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Endocrinology, doi:10.1210/en.2006-0274
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Endocrinology Vol. 147, No. 10 4664-4673
Copyright © 2006 by The Endocrine Society

Antidiabetic Activity of a Highly Potent and Selective Nonpeptide Somatostatin Receptor Subtype-2 Agonist

Mathias Z. Strowski, Doreen E. Cashen, Elizabeth T. Birzin, Lihu Yang, Vandana Singh, Thomas M. Jacks, Krzysztof W. Nowak, Susan P. Rohrer, Arthur A. Patchett, Roy G. Smith and James M. Schaeffer

Medizinische Klinik m. S. Hepatologie, Gastroenterologie, Endokrinologie und Stoffwechsel (M.Z.S., V.S.), Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, 13353 Berlin, Germany; Departments of Animal Pharmacology (D.E.C., T.M.J.), Molecular Endocrinology and Metabolic Disorders, Atherosclerosis and Endocrinology (E.T.B, S.P.R., J.M.S.), and Medicinal Chemistry (L.Y., A.A.P.), Merck Research Laboratories, Rahway, New Jersey 07065; Huffington Center on Aging and Departments of Molecular and Cellular Biology and Medicine (R.G.S.), Baylor College of Medicine, Houston, Texas 77030; and Department of Animal Physiology and Biochemistry (K.W.N.), August Cieszkowski University of Agriculture, 60-637 Poznan, Poland

Address all correspondence and requests for reprints to: Mathias Z. Strowski, M.D., Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Medizinische Klinik m. S. Hepatologie, Gastroenterologie, Endokrinologie und Stoffwechsel, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: mathias.strowski{at}charite.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Somatostatin inhibits both glucagon and insulin secretion. Glucagon significantly contributes to hyperglycemia in type 2 diabetes. Despite its function in the inhibition of glucagon secretion, somatostatin fails to reduce hyperglycemia in type 2 diabetes, due to a parallel suppression of insulin secretion. Five pharmacologically distinct somatostatin receptor subtypes (sst1–sst5) mediate the effects of somatostatin on a cellular level. Pancreatic A cells express sst2, whereas B cells express sst5. In this study, we describe a novel approach to the treatment of type 2 diabetes using a highly sst2-selective, nonpeptide agonist (compound 1). Compound 1 effectively inhibited glucagon secretion from pancreatic islets isolated from wild-type mice, whereas glucagon secretion from sst2-deficient islets was not suppressed. Compound 1 did not influence nonfasted insulin concentration. In sst2-deficient mice, compound 1 did not have any effects on glucagon or glucose levels, confirming its sst2 selectivity. In animal models of type 2 diabetes in the nonfasted state, circulating glucagon and glucose levels were decreased after treatment with compound 1. In the fasting state, compound 1 lowered blood glucose by approximately 25%. In summary, small-molecule sst2-selective agonists that suppress glucagon secretion offer a novel approach toward the development of orally bioavailable drugs for treatment of type 2 diabetes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCAGON AND INSULIN are two major physiological determinants of glucose homeostasis. After food ingestion, the concentration of insulin rises and promotes glucose uptake by the liver, fat, and muscle cells. The postprandial rise in blood glucose concentration suppresses glucagon secretion. In the fasting state, at low glucose levels, the secretion of glucagon increases. Glucagon prevents the further decrease of blood glucose concentration through enhancement of hepatic glycogenolysis and gluconeogenesis and glucose output (1, 2).

Within the intraislet compartment, the synthesis and secretion of glucagon by the pancreatic A cells is also inhibited by insulin and somatostatin (3, 4). The secretion of both insulin and somatostatin increases in response to nutrient ingestion (5). Both hormones, which interact with the corresponding receptors of A cells via endocrine and paracrine mechanisms (6), are thought to participate in a glucose-dependent inhibition of glucagon secretion (5, 7, 8, 9).

