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ENDOCRINE AND DIABETES

A Novel Glucagon Receptor Antagonist, NNC 25-0926, Blunts Hepatic Glucose Production in the Conscious Dog

Molecular Physiology & Biophysics, Vanderbilt University, Nashville, Tennessee (N.R., C.A.E.-G., D.S.E., T.R., D.W.N., A.D.C.); and Novo Nordisk A/S, Malov, Denmark (E.N., M.O.L., L.O.J., K.K., C.L.B.)

Received November 22, 2006; accepted February 13, 2007.

	Abstract

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Elevated glucagon is associated with fasting hyperglycemia in type 2 diabetes. We assessed the effects of the glucagon receptor antagonist (2R)-N-[4-({4-(1-cyclohexen-1-yl)[(3,5-dichloroanilino)carbonyl]anilino}methyl)benzoyl]-2-hydroxy-b-alanine (NNC 25-0926) on hepatic glucose production (HPG) in vivo, using arteriovenous difference and tracer techniques in conscious dogs. The experiments consisted of equilibration (–140 to –40 min), control (40–0 min), and experimental [0–180 min, divided into P1 (0–60 min) and P2 (60–180 min)] periods. In P1, NNC 25-0926 was given intragastrically at 0 (veh), 10, 20, 40, or 100 mg/kg, and euglycemia was maintained. In P2, somatostatin, basal intraportal insulin, and 5-fold basal intraportal glucagon (2.5 ng/kg/min) were infused. Arterial plasma insulin levels remained basal throughout the study in all groups. Arterial plasma glucagon levels remained basal during the control period and P1 and then increased to 70 pg/ml in P2 in all groups. Arterial plasma glucose levels were basal in the control period and P1 in all groups. In P2, the arterial glucose level increased to 245 ± 22 and 172 ± 15 mg/dl in the veh and 10 mg/kg groups, respectively, whereas in the 20, 40, and 100 mg/kg groups, there was no rise in glucose. Net hepatic glucose output was 2 mg/kg/min in all groups during the control period. In P2, it increased by 9.4 ± 2 mg/kg/min in the veh group. In the 10, 20, 40, and 100 mg/kg groups, the rise was only 4.1 ± 0.9, 1.6 ± 0.6, 2.4 ± 0.7, and 1.5 ± 0.3 mg/kg/min, respectively, due to inhibition of glycogenolysis. In conclusion, NNC 25-0926 effectively blocked the ability of glucagon to increase HGP in the dog.

Diabetes is characterized by fasting hyperglycemia and/or abnormal postprandial glycemia (Basu et al., 2004a,b). These abnormalities are associated with increased glucose production, which is the result of both glycogenolysis and gluconeogenesis. Several studies have shown that gluconeogenesis is increased in diabetes due to an increase in the availability of gluconeogenic amino acids, a rise in lipolysis, and the resulting elevation in free fatty acids and glycerol levels (Consoli et al., 1990; Puhakainen et al., 1992; Gastaldelli et al., 2000). Another contributing factor is the increase in gluconeogenic enzymes levels or activity (glucose-6-phosphatase, fructose-1,6-bisphosphatase, and phosphoenolpyruvate carboxykinase) caused by a combination of an absolute or relative increase in glucagon and a reduction in the levels of or resistance to plasma insulin (Pilkis and Granner, 1992). It should be noted, however, that when glucose, insulin, and glucagon concentrations were clamped in normal subjects and those with mild diabetes to levels seen in severely diabetic subjects, excessive endogenous glucose production was entirely accounted for by an increase in glycogenolysis (Basu et al., 2004a). In addition, it has been shown that a lack of suppression of glucagon secretion contributes to postprandial hyperglycemia in subjects with type 2 diabetes, at least in part by altering glycogen metabolism (Shah et al., 2000). Although there are many potential factors responsible for the high rate of glucose production seen in the individual with diabetes, an increased portal vein concentration of glucagon is clearly a factor (Shah et al., 2000; Jiang and Zhang, 2003; Basu et al., 2004b). Therefore, new therapeutic agents capable of blocking the effect of glucagon on glucose production could be effective in lowering fasting hyperglycemia and reducing postprandial glucose excursions in people with type 2 diabetes.

