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J. Biol. Chem., Vol. 276, Issue 28, 25727-25735, July 13, 2001

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

A PHARMACOLOGICAL STUDY WITH THE CHLOROGENIC ACID DERIVATIVE S4048^*

Theo H. van Dijk, Fjodor H. van der Sluijs, Coen H. Wiegman, Julius F. W. Baller, Lori A. Gustafson^§, Hans-Joerg Burger^¶, Andreas W. Herling^¶, Folkert Kuipers, Alfred J. Meijer^§, and Dirk-Jan Reijngoud

From the  Laboratory of Pediatrics, Center for Liver, Digestive and Metabolic Diseases, University Hospital Groningen, Groningen 9700 RB, The Netherlands, the ^§ Department of Biochemistry, Academic Medical Center, Amsterdam 1105 AZ, The Netherlands, and ^¶ Aventis Pharma Deutschland GmbH, Frankfurt aM 65926, Germany

Received for publication, February 8, 2001, and in revised form, April 18, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 kg¹ min¹; 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 versus 33.2 ± 2.9 µmol kg¹ min¹ 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (1). Glc-6-P and/or its pentose-phosphate derivative xylulose 5-phosphate act as signaling compound in the control of gene expression (see Ref. 2 for 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 (3, 4), 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. 2 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 (5) or in urinary N-phenylacetylglutamine (6). 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 from ²H₂O (7) or incorporation of [2-¹³C]glycerol into mass isotopomers of plasma glucose (8, 9). 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 (10). 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 and I + 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) (11). 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 (12, 13). 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?

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

[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.

Methods

In Vitro Experiments Hepatocytes were isolated from 20-24-h-starved male Wistar rats (250 g) by ex situ liver perfusion with collagenase (14). 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).

In Vivo Experiments 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. (15). 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 kg¹ min¹), [2-¹³C]glycerol (9.2 ± 0.5 µmol kg¹ min¹), [1-²H]galactose (4.7 ± 0.2 µmol kg¹ min¹), paracetamol (total dose: 212 ± 10 mg kg¹), and, where indicated, S4048 (total dose: 265 ± 13 mg kg¹) 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.

Metabolite Assays 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 (16). 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 mM MOPS (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 (16). Background glucose was measured in identically treated samples, without addition of amyloglucosidase (16). 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 M MOPS. Glc-6-P was determined fluorometrically with NADP⁺ and Glc-6-P dehydrogenase (16). 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).

Liver Histology 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.

Hepatic mRNA Levels 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 -actin forward primer (AAC ACC CCA GCC ATG TAC G) and reverse primer (ATG TCA CGC ACG ATT TCC C).

Mass Isotopomer Distribution Analysis

Isolation and Derivatization of Plasma Glucose-- 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.

Isolation and Derivatization of p-GlcUA-- 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 N_2. 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.

Gas Chromatography-Mass Spectrometry Procedures-- 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. The m/z 331-337 ions, representing the m₀-m₆ 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 were m/z 331-337, corresponding to the m₀-m₆ mass isotopomers. The ions monitored for glucose-aldonitrile-pentaacetate were m/z 328-334, corresponding to the m₀-m₆ mass isotopomers. The ions monitored for saccharic acid tetraacetate-diethyl ester were m/z 375-381, corresponding to the m₀-m₆ 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 m₀ and less than 2% for m₁ and m₂. To check the range of intensities at the m/z values that allows for reproducible analyses, dilution series were routinely made.

Calculations-- Metabolic fluxes at steady state were calculated essentially according to Hellerstein et al. (10). 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 (17, 18).

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Schematic model of hepatic carbohydrate metabolism. Major metabolic pathways and enzymatic reaction in hepatic carbohydrate metabolism, sharing glucose 6-phosphate as metabolite. These metabolic pathways are as follows: I, de novo synthesis of Glc-6-P; II, glycogenolysis; III, glucose phosphorylation; VI, glucose 6-phosphate hydrolysis; V, glycolysis; VI, glycogen synthesis. The gluconeogenic flux to glucose (gluconeogenesis) is represented by I + IV, and flux to UDP-glucose is shown by I + VI.