In contrast to the tight physiological control of A cell function, the postprandial suppression of glucagon secretion is markedly impaired in type 2 diabetes (10, 11). Glucose not only fails to inhibit glucagon secretion, even a paradoxical increase of circulating glucagon levels after glucose ingestion has been reported (12). Lack of postprandial suppression of glucagon secretion markedly contributes to hyperglycemia in type 2, as well as type 1 diabetes (10, 13, 14, 15).

Because hyperglucagonemia contributes considerably to hyperglycemia in type 2 diabetes, it has been suggested that lowering the glucagon concentration or blocking glucagon action may alleviate hyperglycemia (16). Brand et al. (16) have demonstrated that immunoneutralization of endogenous glucagon by anti-glucagon antibodies markedly reduces hyperglycemia in diabetic animals. In addition, administration of glucagon receptor antagonists and/or reduction of glucagon receptor expression by antisense oligonucleotides decreased hepatic glucose production and lowered the glucose concentration (17). These data suggest that the reduction of circulating glucagon may be an attractive alternative approach to treating type 2 diabetes. However, agents that specifically and selectively inhibit glucagon secretion have not been identified.

Somatostatin is a potent physiological suppressor of glucagon secretion (3, 4). On the cellular level, somatostatin interacts with five pharmacologically distinct G protein-coupled receptor subtypes (sst1–sst5), which have been cloned from humans, rats, and mice (18, 19). Because somatostatin binds to all five receptor subtypes with a similar affinity and because of its short biological half-life, somatostatin receptor subtype-selective agonists have been developed. Some of them are clinically used in the treatment of disorders associated with hormonal hypersecretion.

Somatostatin and agonists have been tested in the therapy of type 2 diabetes. Despite several positive reports, neither somatostatin nor clinically administered agonists have proved to be useful in improving glucose control in patients with type 2 diabetes (20, 21, 22, 23). Because of the relatively modest receptor subtype selectivity, currently used agonists also suppress insulin, which results in either a lack of the effect on glucose or an increase in glucose concentration.

Recently, the molecular basis for the selective regulation of glucagon and insulin secretion by somatostatin receptor subtype-selective agonists has been established. Using antibodies specific for the somatostatin receptors subtype 2 and 5, it has been demonstrated that sst2 is expressed on A cells, whereas sst5 is expressed on B cells (24, 25). Using pancreatic islets isolated from mice carrying a selective deletion of sst2 or sst5, in combination with somatostatin receptor-selective agonists, we and others have demonstrated that sst2 mainly regulates glucagon, whereas sst5 predominantly suppresses insulin secretion (26, 27, 28, 29). Although these data suggest that selective activation of sst2 may decrease glucagon levels, the in vivo relevance of this principle in diabetes has not been evaluated.

Therefore, we have used a potent and highly sst2-selective nonpeptide agonist (compound 1), having more than 3000-fold selectivity over the remaining four receptor subtypes (30), to evaluate its potential in the treatment of type 2 diabetes.

First, we tested its effects on glucagon release from isolated pancreatic islets and in nondiabetic animals with a sst2 deletion to evaluate the in vitro and in vivo selectivity of compound 1. We then characterized the in vivo effects of compound 1 in two different genetic mouse models of type 2 diabetes (ob/ob) and (db/db) mice (31, 32, 33, 34, 35). Finally, we tested the effects of compound 1 in overnight-fasted dogs for its ability to influence fasting blood glucose levels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Somatostatin receptor subtype 2-deficient (sst2–/–) mice (36) and corresponding control male wild-type (WT) control mice (C57BL/6J, 20–35 g, 3–6 months old) were used for both in vitro and in vivo experiments. Leptin gene-deficient male (ob/ob) mice, leptin receptor gene-deficient (db/db) mice (40–55 g, 6–15 wk old), Harlan Sprague Dawley rats (350 g), and adult, male beagles were purchased from Harlan (Indianapolis, IN). Unless otherwise stated, animals were maintained on a 12-h light/12-h dark cycle with free access to food and water. All animal experiments were conducted in accordance with the guidelines of the Rahway Institutional Animal Care and Use Committee and in accordance with German legislation on the protection of animals.

