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J. Biol. Chem., Vol. 277, Issue 32, 28648-28655, August 9, 2002

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Evidence against Glycogen Cycling of Gluconeogenic Substrates in Various Liver Preparations^*

Keld Fosgerau^§, Jens Breinholt^¶, James G. McCormack, and Niels Westergaard^**

From the  Pharmacological Research 2, ^¶ MedChem Research, ^** Hepatic Biochemistry,  Discovery Management, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Maaloev, Denmark

Received for publication, February 15, 2002, and in revised form, May 8, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 ± 0.19 versus 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 versus vehicle, 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 ± 19 versus 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inappropriately elevated endogenous glucose production is established as a major contributor to the fasting hyperglycemia observed in patients with type 2 diabetes (1-4). Endogenous glucose production (EGP)¹ arises via the gluconeogenic pathway or from the breakdown of glycogen. Therefore, inhibition of glycogenolysis and of gluconeogenesis have been regarded as potential therapeutic approaches in the search for novel anti-hyperglycemic drugs for the treatment of this disease (5-10). Glycogen phosphorylase is the rate-controlling enzyme of the glycogenolytic pathway (11), and we have previously reported that 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) is a potent inhibitor of glycogen phosphorylase and glycogen breakdown with an associated anti-hyperglycemic effect (9, 12, 13).

Controversy exists regarding the relative contribution of gluconeogenesis and glycogenolysis to total glucose production in the normal situation and especially in type 2 diabetes (3, 4, 14-16), mainly due to technical difficulties in the quantification of gluconeogenesis (17). Also, the existence of a hepatic "interregulatory" mechanism has been proposed (18-23), further confounding the interpretation of the relative importance of gluconeogenesis and glycogenolysis in hepatic glucose and glycogen metabolism. Thus, basal EGP remained constant when gluconeogenesis was acutely increased by infusion of gluconeogenic precursors (18, 22, 23) or when gluconeogenesis was inhibited with ethanol (19, 20, 24). 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 (25) and in mice (26) and rats (27), giving rise to the concept that a glycogen phosphorylase inhibitor would also lead to inhibition of gluconeogenesis (6).

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 (13). In contrast, Shiota and co-workers (28) 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 (28, 29), in contrast to DAB, which had no effect on glycogen synthesis in hepatocytes (12).

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 rats in 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Animals

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.

Hepatocyte Experiments

Rat hepatocytes were prepared essentially as described by Grunnet et al. (30). 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 (31). Lactate in the medium was determined by using the Sigma lactate reagent.

Measurement of ¹³C Content of Glucose and Lactate in Hepatocyte Medium by ¹³C-Filtered ¹H NMR Using the Flow Injection Technique

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). The ¹³C 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 (39). 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).

Perfusion Experiments

Animals-- 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.

Experimental Protocol-- 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 (32). 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 (33). Livers were perfused in a nonrecirculating set-up, and samples of 2 ml were drawn simultaneously from the inflow and outflow of the liver at t = 0, 5, 10, 15, 20, 25, 30, 35, and 40 min. At t = 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 at t = 43, 52, 55, 58, 61, 64, 67, and 70 min. A steady-state period was defined as the average of values from t = 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.

Lactate Clamps

Animals-- 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.

Experimental Protocol-- 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 µM DAB.² Blood was drawn every third minute for analysis of glucose and lactate and further at t = 0, 7.5, 15, 30, and 45 min for the measurement of NMR parameters. Blood was also drawn every 6 min until t = 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, at t = 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. 3B.

Assays

Blood Glucose and Lactate-- Blood glucose and lactate were measured on-line with a dual glucose/lactate analyzer (YSI 2300 STAT; Yellow Springs Instrument Co.).

Insulin-- 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 (34).

Glucose Radioactivity-- 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.

Glycogen Contents-- 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 0.4 N KOH and subsequent degradation of glycogen with amyloglucosidase (35). Glycogen concentrations are expressed as molar glycosyl units based on absolute hydrolysis of the used glycogen batch.