Effects of antiglucagon antibodies, nonpeptide receptor antagonists, and peptide receptor antagonists on glucagon-induced hyperglycemia in animal models of diabetes have been demonstrated (Unson et al., 1989; Brand et al., 1994; Ling et al., 2002). Previous studies showed that Bay-27-9955 (a nonpeptide antagonist) blocked glucagon-induced increases in glucose production (Petersen and Sullivan, 2001). In addition, Skyrin, a low-molecular-weight nonpeptide, was shown to inhibit glucagon-stimulated cAMP production and glycogenolysis by functionally uncoupling the glucagon receptor from cAMP production. This resulted in a reduction of glucose production by hepatocytes (Parker et al., 2000). Recently, it has been reported that a new compound, N-[3-cano-6-(1,1-dimethylpropyl)-4,5,6,7-tetrahydro-1-benzothien-2-yl]-2-ethylbutamide, blocks glucagon binding to its receptor and thereby antagonizes its biological effects on hepatocytes and on the mouse liver in vivo (Qureshi et al., 2004).

The first aim in the present study was to assess the ability of a novel glucagon receptor antagonist, NNC 25-0926, to block the action of glucagon on hepatic glucose production in vivo in 18-h fasted conscious dogs. The second aim was to then use this tool to assess the role of basal glucagon levels in stimulating hepatic glucose production in the 18-h fasted dog.

	Materials and Methods

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Animal Care and Surgical Procedures. Experiments were performed on 18-h overnight-fasted, conscious mongrel dogs of either sex (19–25 kg). A fast of this duration was chosen because absorption of the diet used in our studies is not complete until 16 to 18 h after a meal. Housing and diet have been described previously (Edgerton et al., 2004). The surgical facility met the standards published by the American Association for the Accreditation of Laboratory Animal Care guidelines, and the protocol was approved by the Vanderbilt University Medical Center Animal Care Committee.

Two weeks before the study, a laparotomy was performed for placement of silastic catheters into the jejunal and splenic veins for intraportal infusion of insulin (Eli Lilly & Co., Indianapolis, IN) and glucagon (Eli Lilly & Co.) and sampling catheters into the femoral artery as well as the portal vein and left common hepatic vein and a renal vein, as described previously (Dobbins et al., 1995). Transonic flow probes (Transonic Systems Inc., Ithaca, NY) were positioned around the hepatic artery and portal vein as described previously (Myers et al., 1991). A catheter was placed in the duodenum for compound administration, as described previously (Moore et al., 1994). All the animals studied met the established criteria for good health: 1) leukocyte count <18,000/mm³, 2) hematocrit >35%, 3) good appetite, and 4) normal stools. On the day of the study, angiocaths were placed in leg veins for [3-³H]glucose (PerkinElmer Life and Analytical Sciences, Boston, MA), indocyanine green (Sigma-Aldrich, St. Louis, MO), peripheral glucose (20% dextrose; Baxter Healthcare, Deerfield, IL), and somatostatin infusion.

Experimental Design. Each experiment consisted of an equilibration period (–140 to –40 min), a basal period (–40 to 0 min), and an experimental period (0–180 min). At –140 min, a priming dose of [3-³H]glucose (33 µCi) was given, followed by a constant infusion of [3-³H]glucose (0.35 µCi/min) and indocyanine green (0.07 mg/min). The small-molecule glucagon receptor antagonist NNC 25-0926 (Novo Nordisk A/S) was dissolved in a vehicle consisting of 13% vitamin E tocopheryl polyethylene glycol 1000 succinate, 1.5% Kollidon 12 pyrogenic free, and 65% sodium phosphate buffer at 0.1 M, pH 7.0, in Milli-Q water (Millipore Corporation, Billerica, MA). At 0 min, the glucagon receptor antagonist NNC 25-0926 was infused intragastrically for 15 min at the required rate. The amount of glucagon receptor antagonist given was based on body weight and the dose desired. Five groups were studied: vehicle (saline), 10, 20, 40, and 100 mg/kg (n = 5, n = 4, n = 4, n = 4, and n = 4, respectively). During the 1st hour following drug administration (first experimental period), glucose was monitored every 5 min to maintain euglycemia using glucose infusion through the saphenous vein as required (20% dextrose). At 60 min (second experimental period), a peripheral infusion of somatostatin (0.8 µg/kg/min) was started to inhibit endogenous insulin and glucagon secretion from the pancreas. At the same time, insulin was infused intraportally at a rate of 200 µU/kg/min (25% below the basal secretion rate), and glucagon was given intraportally at a rate 5-fold basal (2.5 ng/kg/min).

Blood was taken from the femoral artery and the portal and hepatic veins every 20 min during the basal period and every 15 min during the experimental period. The arterial and portal vein blood samples were collected simultaneously 30 s before the collection of the hepatic vein samples to compensate for the transit time of substrates through the liver, thus allowing the most accurate estimates of net hepatic balance. The total volume of blood withdrawn did not exceed 20% of the blood volume of the animal, and 2 volumes of saline was given for each volume of blood withdrawn. All animals were euthanized at the end of the experiment, and the abdomen was opened so that the correct positions of the catheters tips were confirmed.