Reagents
The structure of compound 1 and the binding data to somatostatin receptor subtypes are shown in Fig. 1Go. The synthesis and the pharmacology of the compound 1 has been previously described (30).


Figure 1
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  		FIG. 1. Binding affinities of compound 1 for human somatostatin receptor subtypes (hsst1–hsst5). Compound 1: tert-butyl(ßS)-ß-methyl-N-{[4-(2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)piperidin-1-yl]carbonyl}-D-tryptophyl-L-lysinate.

 
Gey’s balanced salt solution (GBSS), RPMI 1640, penicillin, streptomycin, and fetal bovine serum (FBS) were purchased from Life Technologies, Inc. (Gaithersburg, MD). Liberase RI was obtained from Roche Molecular Biochemicals (Indianapolis, IN). BSA, D-glucose, and L-arginine were from Sigma-Aldrich (St. Louis, MO). Xylazine was from Gemini, Burns Veterinary Supply Inc. (Rockville Center, NY) and ketamin from Ketaset Fort Dodge Laboratories Inc. (Fort Dodge, IO).

Determinations of glucose, glucagon, insulin, and GH concentrations
Plasma and blood glucose concentrations were determined by the glucose-oxidase method. Unless otherwise stated, blood samples were taken by cardiac puncture either 30 min after administration of compound 1 (mice) or 15 min thereafter (rats).

Rat glucagon RIAs were purchased from Linco Research Inc. (St. Charles, MO). For the determination of immunoreactive glucagon (IRG) in rats and dogs, blood was withdrawn at the indicated time points and transferred into tubes supplemented with Trasylol to achieve the final concentration of 500 KIU Trasylol per milliliter. Plasma glucagon in rats and mice was measured using a double-antibody RIA. The specificity for glucagon was approximately 100%, the limit of sensitivity was 10 pg/ml. The intraassay and interassay coefficients of variation were less than 7 and 13%, respectively.

For the detection of IRG in plasma from dogs, the following modification was introduced: antiserum from rat RIA was substituted by the antiserum provided by Dr. Gingerich (Washington University School of Medicine, St. Louis, MO). The cross-reactivity with nonglucagon material was between 10–20 pg/ml.

Canine plasma GH concentration was measured in the laboratory of Dr. H. Chen (Merck Research Laboratories, Rahway, NJ) using material obtained from Dr. A. F. Parlow (Pituitary Hormones and Antisera Center, Harbor-UCLA Medical Center, Torrance, CA). GH RIA had been previously validated for quantification of canine hormones (37). Canine GH was radiolabeled and purified on a PD-10 Sephadex column (Pharmacia, Uppsala, Sweden) and separated from the column using normal rabbit serum. The intraassay and interassay coefficients of variations for the canine GH RIA were 6.8 and 14%, respectively. The sensitivity at 95% binding was 0.8 ng/ml (37).

Insulin concentration in rats, mice, and dogs was determined using a double antibody RIA from Linco Research, Inc. The limit of sensitivity of the assay is 0.1 ng/ml; the specificity for rat and human insulin is 100%. Intraassay and interassay coefficients of variations are 1.4–4.2% or 8.5–9.4%.

Isolation of pancreatic islets and static incubation experiments
All in vitro secretion experiments were performed on pancreatic islets isolated from overnight-fasted adult WT and sst2–/– mice (20–35 g, 3–6 months old) as previously described (29). Briefly, 3 ml of liberase RI solution (33 mg/ml in GBSS) was injected into the main pancreatic duct. Distended pancreas was quickly excised and incubated for 12–14 min at 37 C to allow enzymatic digestion of the exocrine pancreas and connective tissue. The reaction was stopped by the addition of 50 ml ice-cold GBSS containing 10% FBS, and the tissue was mechanically dispersed into small pieces. The digest was then washed several times with GBSS supplemented with 10% FBS and centrifuged for 10 min at 800 x g at room temperature. The islet-rich pellet was resuspended in GBSS, including 10% FBS and antibiotics, and islets were hand-picked using a stereomicroscope. Islets were allowed to recover for 24 h at 37 C in RPMI 1640 medium supplemented with 10% FBS and antibiotics.