Glycogen Radioactivity-- ³H and ¹⁴C in glycogen was determined by boiling tissue samples in 0.4 N 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.

Measurement of ¹³C Content of Glucose and Lactate in Plasma by ¹³C-Filtered ¹H NMR Using the Flow Injection Technique-- 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 (39), and the ¹³C content in position 1 in glucose and position 3 in lactate was measured.

Statistics

Perfusions-- A steady state period was defined at t = 61-70 min by averaging values obtained at t = 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's t test was used to compare steady-state periods when significance was reached by analysis of variance assuming two-sample unequal variance.

Hepatocyte Experiments and Lactate Clamps-- 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hepatocyte Experiments-- Fig. 1 shows 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. 1A) 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. 1B) 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. 1C) 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%).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Effect of DAB on glucose release (A), lactate release (B), and glycogen content (C) in cultured rat hepatocytes. Basal (solid lines) and glucagon-stimulated (dotted lines) glucose production (A), lactate production (B), and glycogen levels (C) in the presence of 0-25 µM DAB. , in the absence of 3 mM glycerol; , in the presence of 3 mM glycerol. 100% corresponded to 436.1 ± 22.8 nmol/mg protein in A 1635.6 ± 220 nmol/mg protein in B, and 1099.1 ± 274.2 nmol/mg protein in C. The horizontal line in C at 312% corresponded to the glycogen levels prior to the 3-h incubation. Results are given as averages ± S.E. with n = 4 different hepatocyte preparations.

NMR Experiments-- 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 (39). 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).

Perfusion Experiments-- Glycogen concentrations were higher in livers perfused with than without DAB (Table II). 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 6-phosphate  glycogen), but had no affect on glycogen synthesis from lactate via the indirect pathway (lactate  glucose  glucose 6-phosphate  glycogen) (Table II). DAB did not affect glycogen synthesis from either glucose or lactate and did not modulate the effect of glucose on pathways of glycogen synthesis. However, glucagon-stimulated hepatic glucose output (HGO) was significantly inhibited by DAB in livers perfused with 0 or 5 mM glucose (*, p < 0.05, Fig. 2, Table III). High levels of glucose (20 mM) alone tended to decrease HGO, but this effect was not significant. We observed no effects of either DAB or glucose on the rate of gluconeogenesis measured as ¹⁴C-lactate incorporation into glucose (p = not significant; Table III).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Effect of DAB and glucose on HGO from the perfused rat liver. Livers from fed rats were perfused with () or without () 2.5 µM DAB in combination with 0 (A), 5 (B), or 20 mM (C) glucose, and HGO was measured by athero-venous differences expressed as µmol/min * g of wet liver (see "Experimental Procedures" for further details). HGO was significantly inhibited by DAB in livers perfused with 0 or 5 mM glucose (p < 0.05) but not in livers perfused with 20 mM glucose. We observed no significant effects of glucose. Results are given as averages ± S.E. with n = 5 perfusions.