Rate of Appearance and Recycling-- 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 (19) as follows,

(Eq. 1)

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,

(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 (10, 20). For the calculation of recycling, two correction factors are introduced (10): the fractional contribution of plasma glucose to UDP-glucose formation c(glc),

(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),

(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,

(Eq. 5)

which is also a measure of glucose/Glc-6-P cycling (10, 20), and the equation,

(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,

(Eq. 7)

and

(Eq. 8)

Rate of Gluconeogenesis-- 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 (8, 21). The gluconeogenic flux into plasma glucose (GNG(glc)) and into UDP-glucose (GNG(UDPglc)) was calculated according to the following,

(Eq. 9)

and

(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 (10),

(Eq. 11)

Glycogenolysis-- The contribution of glycogenolysis to glucose formation (GLY(glc)) and to UDP-glucose formation (GLY(UDPglc)) were calculated according to the following,

(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,

(Eq. 13)

in which c(glc) × total Ra(UDPlgc) is the flux of plasma glucose into the hepatic UDP-glucose pool. In contrast to plasma glucose, the total rate of appearance of UDP-glucose consists of three contributions: gluconeogenic flux from Glc-6-P, glycogenolysis, and the flux of plasma glucose into the UDP-glucose pool. This flux of glycogen into UDP-glucose is a measure of glycogen/glucose 1-phosphate (Glc-1-P) cycling (18).

Individual Fluxes through Enzymes-- The individual fluxes through the enzymes GK, G6Pase, GS, and GP were calculated according to the equation,

(Eq. 14)

in which two contributions to the total flux through GK are considered, i.e. the flux of plasma glucose into UDP-glucose and glucose/Glc-6-P cycling as follows,

(Eq. 15)

(Eq. 16)

and

(Eq. 17)

in which two contributions to the total flux through GP are considered, i.e. glycogenolysis resulting in plasma glucose appearance and glycogen/Glc-1-P cycling.

Statistics-- All values are expressed as mean ± S.D. Statistical significance was determined using Student's t test. p < 0.05 was considered as significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

S4048 Stimulates Glycogenesis and Glycolysis in Isolated Hepatocytes-- Table I summarizes the effects of S4048 on dihydroxyacetone and glucose metabolism in freshly isolated rat hepatocytes. S4048 at 10 µM completely inhibited glucose production from dihydroxyacetone, which was accompanied by an increase in lactate production and glycogen synthesis. In the presence of glucose, S4048 caused a significantly increased lactate production and strongly induced glycogen synthesis. Cellular Glc-6-P concentrations were substantially increased in the presence of S4048, with either glucose or dihydroxyacetone as the substrate.

S4048 Affects Plasma and Hepatic Parameters of Glucose Metabolism in Conscious Rats-- At the start of the experiment, plasma concentrations of glucose and insulin were similar in control and S4048-treated rats (Table II). Plasma glucose concentration slightly increased during the experiment, i.e. by 23%, in control animals. In the animals treated with S4048, plasma glucose concentration dropped from ~4.4 to ~3.5 mM (20%) during the first 3 h of the experiment and remained unchanged thereafter. Insulin concentrations in S4048-treated rats decreased significantly by 56%, in contrast to the control group in which plasma insulin was slightly elevated (+32%). The Glc-6-P content of the liver was significantly higher at the end of the experiment in animals treated with S4048 compared with the control group (+346%), and S4048-treated animals showed an almost 13-fold increase in hepatic glycogen content. At the end of the experiment, liver weight was slightly increased in S4048-treated rats (8.5 ± 0.4 g wet weight versus 9.3 ± 0.4 g wet weight, control versus S4048-treated, respectively).

S4048 Induces Massive Periportal Glycogen Accumulation in the Liver-- Fig. 2A confirms that glycogen was almost absent in the livers from control rats. In livers of S4048-treated rats (Fig. 2B), on the other hand, massive amounts of PAS-positive material were present, indicating a high content of glycogen: most of the glycogen was present in periportal hepatocytes, i.e. the cells surrounding the portal vein.