Islets were washed with GBSS (supplemented with 0.1% BSA), preincubated for 1 h at 37 C, washed again, and incubated in batches of 10 islets per 1 ml GBSS (containing 0.1% BSA) for 2 h at 37 C with 20 mM L-arginine or 20 mM D-glucose in presence or absence of compound 1 at the indicated concentrations. Medium was then aspirated and analyzed using a rat RIA for the concentration of secreted glucagon and insulin. Data are expressed as percent of control, which we defined as glucagon or insulin release observed upon incubation with 20 mM L-arginine or 20 mM D-glucose only. Each data point represents mean ± SEM from three to six independent experiments performed in quadruplicates. Statistical significance was defined as P < 0.05. ***, P < 0.001; **, P < 0.01; *, P < 0.05 vs. controls.

In vivo studies
Adult male sst2–/– and age-matched WT control mice, db/db, and ob/ob mice (40–55 g, 6–15 wk old), and Harlan Sprague Dawley rats (350 g) fed ad libitum received ip or iv injections of 100 µl per 50 g body weight (BW) of vehicle [ethanol in 0.9% sodium chloride (5:95, vol/vol) supplemented with 0.1% BSA] or with compound 1. Unless otherwise stated, animals (n = 6 per group) were anesthetized with ketamine (87 mg/kg BW, im)/xylazine (13 mg/kg BW, im) 30 min after ip injection of compound 1 or vehicle, and then blood was drawn by cardiac puncture. Rats (n = 6) were anesthetized with ketamine/xylazine before administration of compound 1. Rats were injected iv (via the tail vein) either with vehicle or compound 1 at different doses. Blood was taken via cardiac puncture 15 min after administration of compound 1.

Unless otherwise stated, the animals used in this study were nonfasted. Nonfasted plasma glucose levels were similar to that reported by Kaku et al. (38). Experiments were initiated at 0700 h. Because the mice preferentially consume food in the night, the plasma glucose levels were highest in the early morning and gradually declined throughout the day.

To determine whether the activation of sst2 blocks glucagon response during hypoglycemia, overnight-starved WT mice received 0.5 U/kg BW ip human insulin (dissolved in 0.9% NaCl) and compound 1 at the indicated doses. After 30 min, blood was drawn for determination of glucose and plasma glucagon levels.

Oral administration of compound 1 in a total volume of 260 µl was performed via gavage. Vehicle-treated (0.5% sodium lauryl sulfate/0.5% methocel) animals were used as controls. Unless otherwise stated, blood samples were collected in heparinized syringes by cardiac puncture 30 min after the administration of compound or vehicle.

To evaluate the effects of repeated administration of compound 1 on glucose, insulin, and glucagon concentrations in the nonfasted state, animals were injected four times with compound 1 at hourly intervals (ip) 30 min after the final injection (4.5 h after the beginning of the experiment). Animals were anesthetized as previously described.

Blood samples (300 µl) were obtained by heart puncture and drawn into vacutainer plasma separator tubes that contained 60 U of heparin. Recovered plasma was assayed for its concentration of insulin, glucose, and glucagon. Data are expressed as percent change compared with the vehicle-injected age- and weight-matched animals.

The characterization of the effects of compound 1 on glucose, glucagon, insulin, and GH secretion was performed in dogs. Adult, conscious beagles were fasted for 15 h and injected sc either with vehicle or with the indicated doses of compound 1 (volume 0.2 ml/kg BW). Blood was collected from the jugular vein at the indicated time points. Plasma was recovered by centrifugation (30 min at 12,000 x g) and stored at –70 C until analysis.

Data were collected from three (insulin and GH) to six (glucose, glucagon) dogs and are expressed as percent change vs. the average values measured at 15 min and at 0 min before application of compound 1. All animals were closely monitored for signs of toxicity (e.g. hemolysis, respiratory distress, or blood in urine).