Receptor Binding Assays. Glucagon and vasoactive intestinal polypeptide were iodinated according to the chloramines-T method (McConahey and Dixon, 1980). Glucagon-like peptide-1 (7-36)amide and porcine glucose-dependent insulinotropic polypeptide were iodinated by using the lactoperoxidase method (Gow and Wardlaw, 1975). The specific activity of the tracers was approximately 80 MBq/nmol. Assays were carried out in filter microtiter plates. A 50 mM HEPES buffer containing 5 mM EGTA, 5 mM MgCl₂, and 0.005% Tween 20 at pH 7.4 was used. NNC 25-0926 was diluted in dimethyl sulfoxide. Buffer, plasma membrane, varying concentrations of NNC 25-0926, and 50,000 cpm of the respective iodinated peptide were added to the wells. The plates were incubated for 2 h at 30°C. Bound tracer was separated from unbound by vacuum filtration. The plates were washed with buffer and dried. The filters were counted in a gamma scintillation counter.

Analytical Procedures. Immediately after samples were drawn, four 10-µl aliquots of plasma were analyzed for glucose using the glucose oxidase method in a glucose analyzer (Beckman Coulter Inc., Fullerton, CA). Plasma [3-³H]glucose was determined following Somogyi-Nielson deproteinization and [³H₂O] exclusion procedures as described previously (Chu et al., 1997). Blood levels of lactate, alanine, and glycerol were determined according to the methods of Lloyd et al. (1978). Indocyanine green was measured spectrophotometrically at 810 nm to estimate hepatic blood flow (Myers et al., 1991). Immunoreactive insulin and glucagon were measured using a double-antibody radioimmunoassay (Morgan and Lazarow, 1962). Cortisol was measured using a gamma coat radioimmunoassay, canine C-peptide was determined using a disequilibrium double antibody radioimmunoassay, and catecholamines were measured using high-pressure liquid chromatography, as described previously (Chu et al., 1997). NNC 25-0926 was assayed in plasma by high-pressure liquid chromatography-tandem mass spectrometry following protein precipitation. A deuterium (²H) and ¹³C-isotopic-labeled analog of NNC 25-0926 was used as an internal standard.

General Calculations. Hepatic blood flow was determined by two methods: transonic flow probes and indocyanine green. The data shown were calculated using the transonic flows. Plasma glucose values were multiplied by 0.73 to convert them to blood glucose values (Edgerton et al., 2004). Net hepatic balance was determined by subtracting the load_out from load_in, where the load_out = [H] x TF and the load_in = ([A] x AF) + ([P] x PF), and [A], [P], and [H] are the blood or plasma concentrations of the substrates in arterial, portal vein, and hepatic vein, respectively, and AF, PF, and TF refer to hepatic artery, hepatic portal vein, and total hepatic blood or plasma flow, respectively. Positive net balance indicates net output, whereas a negative net balance indicates net uptake by the liver. Net fractional extraction was calculated as net Hepatic balance/load_in. Tracer-determined whole-body glucose production (Ra) and utilization (Rd) were measured using a primed, constant infusion of [3-³H]glucose. Data calculation was carried out using the two-compartment model described by Mari (1992) and canine parameters reported by Dobbins et al. (1994).

Gluconeogenic and Glycogenolytic Flux Calculations. Gluconeogenesis is the synthesis and release of glucose formed from noncarbohydrate precursors. Glucose 6-phosphate produced from flux through the gluconeogenic pathway does not necessarily have to be released as glucose; it can also be stored as glycogen, oxidized, or released as lactate. Therefore, there is a distinction between gluconeogenic flux to glucose 6-phosphate, which is the conversion of precursors to glucose 6-phosphate, and gluconeogenesis per se, which is the release of glucose derived from gluconeogenic flux into the blood. In the present study, we estimated hepatic gluconeogenic flux to glucose 6-phosphate and net hepatic glycogenolytic flux as described previously (Edgerton et al., 2004).