View larger version (139K): [in this window] [in a new window]   Fig. 2.   Effect of S4048 on glycogen accumulation and distribution in the liver. Livers of rats infused with either vehicle or S4048 for 8 h were treated with PAS to stain for glycogen and were examined by light microscopy. A, a representative micrograph of a liver of vehicle-treated rat; B, a representative micrograph for an S4048-treated rat. PV, perivenous area; PP, periportal area.

S4048 Changes Partitioning of Glc-6-P without Altering Gluconeogenic Flux to Glc-6-P-- Fig. 3 shows the effects of S4048 treatment on total glucose production (Fig. 3A) and on total UDP-glucose production (Fig. 3B). The total glucose production rate decreased from 39.9 ± 3.8 µmol kg¹ min¹ in the control animals to 19.6 ± 4.2 µmol kg¹ min¹ in animals treated with S4048. At the same time, the total UDP-glucose production significantly increased from 19.8 ± 1.8 in the control animals to 30.7 ± 1.5 µmol kg¹ min¹ in S4048-treated rats. Compared with control animals, the total gluconeogenic flux into Glc-6-P was not changed significantly in animals treated with S4048 (Fig. 4; 33.3 ± 2.0 versus 33.2 ± 2.9 µmol kg¹ min¹ in control versus S4048-treated, respectively). The flux of de novo synthesized Glc-6-P directed to plasma glucose, however, was significantly decreased in S4048-treated animals as compared with controls (from 20.8 ± 1.7 to 11.6 ± 2.4 µmol kg¹ min¹). In contrast, the flux of newly synthesized Glc-6-P directed to UDP-glucose significantly increased in S4048-treated animals as compared with controls (from 12.5 ± 0.4 to 21.6 ± 0.8 µmol kg¹ min¹). As a consequence, the partitioning of newly synthesized Glc-6-P changed from 62 ± 1% into plasma glucose and 38 ± 1% into glycogen in control rats to 35 ± 5% into plasma glucose and 65 ± 5% into glycogen in S4048-treated rats.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Effects of S4048 treatment in fasted rats on total plasma glucose production (A) and UDP-glucose production (B). The metabolic fluxes were calculated using the equations for total Ra(glc) (IV; Fig. 1) and total Ra(UDPglc) (VI; Fig. 1) in A and B, respectively, as described under "Experimental Procedures." *, significantly different between control and S4048.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Effect of S4048 inhibitor on gluconeogenesis flux and partitioning. The gluconeogenic fluxes are shown directed into the plasma glucose pool (dark gray bar) and into the UDP-glucose pool (light gray bar). The fluxes were calculated using the equations for GNG(glc) (I + IV; Fig. 1) and GNG(UDPglc) (I + VI; Fig. 1), respectively, as described under "Experimental Procedures." *, significantly different between control and S4048

S4048 Affects in Vivo Fluxes through Enzymes Involved in Glc-6-P Metabolism-- In Fig. 5, the values of the various fluxes through enzymes involved in Glc-6-P metabolism are shown, as far as these flux rates could be estimated by the isotopic model used. Administration of S4048 resulted in a decrease of the flux through G6Pase from 39.9 ± 3.8 µmol kg¹ min¹ to 19.6 ± 4.2 µmol kg¹ min¹ and through GK from 10.1 ± 0.4 µmol kg¹ min¹ to 1.6 ± 0.5 µmol kg¹ min¹. Glucose/Glc-6-P cycling decreased from 6.4 ± 0.1 µmol kg¹ min¹ to 0.6 ± 0.3 µmol kg¹ min¹. The flux through GS increased upon administration of S4048 from 19.8 ± 1.8 µmol kg¹ min¹ to 30.7 ± 1.5 µmol kg¹ min¹, whereas the flux through GP was almost unaffected (16.9 ± 5.3 versus 15.6 ± 2.0 µmol kg¹ min¹). Glycogen/Glc-1-P cycling increased from 1.7 ± 2.1 to 7.8 ± 0.6 µmol kg¹ min¹.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Effects of S4048 treatment in fasted rats on the fluxes through hepatic carbohydrate pathways. The metabolic fluxes in vehicle-treated rats are shown in light gray bars, whereas the metabolic fluxes in S4048-treated rats are shown in dark gray bars. Individual fluxes were calculated as described under "Experimental Procedures," using the equations for GK (Fig. 1, III), G6Pase (Fig. 1, IV), GS (Fig. 1, VI), and GP (Fig. 1, II). Glucose/Glc-6-P and glycogen/Glc-1-P recycling were calculated using the equations for r(glc) and GLY(UDPglc), respectively. In Fig. 1, these fluxes are the part of III that enters IV and the part of II that enters VI, respectively.