Statistical analysis
Unless otherwise stated, the data were analyzed by two-way ANOVA with Bonferroni post tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. vehicle-treated animals) was considered as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Somatostatin receptor subtype 2 inhibits glucagon secretion in vitro and in vivo in nondiabetic mice
The pharmacological properties of compound 1 are given in Fig. 1Go. Compound 1 inhibited L-arginine-stimulated glucagon secretion from pancreatic islets isolated from nondiabetic WT mice (IC50, 0.37 nM), but it failed to inhibit glucagon secretion from sst2–/– islets (Fig. 2AGo). Compound 1 inhibited insulin secretion from islets isolated from WT mice at a much lower potency (IC50 > 100 nM) and did not alter insulin secretion from the sst2–/– islets (Fig. 2BGo).


Figure 2
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  		FIG. 2. In vivo and in vitro effects of compound 1 on glucagon secretion in nondiabetic mice. A, Effects of compound 1 on arginine-stimulated glucagon secretion from isolated pancreatic islets obtained from sst2+/+ (WT) and from sst2–/– mice. The IC50 value of compound 1 on glucagon secretion from WT islets was 0.37 nM. The basal glucagon secretion rates were 349 ± 56 (femtograms per milliliter secreted in 1 h per islet) from sst2+/+ islets vs. 545 ± 96 from sst2–/– islets (P > 0.05). Arginine increased the glucagon secretion rate from sst2+/+ islets (2611 ± 179, P < 0.05 vs. basal) and from sst2–/– islets (4913 ± 1151, P < 0.05 vs. basal and P < 0.05 vs. sst2+/+ islets). B, Effects of compound 1 on glucose-stimulated insulin secretion from pancreatic islets isolated from sst2+/+ islets and from sst2–/– islets. The basal insulin secretion rates (picograms per milliliter secreted in 1 h per islet) were 155 ± 35 (sst2+/+ islets) vs. 197 ± 54 (sst2–/– islets) (P > 0.05). Glucose increased the insulin secretion rate from sst2+/+ islets (771 ± 97, P < 0.05 vs. basal) and from sst2–/– islets (915.1 ± 105, P < 0.05 vs. basal and P > 0.05 vs. sst2+/+ islets). The effects of compound 1 (0.01 mg/kg BW, ip) on plasma glucagon, insulin, and glucose levels in sst2+/+ (C) and sst2–/– (D) mice in the nonfasted state are shown. Blood was drawn 30 min after injection of compound 1. Plasma levels of glucose, glucagon, and insulin in untreated and vehicle-injected sst2+/+ animals were 236 ± 37 mg/dl, 49 ± 8 pg/ml, and 383 ± 27 pg/ml, respectively. Plasma levels of glucose, glucagon, and insulin in untreated and vehicle-injected sst2–/– animals were 217 ± 9 mg/dl, 62 ± 17 pg/ml, and 270 ± 16 pg/ml, respectively.

 
We next investigated the effects of compound 1 on circulating glucagon, glucose, and insulin in vivo. Compound 1 (0.01 mg/kg BW, ip) lowered glucagon levels in nonfasted sst2+/+ mice by 58.2 ± 3% (P < 0.003, n = 8) 30 min after injection (Fig. 2CGo). This was accompanied by an 18.7 ± 4.1% (P < 0.02, n = 8) fall in plasma glucose levels (Fig. 2CGo). Insulin levels of the WT animals were not altered (Fig. 2CGo). In the nonfasted state, glucagon, insulin, and glucose levels were not affected in the nondiabetic sst2–/– mice treated with the same dose of compound 1 (Fig. 2DGo).