Lactate Clamps-- The protocol for these experiments is shown in Fig. 3 (see "Experimental Procedures" for further details). Fig. 3B shows that the concentration of DAB used in the lactate clamp experiment was sufficient to significantly reduce a glucagon-induced increase in blood glucose. Plasma lactate levels were clamped at a high physiological level of 5 mM by infusion of exogenous ¹³C-enriched lactate and kept constant during infusion of DAB or vehicle in fed or fasted rats (Fig. 4A). The plasma concentration of insulin following replacement infusion was lower than the basal fasting levels of insulin (Fig. 4B). End point glycogen content was lower in livers from fasted animals compared with fed animals (Table IV, p < 0.001), whereas total lactate infusion volume was higher in fasted rats compared with fed (Table IV, p < 0.001). Infusion of DAB did not affect either plasma lactate and insulin levels or hepatic glycogen content and total lactate infusion volume in fed or fasted rats (Fig. 4 and Table IV, p = not significant). We observed no effects of DAB treatment on plasma levels of glucose regardless of the feeding status (Fig. 4C, p = not significant). In fasted rats, glucose levels rose from 8.6 ± 0.6 to 10.0 ± 0.5 and from 8.1 ± 0.7 to 9.2 ± 0.9 (average ± S.E., Fig. 4), whereas in fed rats glucose levels decreased from 8.2 ± 0.8 to 5.9 ± 0.4 and 8.1 ± 0.5 to 6.1 ± 0.2, with DAB or vehicle treatment in each case, respectively. Plasma lactate and glucose ¹³C enrichments during the lactate clamp in fasted rats in vivo are shown in Table V. In fasted rats, the lactate ¹³C enrichments were constant throughout the DAB infusion period, whereas glucose ¹³C enrichments increased from 0.58 ± 0.10 to 1.43 ± 0.12 and from 0.40 ± 0.15 to 1.23 ± 0.22 with DAB or vehicle treatment, respectively. Thus we observed no effect of DAB (p = not significant) on either glucose or lactate ¹³C enrichments. In fed rats, the ¹³C content in glucose was not different from the natural abundance of 1.108% following enrichment of lactate (data not shown), and we observed no effect of DAB (p = not significant).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Time schedule and protocol for lactate clamp experiments in rats in vivo. A, rats were either fed or fasted overnight before onset of the protocol (see "Experimental Procedures" for further details). At t = 45 min, a continuous infusion of somatostatin (4 µg/kg * min) with basal replacement infusion of insulin (0.4 milliunits/kg * min) was given. Also, a variable infusion of lactate/pyruvate (30% 13C-enriched in C-1 in lactate) was started to clamp plasma lactate levels at 5 mM. At t = 0, following an equilibration period of 45 min, the test period was started with a primed infusion of DAB. Finally, at t = 45 min, the liver was excised, freeze-clamped in N2, and stored at t = 80 °C for later analysis. B, to test the efficacy of the chosen dose of DAB, the following glucagon challenge control experiment was performed in fed rats. Animals were infused with () or without () DAB as outlined above, and then at t = 45 min, the rats received an intraperitoneal injection of glucagon (10 µg/kg), and blood samples were taken every 5 min until t = 90. Results are given as averages ± S.E. with n = 4 animals.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Effect of DAB on plasma lactate (A), insulin (B), and glucose (C) levels in fed and fasted rats in vivo. Plasma lactate levels (A) were clamped at 5 mM and kept constant during the infusion period of either DAB (solid symbols) or vehicle (open symbols) in fed ( or ) and fasted rats ( or ). Plasma levels of insulin are shown in B, and levels of glucose are given in C. DAB infusion did not affect plasma levels of lactate, insulin, or glucose regardless of the feeding status (p = not significant). Results are given as averages ± S.E. with n = 8-9 animals.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The findings that DAB inhibited basal and glucagon-stimulated glucose production from cultured hepatocytes in the absence of glycerol (Fig. 1) as well as glucose production in the perfused liver (Fig. 2) confirmed previously reported effects of this compound (9, 12, 13). Also, it was previously shown that glucose did not affect the inhibitory potency of DAB on glycogen phosphorylase enzyme preparations from rat muscle or pig liver and that glucose plus DAB inhibited glycogen phosphorylase in an additive fashion (9). Here, using the system of the perfused liver, we report that the inhibitory effect of DAB on HGO was not altered by perfusion of rat liver with 5 mM glucose in addition to DAB. The observed decrease in HGO in livers not treated with DAB is believed to be associated with a depletion of the glycogen content following the treatment of livers with low ratios of insulin/glucagon (33).

