Evidence against Glycogen Cycling of Gluconeogenic Substrates in Various Liver Preparations
2002; Elsevier BV; Volume: 277; Issue: 32 Linguagem: Inglês
10.1074/jbc.m201565200
ISSN1083-351X
AutoresKeld Fosgerau, Jens Breinholt, James G. McCormack, Niels Westergaard,
Tópico(s)Metabolism and Genetic Disorders
ResumoThe effect of inhibition of glycogen phosphorylase by 1,4-dideoxy-1,4-imino-d-arabinitol on rates of gluconeogenesis, gluconeogenic deposition into glycogen, and glycogen recycling was investigated in primary cultured hepatocytes, in perfused rat liver, and in fed or fasted rats in vivo clamped at high physiological levels of plasma lactate. 1,4-Dideoxy-1,4-imino-d-arabinitol did not alter the synthesis of glycerol-derived glucose in hepatocytes or lactate-derived glucose in perfused liver or fed or fasted rats in vivo. Thus, 1,4-dideoxy-1,4-imino-d-arabinitol inhibited hepatic glucose output in the perfused rat liver (0.77 ± 0.19versus 0.33 ± 0.09, p < 0.05), whereas the rate of lactate-derived gluconeogenesis was unaltered (0.22 ± 0.09 versus 0.18 ± 0.08,p = not significant) (1,4-dideoxy-1,4-imino-d-arabinitol versusvehicle, μmol/min * g). Overall, the data suggest that 1,4-dideoxy-1,4-imino-d-arabinitol inhibited glycogen breakdown with no direct or indirect effects on the rates of gluconeogenesis. Total end point glycogen content (μmol of glycosyl units/g of wet liver) were similar in fed (235 ± 19versus 217 ± 22, p = not significant) or fasted rats (10 ± 2 versus 7 ± 2, p = not significant) with or without 1,4-dideoxy-1,4-imino-d-arabinitol, respectively. The data demonstrate no glycogen cycling under the investigated conditions and no effect of 1,4-dideoxy-1,4-imino-d-arabinitol on gluconeogenic deposition into glycogen. Taken together, these data also suggest that inhibition of glycogen phosphorylase may prove beneficial in the treatment of type 2 diabetes. The effect of inhibition of glycogen phosphorylase by 1,4-dideoxy-1,4-imino-d-arabinitol on rates of gluconeogenesis, gluconeogenic deposition into glycogen, and glycogen recycling was investigated in primary cultured hepatocytes, in perfused rat liver, and in fed or fasted rats in vivo clamped at high physiological levels of plasma lactate. 1,4-Dideoxy-1,4-imino-d-arabinitol did not alter the synthesis of glycerol-derived glucose in hepatocytes or lactate-derived glucose in perfused liver or fed or fasted rats in vivo. Thus, 1,4-dideoxy-1,4-imino-d-arabinitol inhibited hepatic glucose output in the perfused rat liver (0.77 ± 0.19versus 0.33 ± 0.09, p < 0.05), whereas the rate of lactate-derived gluconeogenesis was unaltered (0.22 ± 0.09 versus 0.18 ± 0.08,p = not significant) (1,4-dideoxy-1,4-imino-d-arabinitol versusvehicle, μmol/min * g). Overall, the data suggest that 1,4-dideoxy-1,4-imino-d-arabinitol inhibited glycogen breakdown with no direct or indirect effects on the rates of gluconeogenesis. Total end point glycogen content (μmol of glycosyl units/g of wet liver) were similar in fed (235 ± 19versus 217 ± 22, p = not significant) or fasted rats (10 ± 2 versus 7 ± 2, p = not significant) with or without 1,4-dideoxy-1,4-imino-d-arabinitol, respectively. The data demonstrate no glycogen cycling under the investigated conditions and no effect of 1,4-dideoxy-1,4-imino-d-arabinitol on gluconeogenic deposition into glycogen. Taken together, these data also suggest that inhibition of glycogen phosphorylase may prove beneficial in the treatment of type 2 diabetes. endogenous glucose production 1,4-dideoxy-1,4-imino-d-arabinitol hepatic glucose output phosphate-buffered saline (trimethylsilyl)-d4-propionate Inappropriately elevated endogenous glucose production is established as a major contributor to the fasting hyperglycemia observed in patients with type 2 diabetes (1DeFronzo R.A. Diabetologia. 1992; 35: 389-397Crossref PubMed Scopus (383) Google Scholar, 2DeFronzo R.A. Diabetes. 