We evaluated the dose-response of compound 1 in rats because of the limited blood volume in mice. In rats fed ad libitum, iv administration of compound 1 dose-dependently decreased glucagon and glucose levels, with the maximum effect being detected after 15 min of treatment without affecting insulin levels (Fig. 3Go, A–C). The lowering of glucagon levels ranged between 66.5 ± 7.3% (0.01 mg/kg BW, P < 0.01, n = 6) and 37.4 ± 6.5% (lowest tested dose of 0.0001 mg/kg BW, P < 0.01, n = 6) (Fig. 3AGo). There was a slight reduction of circulating insulin, possibly due to the fall of plasma glucose levels (Fig. 3BGo). The range of reduction of glucose concentration varied between 31 ± 9% (dose of 0.01 mg/kg BW, P < 0.01, n = 6) and 15 ± 6% (lowest tested dose of 0.0001 mg/kg BW, P < 0.05, n = 6) (Fig. 3CGo). In addition, after oral administration of compound 1 at three different doses into rats, there was a dose-dependent decrease of circulating glucagon levels, paralleled by a reduction in plasma glucose levels (Fig. 3Go, D and F). There was no change in plasma insulin levels after oral administration of compound 1 (Fig. 3EGo). The reduction of plasma glucagon levels ranged between 77 ± 8% (highest tested dose of 10 mg/kg BW, P < 0.001, n = 6) and 45 ± 13% (lowest tested dose of 2.5 mg/kg BW, P < 0.01, n = 6) (Fig. 3DGo). Insulin concentration did not change in response to oral administration of compound 1 (Fig. 3EGo). Glucose levels in the nonfasted state decreased between 34 ± 13% (highest tested dose of 10 mg/kg BW, P < 0.01, n = 6) and 17 ± 8% (lowest tested dose of 2.5 mg/kg BW, P < 0.05, n = 6) (Fig. 3FGo). Thus, compound 1 lowered the concentration of glucagon and glucose after iv and oral route of application.


Figure 3
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  		FIG. 3. In vivo effects of compound 1 on glucagon secretion in nondiabetic nonfasted rats. Characterization of the compound 1 dose response (15 min after iv administration) on plasma glucagon (A), insulin (B), and glucose levels (C) in male Harlan Sprague Dawley rats fed ad libitum. Effects of an oral administration of compound 1 on glucagon (D), insulin (E), and glucose levels (F) in male Harlan Sprague Dawley rats fed ad libitum.

 
Somatostatin receptor subtype 2 decreases circulating glucagon and glucose levels in animal models of type 2 diabetes mellitus
The characteristic hallmarks of type 2 diabetes are obesity, hyperglycemia, insulin resistance, and hyperglucagonemia. We investigated the effects of compound 1 on nonfasted glucagon, glucose, and insulin levels in animal models of type 2 diabetes (ob/ob, db/db mice). In these animals, excessive glucagon secretion contributes to the increased rate of hepatic gluconeogenesis.

Thirty minutes postinjection, compound 1 (0.01 mg/kg BW, ip) reduced plasma glucagon by 73 ± 4.8% (P < 0.001, n = 6) and glucose levels by 37 ± 8.4% (P < 0.004, n = 6) in 6-wk-old ob/ob mice without affecting insulin levels (Fig. 4AGo). Repeated ip administration of compound 1 (four time application of 0.01 mg/kg BW at hourly intervals) reduced plasma glucagon levels by 50.1 ± 8.4% (P < 0.01, n = 6) and glucose levels by 38.7 ± 6.5% (P < 0.01, n = 6) (Fig. 4BGo) after 4.5 h. A higher dose of compound 1 (1 mg/kg BW, ip) decreased nonfasted plasma glucagon levels by 75.2 ± 5.6% (P < 0.0001, n = 6) after 30 min in 15-wk-old male db/db mice paralleled by a 48 ± 4.5% (P < 0.006, n = 6) lowering of plasma glucose levels without accompanying changes in insulin levels (Fig. 4CGo). Oral administration of compound 1 at 1 mg/kg BW and 3 mg/kg BW to ob/ob mice reduced nonfasted glucagon and glucose levels after 30 min (Fig. 4Go, D–F). The maximal inhibition of glucagon was 53 ± 23% vs. control, P < 0.05, n = 6 (Fig. 4DGo). Glucose concentration was reduced maximally by 35 ± 23% vs. control, P < 0.01, n = 6 (Fig. 4FGo). Compound 1 slightly influenced insulin levels (Fig. 4EGo) and did not have any effects on fasting glucagon, insulin, or glucose levels (data not shown).