The relative contribution of gluconeogenesis and glycogenolysis to hepatic glucose production and the existence of a hepatic "interregulation" mechanism (13) of the two pathways has been a matter of controversy due to methodological challenges (14). Thus, in healthy subjects in the postabsorptive phase, the proportion of hepatic glucose production attributed to gluconeogenesis has been reported to range from 40 to 70% (3, 4, 14-16). To gain insights into the mechanisms responsible for the suggested regulation of EGP, we have presently investigated the effects of DAB and gluconeogenic substrates in the systems of primary hepatocytes, perfused rat liver or lactate-clamped rats in vivo.

The addition of glycerol to the incubation medium of cultured rat hepatocytes increased the amount of glucose released, and infusion of lactate in fasted rats in vivo led to an increased glucose production via gluconeogenesis, as reflected in the increased amount of infused lactate in the fasted compared with the fed rats (Table IV). In contrast, infusion of lactate (18) or glycerol (36) in fasted humans did not affect either glucose production or glucose uptake in humans. Further, in humans, the infusion of lactate did not affect relative contributions of gluconeogenesis and glycogenolysis to EGP, indicating an inhibition of gluconeogenesis from endogenous precursors under these circumstances (18).

A possible explanation for these differences may lie in the different levels of glycogen. Thus, in humans with a large capacity for the storage of glucose as glycogen, the process of hepatic glycogen breakdown is ongoing after an overnight fast (18, 36). In contrast, in overnight fasted rats, the glycogen levels are almost empty (Table IV); therefore, glycogen-derived glucose production will be almost absent, which in turn may cause a loss of the interregulation of glucose production (18-20, 36). We cannot exclude the possibility that lactate infusion affected glucose uptake in fed and fasted rats, since this was not measured. However, since the insulin infusion underreplaced normal fasting levels of insulin in our hands, the rate of glucose uptake is not believed to play a significant role.

DAB had no effect on lactate gluconeogenesis in the glucagon-stimulated perfused rat liver and or on lactate gluconeogenesis in fed or fasted rats in vivo. The efficacy of DAB in the lactate infusion protocol in vivo was investigated in a glucagon challenge control experiment as shown in Fig. 3B, demonstrating that the chosen dose of DAB could significantly inhibit glucagon-stimulated glycogen breakdown in rats. Also, DAB had no effect on the rates of glycerol gluconeogenesis in hepatocytes under basal or stimulated conditions (Fig. 1). The lower lactate levels observed in cultured hepatocytes in the presence of DAB was due to a direct inhibition of glycogenolysis, since the levels of [3-¹³C]lactate (derived from [1-¹³C]glucose) declined, whereas no significant effect of DAB was seen on [2-¹³C]lactate production. This also explains why the total lactate levels in the presence of glycerol could not be inhibited to the same extent by DAB as when glycerol was absent. The lower lactate levels found in the presence of glycerol are in agreement with the well known effect of glucagon on pyruvate kinase and the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, causing inhibition of glycolysis and stimulation of gluconeogenesis (37). This is also reflected by the higher levels of [2-¹³C]glucose and lower levels of [2-¹³C]lactate observed in the presence of glucagon. It could then be calculated that under basal conditions in the absence or presence of glycerol, 70 and 54% of the glucose released into the medium was derived from glycogen and 30 and 46% from gluconeogenesis, whereas under stimulated conditions these values were 79 and 21% in the absence of glycerol and 64 and 36% in the presence of glycerol, respectively. The presented data indicate that DAB inhibited glycogenolysis in a direct fashion without any direct or indirect inhibition of gluconeogenesis, thus supporting the notion that the "interregulation" is associated with modulation of gluconeogenesis only and not a general mechanism that maintains glucose output constant (13).

Notably, infusion of lactate in fed rats in vivo led to a decrease of total glucose in plasma. This may be explained by a decrease in the absorption of glucose from the gut and intestine or an effect of anesthesia. Under these circumstances, we detected no glucose production from ¹³C-labeled lactate, and we observed no effect of DAB, suggesting that EGP was low as a result of the postabsorptive state of these animals or possibly a limited effect of DAB on basal glycogenolysis.