The net hepatic uptake of the gluconeogenic precursors alanine, lactate, and glycerol were measured using the arteriovenous difference method (Edgerton et al., 2004). It was assumed that all of the gluconeogenic precursors taken up by the liver were completely converted into glucose 6-phosphate. In these studies, certain gluconeogenic precursors were not measured (e.g., pyruvate, glycine, threonine, serine, glutamine, and glutamate). To the extent that they contributed to gluconeogenic flux, it will be underestimated. To correct for this error and assuming, based on previous studies (Edgerton et al., 2004) suggesting that net hepatic alanine uptake represents a reasonable approximation of the uptake of the unmeasured gluconeogenic precursors, net hepatic alanine uptake was multiplied by 2. Gluconeogenic flux to glucose 6-phosphate was estimated by summing gluconeogenic precursor uptake and dividing by 2 (to convert the data into glucose equivalents). Net hepatic gluconeogenic flux was calculated by subtracting glycolysis from the gluconeogenic flux. Glycolysis was estimated by summing net hepatic lactate output (when it occurred) and glucose oxidation over the course of each experiment. Based on previous studies in dogs, glucose oxidation remains constant during euinsulinemic conditions even when glycemic levels change; therefore, it was assumed to be 0.2 mg/kg/min (Moore et al., 1994). Net hepatic glycogenolysis was determined by subtracting net hepatic gluconeogenic flux from net hepatic glucose balance.

Statistical Analysis. The data were analyzed for differences between vehicle and antagonist groups. Statistical comparisons were carried out using two-way repeated measures ANOVA (SigmaStat; SPSS Inc., Chicago, IL). One-way analysis of variance comparisons were used post hoc when significant F ratios were obtained. Significance was established when P < 0.05 (two-sided test). WinNonlin Professional version 3.1 (Pharsight, Mountain View, CA) was used for noncompartmental analysis of pharmacokinetic parameters and descriptive statistics of NNC 25-0926.

	Results

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NNC 25-0926 is a potent, competitive, glucagon receptor antagonist with an IC₅₀ of 12 nM for inhibition of glucagon binding to cloned human glucagon receptors expressed in baby hamster kidney cells. The compound has a slightly lower affinity for the dog liver glucagon receptor, demonstrating an IC₅₀ of 75 nM. It is also specific for the glucagon receptor in that it binds poorly to related receptors (IC₅₀ values for human GIP receptor, 625 nM; human vasoactive intestinal polypeptide receptor, 429 nM; and human glucagon-like peptide-1 receptor, 21,000 nM). From pharmacokinetic studies conducted in dogs, it was found that NNC 25-0926 has a fairly low oral bioavailability (F p.o. = 18%, t_1/2; = 60 min and clearance = 10 ml/min/kg). Therefore, a relatively high dose range of 10 to 100 mg/kg was used in this study. The plasma concentrations of NNC 25-0926 in the arterial, hepatic, and renal vein were similar, but the level was 2- to 3-fold higher in the hepatic portal vein (Table 1; Fig. 1). Drug levels remained elevated over the 3-h study period in all treated groups. Drug levels increased in a dose-dependent manner with the exception of the 40 mg/kg dose. For some unknown reason that group exhibited lower drug levels than expected. Overall the T_max value occurred between 60 and 100 min with a slightly greater value occurring at the higher doses. In the vehicle and antagonist groups, epinephrine, norepinephrine, and cortisol levels remained basal and unchanged throughout the study, with one exception. In the 40 mg/kg group, one animal had a stress response (Table 2) that elevated the cortisol and epinephrine concentrations in the group as a whole. Even so, the data were not significantly different from those in the other groups. During the first experimental period, glucagon levels showed a tendency to rise in the presence of the antagonist. In response to glucagon infusion, they rose by 70, 80, 68, 93, and 68 pg/ml (change from basal) in the vehicle, 10, 20, 40, and 100 mg/kg groups, respectively, by 15 min (Fig. 2A). Arterial insulin levels (microunits per milliliter) remained basal during the control period and the first experimental period in all groups (7 ± 1, 7 ± 3, 5 ± 2, 9 ± 3, and 6 ± 2, respectively) and then decreased slightly in the second experimental period (4 ± 1, 5 ± 1, 3 ± 1, 4 ± 1, and 4 ± 1, respectively) as a result of the modest under-replacement of the hormone during the pancreatic clamp (Fig. 2B). The C-peptide levels remained basal during the control period and first experimental period in the five groups (0.5 ± 0.1, 0.4 ± 0.1, 0.4 ± 0.1, 0.6 ± 0.1, and 0.4 ± 0.1 pg/ml, respectively). By the end of the second experimental period, C-peptide levels had fallen by 60%, indicating a marked inhibition of endogenous insulin secretion in response to somatostatin infusion (Fig. 2C).

View larger version (16K): [in this window] [in a new window]   Fig. 1. NNC 25-0926 plasma concentration from a typical dog dosed with 10 mg/kg. Each profile is from the four different sampling sites: arterial (), hepatic portal (), hepatic (), and renal vein () during control (–40 to 0 min) and experimental periods (0–180 min).