S4048 Treatment Induces Rapid Changes in Gene Expression-- Expression of genes involved in hepatic carbohydrate metabolism were studied by semiquantitative polymerase chain reaction (Fig. 6). Treatment with S4048 resulted in markedly increased mRNA levels of the genes encoding GLUT2, G6PH and G6PT, GS, and liver-type pyruvate kinase within the 8 h time frame of the experiment. In contrast, GK gene expression was strongly suppressed. As expected on the basis of flux measurements, the mRNA levels of PEP-CK and GP were unaffected.

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Effects of S4048 treatment in fasted rats on gene expression of enzymes involved in Glc-6-P metabolism. A, gel electrophoresis patterns of reverse transcriptase-polymerase chain reaction products of enzymes indicated and of -actin obtained from livers of vehicle-treated (control) or S4048-treated rats (S4048). B, quantification of gel patterns as described under "Experimental Procedures." The intensity ratios of the indicated enzyme over -actin are plotted. Open squares, individual animals in the vehicle-treated group; closed circles, S4048-treated animals. PK, pyruvate kinase.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This study reveals striking, rapid effects of acute pharmacologic inhibition of G6PT by S4048 on hepatic glucose metabolism in fasted rats. The absence of G6PT activity underlies glycogen storage disease type Ib. In the clinical presentation of this inborn error of metabolism, both the primary metabolic effects, due to the absence of the translocase activity, and the metabolic adaptations that occur contribute to the characteristic phenotype observed in these patients; i.e. fasting induced hypoglycemia, hyperlactacidemia, and hyperlipidemia. A similar combination of primary and secondary effects is present in the recently generated G6PH knock-out mice (22).

In view of the pivotal role of Glc-6-P in glucose metabolism, we interpret the changes in hepatic glucose metabolism induced by S4048 as a coordinate response aimed at maintenance of hepatocellular Glc-6-P concentration. Several experimental (23-25) studies and theoretical considerations (26) have emphasized the importance of maintaining constant concentrations of intermediates that are shared by several metabolic pathways. For Glc-6-P metabolism in muscle, Shulman et al. (23, 24) proposed that changes in GS activity did not control glycogen synthesis but, instead, were aimed at maintaining a constant intracellular Glc-6-P concentration. Aiston et al. (25) proposed that activity of G6Pase in hepatocytes changed in such a way that hepatocellular Glc-6-P concentration was maintained during adenoviral G6Pase overexpression in freshly isolated hepatocytes. In line with this proposal, it was shown previously that inhibition of G6PT in rats resulted in an increase in steady state mRNA levels of G6PH (27). From a theoretical point of view, Kacser and Acerenza (26) argued that homeostasis of shared intermediates is necessary for independent regulation of metabolic pathways involved.