1988; 37: 667-687Crossref PubMed Google Scholar, 3Consoli A. Nurjhan N. Capani F. Gerich J.E. Diabetes. 1989; 38: 550-557Crossref PubMed Google Scholar, 4Magnusson I. Rothman D.L. Katz L.D. Shulman R.G. Shulman G.I. J. Clin. Invest. 1992; 90: 1323-1327Crossref PubMed Scopus (613) Google Scholar). Endogenous glucose production (EGP)1 arises via the gluconeogenic pathway or from the breakdown of glycogen. 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Controversy exists regarding the relative contribution of gluconeogenesis and glycogenolysis to total glucose production in the normal situation and especially in type 2 diabetes (3Consoli A. Nurjhan N. Capani F. Gerich J.E. Diabetes. 1989; 38: 550-557Crossref PubMed Google Scholar, 4Magnusson I. Rothman D.L. Katz L.D. Shulman R.G. Shulman G.I. J. Clin. Invest. 1992; 90: 1323-1327Crossref PubMed Scopus (613) Google Scholar, 14Tappy L. Diabetes Metab. 1995; 21: 233-240Google Scholar, 15Diraison F. Large V. Brunengraber H. Beylot M. Diabetologia. 1998; 41: 212-220Crossref PubMed Scopus (47) Google Scholar, 16Tayek J.A. Katz J. Am. J. Physiol. 1996; 270: E709-E717PubMed Google Scholar), mainly due to technical difficulties in the quantification of gluconeogenesis (17Mittelman S.D. Bergman R.N. Curr. Opin. Endocrin. Diabet. 1998; 5: 126-135Crossref Scopus (9) Google Scholar). Also, the existence of a hepatic “interregulatory” mechanism has been proposed (18Haesler E. Schneiter P. 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Physiol. 1995; 15: 581-595Crossref PubMed Scopus (14) Google Scholar, 22Jahoor F. Peters E.J. Wolfe R.R. Am. J. Physiol. 1990; 258: E288-E296PubMed Google Scholar, 23Jenssen T. Nurjhan N. Consoli A. Gerich J.E. J. Clin. Invest. 1990; 86: 489-497Crossref PubMed Scopus (143) Google Scholar) or when gluconeogenesis was inhibited with ethanol (19Puhakainen I. Koivisto V.A. Yki-Järvinen H. Diabetes. 1991; 40: 1319-1327Crossref PubMed Google Scholar, 20Kubota M. Virkamäki A. Yki-Järvinen H. Proc. Soc. Exp. Biol. Med. 1992; 201: 114-118Crossref PubMed Scopus (29) Google Scholar, 24Radziuk J. Pye S. Diabetes Metab. Res. Rev. 2001; 17: 250-272Crossref PubMed Scopus (190) Google Scholar). Collectively, these data suggest that an initial modification of the gluconeogenic rate is followed by compensatory changes in the glycogenolytic rate, thus maintaining a constant EGP. Moreover, a possible futile cycling of gluconeogenic substrates through the glycogen pool has been proposed as the result of studies in isolated hepatocytes (25Massillon D. Bollen M. De Wulf H. Overloop K. Vanstapel F. Van Hecke P. Stalmans W. J. Biol. Chem. 1995; 270: 19351-19356Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) and in mice (26Massillon D. Chen W. Hawkins M. Liu R. Barzilai N. Rosseti L. Am. J. Physiol. 1995; 32: E1037-E1043Google Scholar) and rats (27David M. Petit W.A. Laughlin M.R. Shulman R.G. King J.E. Barrett E.J. J. Clin. Invest. 1990; 86: 612-617Crossref PubMed Scopus (72) Google Scholar), giving rise to the concept that a glycogen phosphorylase inhibitor would also lead to inhibition of gluconeogenesis (6Martin W.H. Hoover D.J. Armento S.J. Stock I.A. McPherson R.K. Danley D.E. Stevenson R.W. Barrett E.J. Treadway J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1776-1781Crossref PubMed Scopus (213) Google Scholar). To address the proposed hepatic interregulatory mechanism, we previously showed that a specific reduction in glucagon-stimulated glycogenolysis due to the application of DAB did not affect the rates of gluconeogenesis in dogs in vivo (13Fosgerau K. Mittelman S.D. Sunehag A. Dea M.K. Lundgren K. Bergman R.N. Am. J. Physiol. 2001; 281: E375-E383Google Scholar). In contrast, Shiota and co-workers (28Shiota M. Jackson P.A. Bischoff H. McCaleb M. Scott M. Monohan M. Neal D.W. Cherrington A.D. Am. J. Physiol. 