View larger version (21K): [in this window] [in a new window]   Fig. 2. Arterial plasma glucagon from basal (picograms per milliliter) (A), insulin (microunits per milliliter) (B), and C-peptide (nanograms per milliliter) (C) during control (–40 to 0 min) and experimental periods (0–180 min) in 18-h fasted conscious dogs exposed either to vehicle () or different concentrations of NNC 25-0926: 10 (), 20 (), 40 (), and 100 mg/mg () during period 1 and maintained with a pancreatic clamp during period 2. Values are means ± S.E.M.; n = 5/vehicle and n = 4/NNC 25-0926 groups. *, P < 0.05 versus vehicle group.

Glucose was infused to maintain euglycemia during the first experimental period in all groups. During this period, the average glucose infusion rate was 0.04 ± 0.07, 0.62 ± 0.46, 0.59 ± 0.37, 0.36 ± 0.26, and 0.77 ± 0.40 mg/kg/min in the vehicle, 10, 20, 40, and 100 mg/kg groups, respectively (Table 3). Consequently, the arterial plasma glucose levels were similar during the control period and first experimental period in all groups. In response to the infusion of glucagon, the arterial plasma glucose level (Fig. 3) increased from 112 ± 4 to 245 ± 22 mg/dl by 180 min in the vehicle group. In the 10 mg/kg group, it rose from 114 ± 4 to 172 ± 15 mg/dl (P < 0.05), whereas in the 20, 40, and 100 mg/kg groups the plasma glucose level did not rise (91 ± 9, 110 ± 14, and 90 ± 5 mg/dl, respectively; P < 0.05 versus control group).

View larger version (23K): [in this window] [in a new window]   Fig. 3. Arterial plasma glucose (milligrams per deciliter) during control (–40 to 0 min) and experimental periods (0–180 min) in 18-h fasted conscious dogs exposed either to vehicle () or different concentrations of NNC 25-0926: 10 (), 20 (), 40 (), and 100 mg/mg () during period 1 and maintained with a pancreatic clamp during period 2. Values are means ± S.E.M.; n = 5/vehicle and n = 4/NNC 25-0926 groups. *, P < 0.05 versus vehicle group.

Net hepatic glucose output was basal in all groups during the control period (Fig. 4A), and it remained unchanged in the first experimental period in the vehicle group (2 mg/kg/min), but it fell in the 10, 20, 40, and 100 mg/kg groups by 1.59 ± 0.58, 1.29 ± 0.47, 1.29 ± 0.50, and 1.66 ± 0.60 mg/kg/min, respectively. In the vehicle group, net hepatic glucose output increased to 9.40 ± 1.61 (7.29) mg/kg/min by 15 min after the 5-fold rise in glucagon level was brought about (Fig. 4A). This increase in net hepatic glucose output was blunted in the presence of the antagonist, so that in the 10, 20, 40, and 100 mg/kg groups, it was only 4.11 ± 0.96 (2.52), 1.68 ± 0.58 (0.39), 2.39 ± 0.69 (1.10), and 1.51 ± 0.27 (-0.15) mg/kg/min, respectively (P < 0.05) (Fig. 4A). The area under the curve for the change in net hepatic glucose balance was reduced in the presence of NNC 25-0926 with a maximal response at 20 mg/kg (Fig. 4B). Tracer determined glucose production confirmed these results (Fig. 5A). Endogenous glucose Ra was similar in all groups during the control period and fell by 0.1, 0.6, 0.1, and 0.8 mg/kg/min by the end of the first experimental period in the 10, 20, 40, and 100 mg/kg groups. The rise in plasma glucagon in the vehicle group increased Ra to 6.5 ± 0.9 (4.13) mg/kg/min by 30 min. The rise in Ra was not as great in the 10 mg/kg group, increasing to 5.0 ± 0.4 (2.27) mg/kg/min (P < 0.05). In the 20, 40, and 100 mg/kg groups, Ra was 2.1 ± 0.20 (–0.90), 2.7 ± 0.6 (0.01), and 2.0 ± 0.3 (–1.10) mg/kg/min, respectively, at 30 min (P < 0.05). Tracer determined glucose use changed minimally in all groups (Fig. 5B). Glucose clearance remained basal in all groups during the control period and first experimental period (2–3 ml/kg/min). During the second experimental period, it fell to 1 ml/kg/min in the vehicle group (Fig. 5C). In the 10-mg/kg group, there was a slight fall at 150 min that continued until the end of the second experimental period. In the 20-, 40-, and 100-mg/kg groups, glucose clearance remained basal.