It has been reported that gluconeogenesis-derived glucose appears to be cycled through the glycogen pool prior to efflux from the liver cells both in vitro (25, 38) and in vivo (26, 27). The magnitude of this cycling is believed to depend on the feeding status (39). In contrast, the absence of glycogen cycling in freshly isolated rat hepatocytes has been reported (30). Here, using the NMR technique and a double labeling approach in cultured primary hepatocytes so that both gluconeogenesis and glycogenolysis could be measured simultaneously (39), we demonstrate that DAB inhibited the release of [1-¹³C]glucose (derived from glycogen) but not [2-¹³C]glucose (derived from [2-¹³C]glycerol). Moreover, no labeling of glucose residues in the C-2 position in glycogen was found (data not shown), although gluconeogenesis flux was clearly taking place. Also, we observed no effects of DAB on the synthesis of glycogen from lactate or glucose in the perfused rat liver or on total glycogen content as well as ¹³C-labeled lactate incorporated into glycogen (data not shown) in fed or fasted rats in vivo. These findings strongly suggest that cycling of gluconeogenic-derived glucose through the glycogen pool did not take place, and the data exemplifies how having a precise pharmacological tool like DAB can allow important physiological questions to be approached.

Previously, it was demonstrated that glycogen synthesis in primary cultures of hepatocytes was not affected by DAB (12). In contrast, BAY R 3401, a different glycogen phosphorylase inhibitor, was reported to promote the deposition of gluconeogenic carbon as glycogen (28, 29). In the present study, we showed that DAB did not promote the deposition of gluconeogenic (lactate) carbon as glycogen in the perfused rat liver or in fed or fasted rats in vivo, thus demonstrating a difference in the mechanism of action between the compounds DAB and BAY R 3401. Furthermore, DAB and BAY R3401 have been reported to bind to different sites of glycogen phosphorylase (5). This difference suggests that stimulation of the glycogenic process following inhibition of glycogen phosphorylase is not a general mechanism.

In conclusion, this paper describes DAB as a potent inhibitor of glycogen breakdown and consequently endogenous glucose production with no consequential effects on gluconeogenic deposition into glycogen or on rates of gluconeogenesis. Also, using DAB we demonstrate no substantial rate of glycogen cycling as investigated in primary cultures of hepatocytes, perfused rat liver, and fed or fasted rats clamped at a high physiological level of lactate. Taken together, these data suggest that inhibition of glycogen phosphorylase may prove beneficial in the treatment of type 2 diabetes by lowering rates of EGP, but by still allowing glucagon-induced gluconeogenic glucose production to avoid risk of hypoglycemia.

	ACKNOWLEDGEMENTS

The expert technical assistance of K. E. Pedersen, M. O. N. Jensen, H. Petersen, and E. G. Mortensen is gratefully acknowledged. Also, we thank Drs. B. Andersen and V. Diness for comments and fruitful discussions of the present work and Dr. S. H. Hansen for development of the NMR methodology.

	FOOTNOTES

^* 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: Pharmacological Research 2, Novo Nordisk Park G8 1.03, DK-2760 Maaloev, Denmark. Tel.: 45-4443-7601; Fax: 45-4443-4537; E-mail: kf@novonordisk.com.

Present address: Combio A/S, c/o Carlsberg Laboratories, Gamle Carlsbergvej 10, DK-2500 Valby, Denmark.

Published, JBC Papers in Press, May 31, 2002, DOI 10.1074/jbc.M201565200

² P. McKay and L. Yndal, unpublished observation.

	ABBREVIATIONS

The abbreviations used are: EGP, endogenous glucose production; DAB, 1,4-dideoxy-1,4-imino-D-arabinitol; HGO, hepatic glucose output; PBS, phosphate-buffered saline; TSP, (trimethylsilyl)-d4-propionate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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