The validity of the isotopic model and the MIDA approach has been substantiated in various studies, although some controversy still remains (28-40). Like any method, the MIDA approach is based on certain assumptions. Several of these assumptions have been addressed both experimentally (36, 39-42) and theoretically (43), and the outcomes of these studies have been critically reviewed (43, 44). The methodology tolerates a wide range of label disequilibrium in the triose phosphate pool. It may be sensitive to isotope gradients in the triose phosphate pool across the liver (i.e. those in periportal and perivenous cells), but the existence of such a gradient has not yet been proven experimentally. On the contrary, recent data by Siler et al. (33) make the existence of such a gradient unlikely. Although the applied [2-¹³C]glycerol infusion rates are high in comparison with the usual infusion rates in in vivo tracer experiments, only minor confounding effects are to be expected due to [2-¹³C]glycerol. Previs et al. (40) have shown in 30-h fasted mice that steady state concentrations of glycerol in plasma started to increase at a glycerol infusion rate of 60 µmol kg¹ min¹ and that the endogenous glucose production started to increase at 120 µmol kg¹ min¹. In our experiments in rats, fasted for 24 h, a [2-¹³C]glycerol infusion rate of less than 10 µmol kg¹ min¹ was used. The calculated isotope mole percent enrichment of the "true triose phosphate" precursor pool for de novo Glc-6-P synthesis (p value) was about 15% in our experiments, indicating that the [2-¹³C]glycerol infusion contributed only moderately to the total production rate of intracellular triose phosphate. Finally, direct comparison of independent isotopic methods to estimate gluconeogenesis has yielded either very similar or slightly lower values for the MIDA method (41, 45). For our comparative study, these concerns are of lesser importance. We studied changes in de novo synthesis of Glc-6-P and partitioning of newly synthesized Glc-6-P brought about by acute inhibition of hepatic glucose production. Measurements were done under very similar conditions, and, as a consequence, the results obtained reflect actual changes in Glc-6-P metabolism.

Quantitatively, the changes in the calculated fluxes through GS and GP brought about by S4048 were almost equal to the measured amount of glycogen found in livers of S4048-treated rats at the end of the experiment. Glycogen accumulation is the net result of the opposing fluxes through GS and GP. In the presence of S4048, the difference between the flux through GS (~30 µmol kg¹ min¹) and GP (~15 µmol kg¹ min¹) equals ~15 µmol kg¹ min¹. At the end of the experiment, this results in 7200 µmol kg¹ or ~225 µmol g wet weight¹ of glycogen (liver weight was ~34 g wet weight kg¹), matching the measured amount of glycogen formed (~225 µmol g wet weight¹; Table II). The increased net glycogen synthesis (~15 µmol kg¹ min¹) was, however, less than the decrease in endogenous glucose production (~20 µmol kg¹ min¹) brought about by S4048. The remainder of the decrease in total glucose production (~5 µmol kg¹ min¹) can be accounted for by the decrease in glucose/Glc-6-P cycling (cf. Equation 7), which decreased from ~6 µmol kg¹ min¹ to ~1 µmol kg¹ min¹ in the presence of S4048.

The de novo synthesis of Glc-6-P was unaffected by inhibition of G6PT. When gluconeogenesis would have been calculated based on the fractional contribution to plasma glucose alone, our results would have led us to conclude that gluconeogenesis was inhibited in parallel with inhibition of glucose production. By analyzing both plasma glucose and urinary p-GlcUA, however, we were able to show that the decrease in hepatic glucose production was not associated with a decrease in the gluconeogenic flux to Glc-6-P but with a predominant partitioning of newly synthesized Glc-6-P into glycogen. Thus, no feedback inhibition on the gluconeogenic flux by its product Glc-6-P was observed in the 8-h time frame of the experiment. Inhibition of G6PT decreased plasma glucose and insulin concentrations as well. The rate of de novo synthesis of Glc-6-P was also not increased in the face of decreased plasma glucose and insulin concentration. Gene expression of PEP-CK was found to be unaffected, in parallel with the unaffected gluconeogenic flux to Glc-6-P. The role of PEP-CK in controlling the gluconeogenic flux is a matter of controversy. Although PEP-CK has been claimed to be rate-limiting in gluconeogenesis (46), measurements until now did not substantiate this claim. In hepatocytes from fasted rats, PEP-CK exerted only minor control over gluconeogenesis from lactate (47). Recent data by the group of Magnuson (48), using an allelogenic CreloxP gene targeting strategy to inactivate PEP-CK specifically in mouse liver, substantiate these observations. Hepatic glucose production did not diminish until activity of PEP-CK in liver reached levels below 90-95% of its initial activity. Hormone-stimulated PEP-CK gene expression can be repressed by high extracellular glucose concentrations in vitro after intracellular metabolism of glucose (49, 50). We did not observe such a regulation. In our in vivo experiments, the high intracellular Glc-6-P concentration was, however, accompanied by low plasma glucose and insulin concentrations, which might contribute to the observed difference in outcome of the in vitro findings and our study (49, 50).