View larger version (24K): [in this window] [in a new window]   Fig. 4. A, net hepatic glucose output (milligrams per kilogram per minute) during control (–40 to 0 min) and experimental periods (0–180 min) in 18-h fasted conscious dogs exposed either to vehicle () or different concentrations of NNC 25-0926: 10 (), 20 (), 40 (), and 100 mg/mg () during period 1 and maintained with a pancreatic clamp during period 2. B, AUC, net hepatic glucose output change over 60 to 180 min (milligrams per kilogram per 120 min). Values are means ± S.E.M.; n = 5/vehicle and n = 4/NNC 25-0926 groups. *, P < 0.05 versus vehicle group.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Tracer-determined glucose production (milligrams per kilogram per minute) (A), glucose use (milligrams per kilogram per minute) (B), and glucose clearance (milligrams per kilogram per minute) (C) during control (–40 to 0 min) and experimental periods (0–180 min) of studies conducted on 18-h fasted conscious dogs exposed either to vehicle () or different concentrations of NNC 25-0926: 10 (), 20 (), 40 (), and 100 mg/mg () during period 1 and maintained with a pancreatic clamp during period 2. Values are means ± S.E.M.; n = 5/vehicle and n = 4/NNC 25-0926 groups. *, P < 0.05 versus vehicle group.

Lactate, glycerol, and alanine are quantitatively the most important gluconeogenic precursors. There were no significant differences in arterial blood levels or net hepatic uptake rates of alanine or glycerol between groups (Table 4). Net hepatic lactate output was 5 µmol/kg/min in the vehicle group during the control period and first experimental period, after which it increased to 13 µmol/kg/min 15 min after the rise in plasma glucagon was brought about. A similar rise (14–22 µmol/kg/min) occurred in the 10 mg/kg group, whereas no change occurred in the 20, 40, and 100 mg/kg groups. By the end of the experimental period, net hepatic lactate uptake was present in the 20, 40, and 100 mg/kg group, whereas the vehicle and 10-mg/kg groups still displayed net lactate output (Fig. 6). Because overall gluconeogenic precursor uptake did not change in any group there was no difference in net hepatic gluconeogenic flux between groups as estimated by the arteriovenous difference method (Fig. 7A).

View larger version (30K): [in this window] [in a new window]   Fig. 6. Lactate arterial blood levels (micromoles per milliliter) (A) and net hepatic lactate balance (micromoles per kilogram per minute) (B) during control (–40 to 0 min) and experimental periods (0–180 min) of studies conducted on 18-h fasted conscious dogs exposed either to vehicle () or different concentrations of NNC 25-0926: 10 (), 20 (), 40 (), and 100 mg/mg () during period 1 and maintained with a pancreatic clamp during period 2. Values are means ± S.E.M.; n = 5/vehicle and n = 4/NNC 25-0926 groups. *, P < 0.05 versus vehicle group.

View larger version (28K): [in this window] [in a new window]   Fig. 7. Gluconeogenic flux (milligrams per kilogram per minute) (A) and net hepatic glycogenolytic flux (milligrams per kilogram per minute) (B) during control (–40 to 0 min) and experimental periods (0–180 min) of studies conducted on 18-h fasted conscious dogs exposed either to vehicle () or different concentrations of NNC 25-0926: 10 (), 20 (), 40 (), and 100 mg/mg () during period 1 and maintained with a pancreatic clamp during period 2. Values are means ± S.E.M.; n = 5/vehicle and n = 4/NNC 25-0926 groups. *, P < 0.05 versus vehicle group.

Net hepatic glycogenolytic flux fell in the first experimental period in the 20, 40, and 100 mg/kg groups (Fig. 7B). In response to the 5-fold rise in glucagon level, net hepatic glycogenolytic flux increased to 9.73 ± 1.84 (7.98) mg/kg/min 15 min after the rise in glucagon in the vehicle group and then waned with time. The glucagon-stimulated increase in net hepatic glycogenolytic flux was inhibited in the 10 mg/kg group, increasing to only 5.53 ± 1.03 (3.23) mg/kg/min (P < 0.05), and it was almost completely eliminated in the 20, 40, and 100 mg/kg groups at 75 min [1.45 ± 0.67 (0.12), 2.27 ± 0.56 (0.77), and 1.69 ± 0.31 (–0.53) mg/kg/min, respectively; P < 0.05].