Figure 4
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  		FIG. 4. Effects of compound 1 on glucose levels in ad libitum-fed animal models of type 2 diabetes. A, Effects of a single injection of low-dose (0.01 mg/kg BW, ip) compound 1 on plasma glucagon, insulin, and glucose levels in 6-wk-old ob/ob mice fed ad libitum. Plasma levels of glucose, glucagon, and insulin in untreated and vehicle-injected nonfasted animals were 729 ± 93 mg/dl, 97.3 ± 14.5 pg/ml, and 3.97 ± 0.8 ng/ml, respectively. Blood was drawn 30 min after the injection. B, Effects of repeated administration of compound 1 (0.01 mg/kg BW, ip, four times at hourly intervals) on plasma levels of glucagon, insulin, and glucose in 6-wk-old ad libitum-fed ob/ob mice. Blood was drawn 4.5 h after the first injection (30 min after the final injection). C, Effects of high-dose compound 1 (1 mg/kg BW, ip) on plasma levels of glucagon, insulin, and glucose in 15-wk-old db/db mice fed ad libitum. Blood was drawn 30 min after the injection of compound 1. Plasma levels of glucose, glucagon, and insulin in untreated and vehicle injected animals were 689 ± 53 mg/dl, 299 ± 23 pg/ml, and 908 ± 176 pg/ml, respectively. Data are expressed as the percent change compared with the vehicle-injected age- and weight-matched animals. Effects of the oral administration of compound 1 at 1 mg/kg BW and 3 mg/kg BW on plasma glucagon (D), insulin (E), and glucose (F) levels in 6-wk-old male ob/ob mice fed ad libitum. Blood was drawn 30 min after the injection.

 
Somatostatin receptor subtype 2-selective agonist moderately lowers fasting blood glucose levels
We administered compound 1 (0.5, 0.05, and 0.005 mg/kg BW, sc) to overnight-fasted, nondiabetic beagles to determine whether inhibition of glucagon secretion reduces fasting blood glucose levels. Compound 1 lowered fasting blood glucose levels in a dose- and time-dependent manner (Fig. 5AGo). The intermediate dose of compound 1 (0.05 mg/kg BW) maximally lowered the fasting blood glucose levels by 24.8 ± 6% (P < 0.05, n = 6) at the 45-min time point (Fig. 5AGo). There was no further decrease in blood glucose levels at the higher dose of compound 1 (0.5 mg/kg BW). The fall in fasting blood glucose levels was accompanied by a reduction of circulating immunoreactive plasma glucagon; the maximum suppression was 43 ± 6.5% observed at a dose of 0.5 mg/kg BW at the 90-min time point (Fig. 5BGo). We observed a tendency toward decreased insulin concentration; however, the effect did not reach a statistical significance (Fig. 5CGo).


Figure 5
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  		FIG. 5. Effects of compound 1 on fasting glucose levels in nondiabetic animals. Effects of compound 1 on fasting blood glucose levels (A), plasma IRG (B), insulin (C), and GH levels (D) in nondiabetic dogs. Male dogs were randomly assigned to the indicated treatment group. Blood was withdrawn from the jugular vein 15 min prior injection with compound 1 or vehicle. Fasting blood glucose, plasma glucagon, insulin, and GH levels were 97 ± 3.6 mg/dl, 69.8 ± 5 pg/ml, 7.48 ± 3 ng/ml, and 1.39 ± 0.24 ng/ml, respectively.