1997; 273: E866-E879Google Scholar) using the compound BAY R 3401 to inhibit glycogenolysis reported that maximal estimates of gluconeogenesis were higher in the drug-treated groups than in the placebo-treated. The difference in these findings may be explained as being due to a difference in the mechanism of action of the compounds DAB and BAY R 3401, since it was reported that BAY R 3401 promoted deposition of gluconeogenic carbon as glycogen (28Shiota M. Jackson P.A. Bischoff H. McCaleb M. Scott M. Monohan M. Neal D.W. Cherrington A.D. Am. J. Physiol. 1997; 273: E866-E879Google Scholar, 29Bergans N. Stalmans W. Goldmann S. Vanstapel F. Diabetes. 2000; 49: 1419-1426Crossref PubMed Scopus (51) Google Scholar), in contrast to DAB, which had no effect on glycogen synthesis in hepatocytes (12Andersen B. Rassov A. Westergaard N. Lundgren K. Biochem. J. 1999; 342: 543-548Crossref Google Scholar). In the present study, we have investigated the effects of DAB on the gluconeogenic pathway using lactate and glycerol as substrates as well as the effects of DAB on glycogen synthesis and gluconeogenic substrate cycling through glycogen as assessed by NMR methodology in the systems of primary hepatocytes, perfused rat liver, and lactate-clamped ratsin vivo. We conclude that inhibition of glycogenolysis with DAB has no effect on gluconeogenesis from lactate or glycerol or on glycogen synthesis, thus suggesting that inhibition of glycogenolysis may prove beneficial for the treatment of type 2 diabetes. Male and female Sprague-Dawley rats were obtained from Møllegård Breeding Centre (Denmark). Prior to the experiments, animals were housed at ∼25 °C and constant humidity and subjected to a standard light (6 a.m. to 6 p.m.)/dark (6 p.m. to 6 a.m.) cycle and free access to normal rat chow and water. Rat hepatocytes were prepared essentially as described by Grunnet et al. (30Grunnet N. Jensen S. Dich J. Arch. Biochem. Biophys. 1994; 309: 18-23Crossref PubMed Scopus (6) Google Scholar). The isolated cells, of which more than 85% excluded trypan blue, were suspended in basal medium (Medium 199; Invitrogen) containing glucose (5.5 mm) supplemented with fetal calf serum (4%; Invitrogen), insulin (1 nm; Novo Nordisk A/S), and dexamethasone (100 nm; Merck). The cell suspension (1 ml of 0.33 million/ml suspension) was plated onto collagen-coated (Sigma) 12-well Petri dishes (NUNC A/S) or 60-mm Petri dishes (4 ml of 0.55 million/ml suspension) for NMR studies. After 3 h, the medium was changed to a medium with a composition as described above except that the serum was omitted. To study the effect of DAB on glycogenolysis, medium was changed after 24 h to basal medium containing 15 mm glucose or [1-13C]glucose (Cambridge Isotope Laboratories) for NMR experiments and 10 nm insulin in order to build up glycogen stores. After an additional 20-h incubation under these conditions, the hepatocytes were washed twice with prewarmed buffer A (pH 7.4 at 37 °C) containing NaCl (117.6 mm), KCl (5.4 mm), Mg2SO4 (0.82 mm), KH2PO4 (1.5 mm), HEPES (20 mm), NaHCO3 (9 mm), human serum albumin (0.1% w/v), and CaCl2 (2.25 mm) and subsequently incubated in 1 or 3 ml (60-mm dishes) of buffer A in the presence or absence of 3 mm glycerol or [2-13C]glycerol (Cambridge Isotope Laboratories) for the NMR experiments, with or without 0–25 μm DAB (Novo Nordisk A/S) and with or without 1–2.0 nm glucagon (Novo Nordisk A/S) for 3 h. Basal and glucagon-stimulated glycogenolysis was measured as glucose (see assay below) released into buffer A. Glycogen levels were determined after washing the cells twice with ice-cold saline and by using amyloglucosidase (Roche Molecular Biochemicals) digestion and subsequent glucose measurement as above (31Kunst A. Draeger B. Ziegenhorn J. Bergmayer H.U. Bergmayer J. Grassl M. Methods of Enzymatic Analysis. II. Verlag Chemie, Weinheim, Germany1984: 154Google Scholar). Lactate in the medium was determined by using the Sigma lactate reagent. For the NMR