	Discussion

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The purpose of this study was to assess the ability of a novel glucagon receptor antagonist (NNC 25-0926) to block the action of glucagon on hepatic glucose production in vivo in the 18-h fasted conscious dog. In the present study, a physiological rise in plasma glucagon increased net hepatic glucose output and caused a rise in arterial plasma glucose. These effects were blunted by a low dose of drug (10 mg/kg) and eliminated with higher doses. The compound decreased hepatic glucose production by inhibiting the effect of glucagon on glycogenolysis. Having established the ability of the compound to block the action of glucagon, we were then able to use it to assess the role of basal glucagon in stimulating hepatic glucose output after an 18-h fast in the dog. Inhibition of basal glucagon action caused a decrease of 1.2 mg/kg/min (60%) in net hepatic glucose output, confirming the importance of glucagon in maintaining the supply of glucose to the tissues of the body under fasting conditions.

Several studies have been carried out to examine the effects of blocking the stimulatory effects of glucagon on hepatic glucose production. Glucagon receptor knockout mice (GCGR^–/–) showed decreased blood glucose levels, hypertrophy of the pancreas, and hyperglucagonemia as a compensatory mechanism for the inhibition of glucagon receptor binding (Gelling et al., 2003). Published reports on the use of glucagon receptor antisense oligonucleotides indicate a reversal in the diabetic phenotype that comes about because of a decrease in hepatic glucose production and improved pancreatic -cell function in rodent models of type 2 diabetes (Liang et al., 2004; Sloop et al., 2004). In the present study, the effect of a new nonpeptide glucagon receptor antagonist, NNC 25-0926, was examined for both its pharmacokinetic and pharmacodynamic properties.

The pharmacokinetic analysis of the compound showed that the plasma NNC 25-0926 concentration remained elevated throughout the entire experimental period in all groups. The C_max occurred between 1 and 2 h after dosing, and there was a dose-dependent increase in its value. The data indicated that the concentration of the compound in plasma was 2- to 3-fold higher in the portal vein than in the other vessels. This difference in the concentration of the compound between the vessels can be explained by the fact that the drug was delivered into the portal vein (following absorption), and it was cleared by the liver. For an unknown reason, the C_max values in two of the dogs in the 40 mg/kg group were similar to the mean C_max value seen in the 10 mg/kg group. It should be noted that the low C_max values in those two dogs were associated with proportionally reduced bioactivity.

Under normal physiological conditions, glucagon increases hepatic glucose production and decreases hepatic glucose uptake and has no effect on glucose uptake by muscle or fat. In the present study, the glucagon receptor antagonist blocked the ability of glucagon to increase hepatic glucose production and to decrease hepatic glucose uptake (manifested as a drop in glucose clearance). The combination of both these actions caused a reduction in the plasma glucose excursion. Because glucagon had no effect on glucose uptake by muscle or fat, the antagonist had no effect on glucose use.

To explain the inhibition of hepatic glucose production caused by NNC 25-0926, hepatic gluconeogenic and glycogenolytic rates were measured. Although the rate of hepatic gluconeogenic flux was not altered by the glucagon receptor antagonist, the glucagon-induced increase in net hepatic glycogenolysis was significantly suppressed. Therefore, the mechanism by which the glucagon receptor antagonist blunted hepatic glucose production was through an inhibition of the effect of the hormone on glycogenolysis. The effect of glucagon is time- and concentration-dependent. The present study confirms results from previous experiments showing that a 5-fold elevation of glucagon for 2 h results in increased glycogenolysis but no change in gluconeogenic flux (Cherrington et al., 1981; McGuinness et al., 1994). The lack of effect of glucagon on the gluconeogenic rate can be explained by the fact that the effect of the hormone on gluconeogenesis is limited by its inability to increase the release of gluconeogenic precursors from nonhepatic tissues. During glucagon infusion, transcription of the gluconeogenic enzymes increases, but this up-regulation is counteracted by the fact that there is a decrease in availability of gluconeogenic precursors in plasma. As a result, there is no change in the gluconeogenic flux rate. Chronic infusion of glucagon has, in contrast, been shown to increase gluconeogenesis (McGuinness et al., 1994). This occurs as a result of increasing the availability of gluconeogenic substrates and by augmenting the activity of rate-limiting gluconeogenic enzymes (Burcelin et al., 1996). Thus, it is possible that a chronic infusion of NNC 25-0926 may cause a decrease in gluconeogenesis as well as in glycogenolysis. A recent study has shown that chronic administration of NNC 25-0926 to mice that were fed a high fat diet led to improved -cell function, insulin sensitivity, and glucose tolerance. However, these authors did not measure the effects of the chronic infusion of NNC 25-0926 on gluconeogenesis or glycogenolysis (Nishimura Erica et al., 2006)

