µl of BSTFA/pyridine/chlorotrimethylsilane (5:1:0.07 (v/v)) was added and incubated for 120 min at 90 °C. After drying, 1 ml of ethyl acetate was added. The dry residue of the second fraction was oxidized to saccharic acid by reacting with 35 µl of sodium nitrite (0.4 g/ml water) and 70 µl of nitric acid (32.5% in water) at 130 °C for 25 min, followed by drying at 60 °C under N2. After cooling, saccharic acid was derivatized to its tetraacetate-diethylester by adding 400 µl of ethanol/acetylchloride (10:1 (v/v)) and incubating for 45 min at room temperature, followed by drying at 60 °C under N2. To the dry residue, 150 µl of pyridine/acetic anhydride (1:2 (v/v)) was added and incubated for 30 min at 60 °C, followed by drying at 60 °C under N2. The dry residue was dissolved in 50 µl of ethyl acetate and transferred to an injection vial. All samples were analyzed by gas chromatography-mass spectrometry. Derivatives were separated on a HP 5890 gas chromatograph (Hewlett-Packard, Palo Alto, CA) using an AT-5 20 m × 0.18 mm inner diameter (0.4-µm film thickness) capillary column (Alltech, Breda, the Netherlands). The GC temperature profile for p-GlcUA tetratrimethylsilyl-ethyl ester was as follows: the initial temperature was 250 °C for 2 min and rose then to 280 °C at a rate of 25 °C/min. The column was held at 280 °C for 10 min. The compound eluted at 10.0 min. Them/z 331–337 ions, representing them 0–m 6 mass isotopomers, were monitored in electron impact mode. The same gas chromatography temperature profile was used for glucose-pentaacetate, glucose-aldonitrile-pentaacetate, and saccharic acid tetraacetate-diethyl ester derivatives. The initial temperature was 80 °C for 1 min and rose then to 280 °C at a rate of 20 °C/min. The column was held at 280 °C for 5 min. The compounds eluted at 8.1, 10.6, and 10.9 min, respectively. Chemical ionization with methane was used. The ions monitored for glucose-pentaacetate werem/z 331–337, corresponding to them 0–m 6 mass isotopomers. The ions monitored for glucose-aldonitrile-pentaacetate werem/z 328–334, corresponding to them 0–m 6 mass isotopomers. The ions monitored for saccharic acid tetraacetate-diethyl ester werem/z 375–381, corresponding to them 0–m 6 mass isotopomers. The accuracy of the measurement was checked by injection of a standard sample after every eight experimental samples. The series were rejected when the reproducibility of the measurement of the standard sample was less than 1% for m 0 and less than 2% form 1 and m 2. To check the range of intensities at the m/z values that allows for reproducible analyses, dilution series were routinely made. Metabolic fluxes at steady state were calculated essentially according to Hellerstein et al. (10Hellerstein M.K. Neese R.A. Linfoot P. Christiansen M. Turner S. Letscher A. J. Clin. Invest. 1997; 100: 1305-1319Crossref PubMed Scopus (169) Google Scholar). The isotopic model of hepatic glucose metabolism is very similar to the one shown in Fig. 1, with the exception that glycolysis (Fig. 1,V) is absent. In this model, two metabolic pathways give rise to plasma glucose and hepatic UDP-glucose formation,i.e. the gluconeogenic flux to Glc-6-P (Fig. 1,I) and glycogenolysis (Fig. 1, II). At steady state, glycogenesis (Fig. 1,VI) equals the formation of UDP-glucose (17Magnusson I. Rothman D.L. Jucker B. Cline G.W. Shulman R.G. Shulman G.I. Am. J. Physiol. 1994; 266: E796-E803PubMed Google Scholar, 18Hellerstein M.K. Metabolism. 2000; 49: 1375-1378Google Scholar). Rates of appearance of glucose into the plasma glucose pool (Ra(glc)) and into the UDP-glucose pool (Ra(UDPglc); via p-GlcUA) were calculated by isotope dilution (19Wolfe R.R. Principles and Practice of Kinetic Analysis. 