Acute inhibition of G6PT in vivo raised hepatocellular Glc-6-P concentration and abolished glucose/Glc-6-P cycling. The simultaneous action of G6PH, G6PT, and GK represents a homeostatic mechanism aimed at maintaining a constant intracellular Glc-6-P concentration (25). GK enzyme activity does not experience feedback inhibition by Glc-6-P (see Ref 51. and references therein), so that Glc-6-P in excess of metabolic demands must be hydrolyzed by G6PH. Inhibition of G6PT interferes with this homeostatic mechanism. In isolated hepatocytes, inhibition of G6PT also increased glucose incorporation into glycogen and glycogenolysis. This emphasizes the importance of glucose/Glc-6-P cycling in hepatocellular glucose metabolism. As has been reported previously, high intracellular concentrations of Glc-6-P markedly stimulated expression of the gene encoding G6PH (27). This was confirmed in the present study. GK gene expression, on the other hand, is strongly reduced by S4048 treatment. This suggest that at high intracellular Glc-6-P concentrations, a negative control system is operational to down-regulate GK expression, quite different from in vitro studies on GK gene expression (3, 4). In the latter studies, GK gene expression was found not to depend on intracellular glucose metabolism. Irrespective of the very low GK mRNA levels, some glucose phosphorylation did still occur, as is evident from our calculations. It is important to realize that t_1/2 of the GK protein is relatively long (30 h; cf. Ref. 51) in comparison with the duration of the experiment. The role of increased expression of the gene encoding for GLUT2 in maintaining a constant hepatocellular Glc-6-P concentration is not clear, particularly since very recent data show that glucose production from pyruvate is not affected in hepatocytes isolated from GLUT2 knock-out mice (52). The absence of GLUT2 did, however, lead to a sustained elevated intracellular Glc-6-P. This indicates a role of GLUT2 in regulating intracellular Glc-6-P concentration by exporting cytosolic glucose, thereby preventing rephosphorylation of glucose by GK.

Glycogen synthesis was strongly stimulated upon inhibition of G6PT. This was accompanied by an increased glycogen/Glc-1-P cycling. Apparently, both GS and GP were simultaneously active. Glc-6-P is an allosteric activator of GS b in hepatocytes and also activates GS phosphatase, while glucose is a competitive inhibitor of GP a activity and promotes the dephosphorylation and inactivation of GP (1). Inhibition of G6PT raised the hepatocellular concentration of Glc-6-P. This may have stimulated GS b activity and/or promoted its dephosphorylation into its active a form by glycogen-associated protein phosphatase-1. As a consequence, flux through GS increased. On the other hand, inhibition of G6PT also decreased the concentration of plasma glucose, so that the activity of GP a may remain high. The observations on glycogen/Glc-1-P cycling are in line with studies by others with in vivo ¹³C MRS on the simultaneous synthesis and degradation of liver glycogen during a D-[1-¹³C]glucose infusion in fasted and fed rats (53) and humans (17). The continuous degradation and synthesis of glycogen adds to Glc-6-P homeostasis. Newsholme and Crabtree (54) have argued that, in the presence of substrate cycling, large fluctuations in concentrations of intermediates can be dampened by relatively small changes in the rates of the opposing reaction, constituting the substrate cycle. In our study, GS gene expression was increased, while gene expression of GP was unaffected. The physiological importance of these changes in regulation of glycogen metabolism is not yet clear, but they may point to a control loop, at the level of gene expression, by which Glc-6-P stimulates its own deposition into glycogen, which adds to the proposed homeostatic mechanism.