experiments, 250 μl of the hepatocyte incubation medium was first taken to determine glucose and lactate as above. The remaining 2750 μl was lyophilized and redissolved in 500 μl of phosphate buffer (6.7 mm, pH 7.4) containing sodium (trimethylsilyl)-d-4-propionate (TSP) (20 mm; Cambridge Isotope Laboratories) as an internal standard and D2O (10%; 99 atom % deuterium; Aldrich) for NMR analysis. For cellular glycogen content, the cells were first hydrolyzed in 750 μl of amyloglucosidase buffer, of which 500 μl was then lyophilized and redissolved for NMR spectroscopy as described above for the medium samples. Samples were transferred to 96-well plates for automated flow injection NMR analysis. Gradient selected one-dimensional heteronuclear single quantum coherence spectra were acquired at 600.13 MHz 1H frequency on a Bruker DRX600 instrument (Bruker, Rheinstetten, Germany) equipped with a 160-μl single cell-selective inverse flow injection (1H,13C) z-gradient probe head (Bruker). The sample was placed in the flow cell by means of a Gilson 215 liquid handler robot (Gilson Inc., Middleton, WI). The13C content in positions 1 and 2 of glucose and positions 2 and 3 of lactate was measured by recording the integral values of the corresponding 1H signals relative to the integral of the TSP peak in the one-dimensional heteronuclear single quantum coherence spectra (39Hansen S.H. McCormack J.G. NMR Biomed. 2002; (in press)PubMed Google Scholar). Absolute quantification was performed by acquiring one-dimensional heteronuclear single quantum coherence spectra of glucose and lactate reference samples of known concentrations, and enrichment was calculated from the total pool sizes determined by biochemical assays or by 1H NMR spectroscopy. We observed a good correlation between the NMR method and the biochemical method (data not shown). The rats were kept as described above. Fed female Sprague-Dawley rats (8–10 weeks, 218 ± 13 g) were divided into six groups (n = 5/group), and the livers were perfused as described below. Rats were anesthetized with 3.5 ml/kg of a freshly prepared mixture containing 100 μl of Hypnorm (Jansson Cilag) and 100 μl of Dormicum (local pharmacy), and 200 μl of H2O and livers were perfused in situ through the portal vein at a constant flow rate of 18 ml/min with a Krebs-Ringer bicarbonate buffer equilibrated with O2/CO2 (19:1) to a pH of the perfusate at 7.4 at 37 °C (32Exton J.H. Methods Enzymol. 1975; 39: 25-36Crossref PubMed Scopus (39) Google Scholar). After 10 min of preperfusion, the experiment was initiated at t = 0 by a change to an equilibrated Krebs-Ringer buffer containing lactate (1.67 mm), pyruvate (0.33 mm), insulin (10 microunits/ml), glucagon (88 pg/ml), and [6-3H]glucose (0.03 μCi/ml; Amersham Biosciences), with or without DAB (2.5 μm) and at three glucose concentrations (0, 5, or 20 mm) (i.e. in six groups). The low insulin/glucagon ratio used has previously been reported to stimulate net glucose output in perfused rat liver (33Shiota M. Green R. Colburn C.A. Mitchell G. Cherrington A.D. Metabolism. 1996; 45: 481-485Abstract Full Text PDF PubMed Scopus (9) Google Scholar). Livers were perfused in a nonrecirculating set-up, and samples of 2 ml were drawn simultaneously from the inflow and outflow of the liver att = 0, 5, 10, 15, 20, 25, 30, 35, and 40 min. Att = 40 min, 0.1 μCi/ml [U-14C]lactate (Amersham Biosciences) was added to the Krebs-Ringer buffer, and the perfusion was changed to a recirculating set-up (200 ml). Samples of 2 ml were drawn from the inflow and outflow of the liver att = 43, 52, 55, 58, 61, 64, 67, and 70 min. A steady-state period was defined as the average of values fromt = 61–70 min, where conditions were assumed to be constant. Perfusate samples were collected on ice and stored at −80 °C for later analysis. Finally, at t = 70 min, the liver was rapidly excised, freeze-clamped in N2 and stored at −80 °C for later analysis. The rats were kept as described above. Four groups of male weight-matched Sprague-Dawley rats aged 10–12 weeks were studied: group 1, fasted, DAB-treated (n = 9, weight 352 ± 11 g expressed as mean ± S.D.); group 2, fasted, vehicle (n = 9, 354 ± 11 g); group 3, fed, DAB-treated (n = 8, 361 ± 14 g); and group 4, fed, vehicle (n = 9, 367 ± 11 g). At the day of the experiment, the rats were anesthetized with isofluran, and two catheters were implanted. One catheter was set in the right vena jugularis for infusion (sp210 syringe pump; World Precision Instruments, Aston, UK) of somatostatin (SRIF), insulin, and lactate/pyruvate. A second catheter was set in the right vena femoralis for infusion of donor erythrocytes (hematocrit 62 ± 5% in PBS buffer; 7.1 ± 0.2 ml) and DAB or vehicle. After surgery, the animals were rested for 30 min before the start of infusions. At t = −45 min, a continuous infusion of somatostatin (4 μg/kg * min) was given for suppression of endogenous insulin production, and a basal replacement infusion of insulin (0.4 milliunits/kg * min) was given. Also, a variable infusion of lactate/pyruvate (5:1, 1.25 m, 30%13C-enriched in C-3 in lactate) was started to clamp plasma lactate levels at 5 mm. At t = 0, following this equilibration period of 45 min, the test period was started with a primed infusion of DAB (1.38 mg/kg + 13.1 μg/kg * min) or saline (vehicle). The selected dose of DAB (1.97 mg/kg) was based on preliminary experiments in rats to obtain a constant plasma concentration of 5 μmDAB. 2P. McKay and L. Yndal, unpublished observation. Blood was drawn every third minute for analysis of glucose and lactate and further att = 0, 7.5, 15, 30, and 45 min for the measurement of NMR parameters. Blood was also drawn every 6 min untilt = 0 and further at t = 7.5, 12, 15, 21, 27, 30, 33, 39, and 45 min for determination of insulin. Blood was collected in EDTA-coated tubes and centrifuged immediately (Eppendorf centrifuge 5417 R; Radiometer). Plasma was either kept on ice and processed the same day or kept at −80 °C until assayed. Finally, att = 45, the liver was rinsed with saline injected through the portal vein, excised, freeze-clamped in N2, and stored at t = −80 °C for later analysis. The outline of the infusion protocol is shown in Fig. 3. In order to evaluate the DAB infusion protocol, the following control experiment was performed. Fed rats were infused with or without DAB as outlined above, and then at t = 45, the animals received an intraperitoneal injection of glucagon (10 μg/kg), and blood samples were taken every 5 min until t = 90. The result is shown later in Fig. 3 B. Blood glucose and lactate were measured on-line with a dual glucose/lactate analyzer (YSI 2300 STAT; Yellow Springs Instrument Co.). Insulin was measured with an enzyme-linked immunosorbent assay method based on two murine monoclonal antibodies that bind to different epitopes on the insulin molecule (34Andersen L. Dinesen B. Jørgensen P.N. Poulsen F. Røder M.E. Clin. Chem. 1993; 39: 578-582Crossref PubMed Scopus (543) Google Scholar). Inflow and outflow perfusate samples were placed on a tandem column of 1 ml of cation exchanger (Dowex 50; Sigma) over 2 ml of anion binder (Dowex 1-X8 100–200 mesh (acetate); Sigma) for separation of [14C]glucose from [14C]lactate and counted in a scintillation counter (Tri-carb 4530; Packard Instrument Co.). The anion exchanger in the chloride form was exchanged to the acetate form by subsequent treatment with 0.5 m NaOH and 0.2 m HAc prior to the assay. The glycogen contents of the freeze-clamped liver samples were determined enzymatically as μmol of glycosyl units per gram of wet weight after boiling the tissue in 0.4n KOH and subsequent degradation of glycogen with amyloglucosidase (35Katz J. Golden S. Wals P.A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 3433-3437Crossref PubMed Scopus (107) Google Scholar). Glycogen concentrations are expressed as molar glycosyl units based on absolute hydrolysis of the used glycogen batch. 