2nd Ed. Wiley-Liss, New York1992: 117-132Google Scholar) as follows, Ra(glc)=(MPE(glc;m6)infuse/MPE(glc;m6)plasma−1)(Eq. 1) ×infusion(glc;m6)in which MPE(glc;m6)infuse and MPE(glc;m6)plasma are the mole percent enrichments of [U-13C]glucose in the infusate and plasma, respectively, and infusion(glc;m6) is the infusion rate of [U-13C]-glucose, and the following,Ra(UDPglc)=(MPE(gal;m1)infuse/MPE(pGlcUA;m1)urine−1)×infusion(gal;m1)(Eq. 2) in which MPE(gal;m1)infuse and MPE(pGlcUA;m1)urine are the mole percent enrichments of [1-2H]galactose in the infusate and p-GlcUA in urine, respectively, and infusion(gal;m1) is the infusion rate of [1-2H]galactose. Ra(UDPglc) was calculated based on the assumption of a constant and complete entry of galactose into the hepatic UDP-glucose pool and that the label distribution in urinary p-GlcUA reflects the label distribution in UDP-glucose. The contribution of recycling should be added to these rates of appearance to obtain the total rates of appearance (10Hellerstein M.K. Neese R.A. Linfoot P. Christiansen M. Turner S. Letscher A. J. Clin. Invest. 1997; 100: 1305-1319Crossref PubMed Scopus (169) Google Scholar, 20Giaccari A. Rossetti L. J. Clin. Invest. 1992; 89: 36-45Crossref PubMed Scopus (75) Google Scholar). For the calculation of recycling, two correction factors are introduced (10Hellerstein M.K. Neese R.A. Linfoot P. Christiansen M. Turner S. Letscher A. J. Clin. Invest. 1997; 100: 1305-1319Crossref PubMed Scopus (169) Google Scholar): the fractional contribution of plasma glucose to UDP-glucose formation c(glc),c(glc)=MPE(pGlcUA;m6)urine/MPE(glc;m6)plasma(Eq. 3) in which MPE(pGlcUA;m6)urine and MPE(glc;m6)plasma are the mole percent enrichments of urinary p-GlcUA and plasma glucose, respectively, during an infusion of [U-13C]glucose and the fractional contribution of UDP-glucose to plasma glucose formation c(UDPglc),c(UDPglc)=MPE(glc;m1)plasma/MPE(pGlcUA;m1)urine(Eq. 4) in which MPE(glc;m1)plasma and MPE(pGlcUA;m1)urine are the mole percent enrichments of urinary p-GlcUA and plasma glucose, respectively, during an infusion of [1-2H]galactose. Recycling of glucose (r(glc)) and UDP-glucose (r(UDPglc)) were calculated according to the following,r(glc)=(c(glc)/(1−c(glc)))×Ra(glc)(Eq. 5) which is also a measure of glucose/Glc-6-P cycling (10Hellerstein M.K. Neese R.A. Linfoot P. Christiansen M. Turner S. Letscher A. J. Clin. Invest. 1997; 100: 1305-1319Crossref PubMed Scopus (169) Google Scholar, 20Giaccari A. Rossetti L. J. Clin. Invest. 1992; 89: 36-45Crossref PubMed Scopus (75) Google Scholar), and the equation,r(UDPglc)=(c(UDPglc)/(1−c(UDPglc)))×Ra(UDPglc)(Eq. 6) Total rates of appearance of glucose into the plasma glucose pool (total Ra(glc)) and into the hepatic UDP-glucose pool (total Ra(UDPglc)) were calculated according to the following,total Ra(glc)=Ra(glc)+r(glc)(Eq. 7) andtotal Ra(UDPglc)=Ra(UDPglc)+r(UDPglc)(Eq. 8) The fractional gluconeogenic flux into both plasma glucose (f(glc)) and hepatic UDP-glucose (f(UDPglc); as measured in urinary p-GlcUA) were calculated by MIDA as described in detail elsewhere (8Neese R.A. Schwarz J.-M. Faix D. Turner S. Letscher A. Vu D. Hellerstein M.K. J. Biol. Chem. 1995; 270: 14452-14463Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 21Hellerstein M.K. Neese R.A. Am. J. Physiol. 1992; 263: E988-E1001PubMed Google Scholar). The gluconeogenic flux into plasma glucose (GNG(glc)) and into UDP-glucose (GNG(UDPglc)) was calculated according to the following, GNG(glc)=f(glc)×total Ra(glc)(Eq. 9) andGNG(UDPglc)=f(UDPglc)×total Ra(UDPglc)(Eq. 10) Total gluconeogenic flux (total GNG) is the sum of both components corrected for the exchange of label between the plasma glucose and hepatic UDP-glucose pools (10Hellerstein M.K. Neese R.A. Linfoot P. Christiansen M. Turner S. Letscher A. J. Clin. Invest. 1997; 100: 1305-1319Crossref PubMed Scopus (169) Google Scholar),total GNG=(1−c(glc))×GNG(glc)+(1−c(UDPglc))×GNG(UDPglc)(Eq. 11) The contribution of glycogenolysis to glucose formation (GLY(glc)) and to UDP-glucose formation (GLY(UDPglc)) were calculated according to the following, GLY(glc)=(1−f(glc))×total Ra(glc)(Eq. 12) in which the contribution of glycogenolysis to the total rate of appearance of glucose in plasma is equal to the part, which does not derive from gluconeogenesis, and the equation,GLY(UDPglc)=(1−f(UDPglc))×total Ra(UDPglc)−c(glc)×total Ra(UDPglc)(Eq