There was no change in glycerol levels in the vehicle group or in the treatment groups; therefore NNC 25-0926 had no effect on any action of glucagon on lipolysis. These data are consistent with earlier data suggesting that the effect of glucagon on lipolysis is minimal (Jensen et al., 1991; Gravholt et al., 2001) Normally, glucagon increases net hepatic alanine extraction, and this causes a fall in plasma alanine that then in turn reduces net hepatic alanine uptake and returns it to its basal rate. Glucagon increased the fractional extraction of alanine by the liver in the vehicle group, and this effect was blocked in the treatment groups. This indicates that the effect of glucagon on alanine transport into the hepatocyte (alanine fractional extraction) was not observed in the presence of the antagonist. When the effect of glucagon on alanine transport into the liver was blocked, so, too, was the fall in plasma alanine.

When the breakdown of glycogen into glucose 6-phosphate occurs at a rate too great for flux through glucose-6-phosphatase, lactate leaves the liver following carbon flux through glycolysis, leading to an increase in net hepatic lactate output. In the present study, there was an increase in net hepatic lactate output in the vehicle and the 10 mg/kg groups, but there was no change in net hepatic lactate output in the 20, 40, and 100 mg/kg groups, confirming that the drug inhibits glycogenolysis. When the effect of glucagon on glycogenolysis was blocked, its effect on net hepatic lactate output was also eliminated.

At doses of 20 and 100 mg/kg, basal net hepatic glucose output was reduced from 1.6 ± 0.5 and 2.2 ± 0.6 mg/kg/min to 0.6 ± 0.2 and 0.9 ± 0.6 mg/kg/min, respectively, by the end of the first experimental period. Basal endogenous Ra decreased from 3.1 ± 0.4 and 3.1 ± 0.2 to 2.2 ± 0.4 and 1.8 ± 0.9 mg/kg/min, respectively, over the same time period. Glucose infusion was thus required to maintain euglycemia. The data from the 40 mg/kg group were from the two animals that had low C_max values. These data confirm that basal glucagon is responsible for a significant portion of basal hepatic glucose output. This is in line with earlier data obtained in studies in which somatostatin was used to selectively block glucagon secretion (Cherrington et al., 1979). Raju and Cryer (2005) recently concluded that the maintenance of a normal postabsorptive plasma glucose level is regulated by increments or decrements of insulin alone and that the role of glucagon is only evident when plasma glucose concentrations are below physiological levels or during overt hypoglycemia (Raju and Cryer, 2005). On the contrary, the present study shows that the basal glucose production rate was decreased due to an inhibition of glucagon action under euglycemic conditions at a time when there was no change in the insulin level. Given that in the absence of hyperinsulinemia the plasma glucagon levels can rise in response to small decrements in plasma glucose (Flattem et al., 2001), it is clear that the hormone is an important regulator of the fasting glucose level.

In conclusion, NNC 25-0926 inhibits the ability of glucagon to increase plasma glucose levels by blunting hepatic glucose production. The mechanism by which this inhibition occurs is through an effect on hepatic glycogenolysis. By using a maximally effective dose of the glucagon receptor antagonist (20 mg/kg), fasting plasma glucagon was confirmed to be responsible for a significant portion (60%) of basal hepatic glucose production. It is now important to determine whether chronic inhibition of glucagon action in individuals with type 2 diabetes would result in a long-term improvement in glycemia.

	Acknowledgements

 
We thank Margaret Lautz, Jon Hastings, Angelina Penaloza, Wanda Snead, Eric Allen, Patrick Donahue, Michael P. Andersen, and Kirsten Meeske for excellent technical support.

	Footnotes

 
This research was supported by a grant from the Novo Nordisk A/S. This work was presented in part at the 41st Annual Meeting of the European Association for the Study of Diabetes; 2005 Sept; Athens, Greece.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.115717.

ABBREVIATIONS: NNC 25-0926, (2R)-N-[4-({4-(1-cyclohexen-1-yl)[(3,5-dichloroanilino)carbonyl]anilino}methyl)benzoyl]-2-hydroxy-b-alanine; Ra, tracer-determined whole-body glucose production; Rd, tracer-determined whole-body glucose utilization; AUC, area under the curve; P1, period 1; P2, period 2; Bay-27-9955, (+)-3,5-diisopropyl-2-(1-hydroxyethyl)-6-propyl-4'-fluoro-1,1'-biphenyl.

Address correspondence to: Dr. Alan D. Cherrington, Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 704 Robinson Research Bldg., Nashville, TN 37232-0615. E-mail: alan.cherrington{at}vanderbilt.edu

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