3H and 14C in glycogen was determined by boiling tissue samples in 0.4n KOH. Glycogen was precipitated by the addition of ethanol to 70% (v/v). After 2 h on ice, the precipitate was collected by centrifugation, washed twice by 70% ethanol, and hydrolyzed by boiling for 2 h with 0.2 m sulfuric acid. Radioactivity in the hydrolysate was measured by liquid scintillation counting. Plasma samples were centrifuged, and 200 μl was transferred to 96-well plates. Phosphate buffer (50 mm, pH = 7.4, 200 μl) containing TSP (50 mm) as internal reference was added. Gradient-selected one-dimensional heteronuclear single quantum coherence experiments were acquired as described for the hepatocyte medium samples (39Hansen S.H. McCormack J.G. NMR Biomed. 2002; (in press)PubMed Google Scholar), and the 13C content in position 1 in glucose and position 3 in lactate was measured. A steady state period was defined att = 61–70 min by averaging values obtained att = 61, 64, 67, and 70 min, and a two-way analysis of variance (Prism 3.0) was used to test for the effects of DAB and glucose on all rat liver outcome variables. An unpaired Student'st test was used to compare steady-state periods when significance was reached by analysis of variance assuming two-sample unequal variance. The study groups were compared by a two-way analysis of variance (Prism 3.0) and subjected to an unpaired Student's t test when significance was reached by analysis of variance assuming two-sample unequal variance. Fig. 1shows the effects of DAB on glucose release and lactate release and glycogen content in cultured rat hepatocytes incubated under the basal or glucagon-stimulated condition and in the absence or presence of 3 mm glycerol. Data are expressed relative to basal conditions in the absence of DAB, which equals 100%. Glucose release (Fig. 1 A) was inhibited dose-dependently by DAB under basal conditions to about 30% of the initial value. Glucagon increased the glucose release 2.7-fold, and this could be inhibited by DAB to 57%, which was significantly above the basal condition (p < 0.05). The addition of glycerol increased the glucose release 2.5-fold alone and 5.2-fold in the presence of glucagon compared with the basal conditions. DAB did not inhibit glucose release in the presence of glycerol, with or without glucagon, to the level seen in the absence of glycerol (p < 0.05). Glucagon decreased lactate release (Fig. 1 B) to 50–70% of the initial value without DAB. However, independently of glucagon, DAB was not able to inhibit lactate release to the same extent with glycerol as without glycerol. In the absence of DAB, the glycogen content (Fig.1 C) fell from the initial value of 312% to 100% under basal conditions and 50% in the presence of glucagon independent of glycerol. The addition of DAB under all incubation conditions dose-dependently prevented glycogen degradation up to 225–275%, corresponding to 70–85% of the initial value (312%). Cells were preincubated with [1-13C]glucose to build up the 13C-labeled glycogen content and subsequently incubated under basal or glucagon-stimulated conditions and with 3 mm[2-13C]glycerol. C-1-labeled glucose and C-3-labeled lactate in the medium are derived from C-1-labeled glucose residues in glycogen, whereas glucose and lactate labeled in the C-2 position are derived from C-2-labeled glycerol (39Hansen S.H. McCormack J.G. NMR Biomed. 