In freshly isolated hepatocytes, in short term incubations, glycolysis was strongly stimulated in the presence of S4048, and glucose was more effectively converted into lactate. These observations point to the importance of glucose/Glc-6-P cycling in glucose metabolism in this in vitro experimental system. Likewise, in vivo treatment of rats with S4048 resulted in an increased plasma lactate concentration, and the expression of the gene encoding liver-type pyruvate kinase was markedly up-regulated. Regulation of liver-type pyruvate kinase critically depends on glucose metabolism. Both Glc-6-P and xylulose 5-phosphate have been implicated in this regulation (see Ref. 2 for a review).

Results of a number of studies on glycogen synthesis are in line with the proposed notion that the gluconeogenic flux to Glc-6-P is not subjected to acute changes (cf. Ref. 55) under various experimental conditions. For instance, during refeeding after a period of fasting, glycogen is synthesized by two metabolic routes: a "direct" one (Glc  Glc-6-P  UDP-Glc  glycogen; Fig. 1, III + VI) and an "indirect" one (Glc  C₃-compound  Glc-6-P  UDP-Glc  glycogen; Fig. 1, III + V + I + VI) (55). After glucose phosphorylation and glycolysis, the "indirect" pathway is identical to the gluconeogenic flux to Glc-6-P with subsequent partitioning of newly synthesized Glc-6-P into glycogen. Partitioning of Glc-6-P will determine whether newly synthesized Glc-6-P will go to either glucose production or glycogen synthesis. This partitioning is a function of the relative activities of the enzymes involved in Glc-6-P metabolism. In case of NIDDM, with inappropriately high hepatic glucose production, this partitioning mechanism may be perturbed. In fact, it has been reported that in patients with NIDDM the activity ratio of G6Pase over GK was increased (56). Increasing the activity ratio of G6Pase over GK by adenovirus-mediated overexpression of the gene encoding G6PH was associated with increased hepatic glucose production in conscious rats (57). Overexpressing the gene encoding for the GK and thereby decreasing the activity ratio of G6Pase over GK resulted in a decreased hepatic glucose production in conscious rats (58).

In summary, this study showed that acute pharmacologic inhibition of G6PT resulted in a marked increase in hepatocellular Glc-6-P and glycogen without affecting the gluconeogenic flux to Glc-6-P. The expression of genes of enzymes in glucose cycling, glycogen synthesis, and glycolysis was changed in such a way to maximize the ability to deposit newly synthesized Glc-6-P into glycogen in order to maintain cellular Glc-6-P homeostasis.

	ACKNOWLEDGEMENTS

We thank Rick Havinga and Theo Boer for excellent technical assistance.

	FOOTNOTES

^* This study was supported by Dutch Diabetes Research Foundation Grant 96.604.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 31-50-361-3295; Fax: 31-50-361-1746; E-mail: d.j.reijngoud@med.rug.nl.

Published, JBC Papers in Press, May 9, 2001, DOI 10.1074/jbc.M101223200

	ABBREVIATIONS

The abbreviations used are: Glc-6-P, glucose 6-phosphate; GK, glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.2), G6Pase, glucose-6-phosphatase; G6PH, glucose-6-phosphatase hydrolase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9); GLUT2, glucose transporter type 2, G6PT, glucose-6-phosphatase translocase; GP, glycogen phosphorylase (glycogen 1,4--D-glucan:orthophosphate -D-glucosyltransferase, EC 2.4.1.1); GS, glycogen synthase (UDP-glucose:glycogen 4--D-glucosyltransferase, EC 2.4.1.11); PEP-CK, phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxylyase (transphosphorylating), EC 4.1.1.32); Glc-1-P, glucose-1-phosphate; p-GlcUA, paracetamol-glucuronide; MOPS, 4-morpholinepropanesulfonic acid N,O-Bis(trimethylsilyl)trifluoroacetamide; MIDA, mass isotopomer distribution analysis; MRS, magnetic resonance spectroscopy.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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