2002; (in press)PubMed Google Scholar). This therefore allowed us to study the effect of DAB on glucose production from gluconeogenesis (from glycerol) and from glycogenolysis in a more detailed manner and simultaneously. The effect of 20 μm DAB on total glucose, lactate, and glycogen content (Table I) corresponded well with the data presented in Fig. 1 with a few exceptions. DAB clearly inhibited the release of [1-13C]glucose but noticeably not [2-13C]glucose into the medium to the same extent under basal or glucagon-stimulated conditions with glycerol. Also, the amount of [3-13C]lactate released into the incubation medium was inhibited by DAB, with a more pronounced effect under stimulated conditions. DAB did not inhibit the release of [2-13C]lactate (p = not significant); however, the presence of glucagon reduced the amount of [2-13C]lactate released (p < 0.05). Stimulation of hepatocytes with glucagon caused less [1-13C]glucose to be retained in glycogen, corresponding with increased amounts of labeled glucose in the medium. However, DAB retained 1-13C-labeled glucose in glycogen to the same extent under basal and stimulated conditions, and enrichment of the C-1 position in glycogen was independent of the presence of glucagon and DAB. No enrichment at the C-2 position was found in glycogen (data not shown).Table IEffect of DAB on glycogenolysis and gluconeogenesis in primary rat hepatocytesTotal GluTotal Lac[1-13C]Glu[2-13C]Glu[2-13C]Lac[3-13C]Lacnmol/mg proteinMediumBasal2059.3 ± 172.11295.2 ± 343.8913.0 ± 128.4410.0 ± 22.9438.0 ± 111.0185.4 ± 56.5(44.3 ± 6.2)(19.9 ± 1.1)(33.5 ± 2.7)(14.3 ± 1.8)Basal + DAB885.3 ± 106.2**570.8 ± 98.1142.4 ± 13.9***461.3 ± 37.2247.0 ± 67.345.0 ± 8.3*(16.1 ± 1.6)(52.1 ± 4.2)(43.2 ± 5.1)(7.4 ± 1.0)Stim3124.7 ± 335.8567.9 ± 110.01362.6 ± 173.7699.3 ± 46.6140.1 ± 37.479.9 ± 22.7(43.6 ± 5.6)(22.4 ± 1.5)(24.6 ± 3.3)(14.1 ± 1.7)Stim + DAB1184.6 ± 156.0***292.4 ± 11.7*182.5 ± 19.7***630.8 ± 47.564.7 ± 12.827.3 ± 3.1*(15.4 ± 1.7)(53.3 ± 4.0)(22.1 ± 3.3)(9.3 ± 1.0)CellBasal1804.4 ± 323.4922.6 ± 186(51.1 ± 10.3)Basal + DAB3434.2 ± 547.6*1854.5 ± 319.2*(54.0 ± 9.3)Stim1135.6 ± 261.6567.6 ± 100.1(50.0 ± 8.9)Stim + DAB3381.4 ± 452.6**1866.0 ± 276.9**(55.2 ± 8.2)Cells were incubated 24 h with [1-13C]glucose to build up a 13C-labeled glycogen pool. Subsequently, the cells were incubated with 3 mm [2-13C]glycerol under basal or glucagon-stimulated (Stim) conditions (see “Experimental Procedures”). C-1-labeled glucose (Glu) and C-3-labeled lactate (Lac) in the media are derived from C-1-labeled glucose residues in glycogen, whereas glucose and lactate labeled in the C-2 position are derived from C-2-labeled glycerol. Data in parenthesis indicate percentage of13C enrichment. The concentration of DAB was 20 μm. The amount of glucose and [1-13C]glucose in the cells reflects glycogen levels. Results are given as averages ± S.E. with n = 6 different hepatocyte preparations. ***, p < 0.001; **, p < 0.01; and *,p < 0.05 compared to the same condition in the absence of DAB. Open table in a new tab Cells were incubated 24 h with [1-13C]glucose to build up a 13C-labeled glycogen pool. Subsequently, the cells were incubated with 3 mm [2-13C]glycerol under basal or glucagon-stimulated (Stim) conditions (see “Experimental Procedures”). C-1-labeled glucose (Glu) and C-3-labeled lactate (Lac) in the media are derived from C-1-labeled glucose residues in glycogen, whereas glucose and lactate labeled in the C-2 position are derived from C-2-labeled glycerol. Data in parenthesis indicate percentage of13C enrichment. The concentration of DAB was 20 μm. The amount of glucose and [1-13C]glucose in the cells reflects glycogen levels. Results are given as averages ± S.E. with n = 6 different hepatocyte preparations. ***, p < 0.001; **, p < 0.01; and *,p < 0.05 compared to the same condition in the absence of DAB. Glycogen concentrations were higher in livers perfused with than without DAB (TableII). At 20 mm glucose, this effect of DAB was significant (p < 0.05). Glucose concentration did not affect the glycogen content or the ability of DAB to inhibit glycogen breakdown (p = not significant). Glucose enhanced (p < 0.001) glycogen synthesis via the direct pathway (glucose → glucose
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