 
At low glucose levels GH secretion increases and stimulates endogenous glucose production (39). Because somatostatin inhibits GH secretion, we tested the effects of compound 1 on GH levels in fasting dogs. After an overnight fasting period, GH secretion in dogs increased by approximately 15% (data not shown). We did not observe any statistically significant effects of compound 1 (at 0.5, 0.05, and 0.005 mg/kg BW) on GH levels (Fig. 5DGo). Thus, in nondiabetic dogs, compound 1 maximally lowered fasting glucose levels by approximately 25%. Moreover, the reduction of blood glucose levels in beagles was not due to decreased circulating GH.

Somatostatin receptor subtype 2-selective agonist decreases glucagon secretion during insulin-induced hypoglycemia
Finally, we examined whether sst2-selective agonist blocks glucagon secretion in response to insulin-induced hypoglycemia. Overnight-fasted mice were injected with 0.5 U/kg BW human insulin (ip), and coinjected with compound 1 at doses of 0.001, 0.01, or 0.1 mg/kg BW (ip). As expected, administration of insulin resulted in a reduction of blood glucose levels after 30 min by approximately 30% (Table 1Go). Compound 1, at the highest tested dose of 0.1 mg/kg BW, diminished the increase of circulating glucagon in response to hypoglycemia by approximately 30%. The decrease of circulating glucagon observed at the highest tested dose of compound 1 was accompanied by a fall of blood glucose concentration. The lower doses of compound 1 (0.01 and 0.001 mg/kg BW) slightly decreased glucagon concentration, without reaching a statistically significant difference. However, at the intermediate dose of compound 1 (0.01 mg/kg BW), the decrease of blood glucose concentration was not paralleled by a fall of glucagon levels. These results suggest that sst2 may prevent the rise of glucagon during starvation/hyperinsulinemia-induced hypoglycemia and that other extrapancreatic effects (e.g. inhibition of sympathoadrenergic system or reduction of ACTH/cortisol axis) may contribute to the sst2-dependent lowering of blood glucose concentration.


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  		TABLE 1. Effects of the sst2-selective agonist (compound 1) on glucagon response during insulin-induced hypoglycemia in mice after overnight starvation

 
Taken together, the partial inhibition of glucagon rise in response to hypoglycemia by the sst2-selective agonist appears to be a dose-dependent effect.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our study demonstrates, for the first time, a link between a somatostatin receptor subtype 2-mediated inhibition of glucagon secretion and reduction in hyperglycemia. The results of our study strongly accentuate the contribution of hyperglucagonemia to hyperglycemia in type 2 diabetes. The reduction of hyperglucagonemia by an sst2-selective agonist improves glucose control in experimental animal models of type 2 diabetes. Thus, the principle of lowering the concentration of circulating glucagon may potentially provide an approach to alleviate hyperglycemia in type 2 diabetes.

In our previous study we demonstrated, using a different sst2-selective agonist, that sst2 regulates glucagon secretion in vitro (28). In this study, we use a different sst2-selective nonpeptidal agonist, compound 1 (Fig. 1Go), which has more than 3000-fold selectivity over the other four known somatostatin receptor subtypes (30).

In this study, we report that compound 1 inhibits glucagon secretion from isolated murine islets with an IC50 value of 0.37 nM, which is similar to that observed in experiments using pancreatic islets isolated from rats (30). Compared with the sst2-selective agonist used in a previous in vitro study (28), compound 1 appears to be more potent at inhibiting glucagon secretion from isolated murine islets.

Compound 1 has been shown to display a higher binding affinity to sst2. The reported binding potency (Ki) for compound 1 was 0.01 nM (30), whereas the Ki value of the formerly used sst2-selectve agonist was 0.05 nM (30, 40, 41). Thus, the higher potency at inhibiting glucagon secretion from murine and rat pancreatic islets is due to higher affinity of compound 1 to sst2. In addition, the potent inhibition glucagon secretion from WT islets and the lack thereof from islets carrying a deletion of sst2 indicates that compound 1 interacts selectively with sst2 in a system of isolated pancreatic islets.

In vivo, we observed an effective reduction of nonfasted glucagon concentration in healthy, nondiabetic sst2+/+ mice. In contrast, no statistically significant effects were detected in sst2-null mice. In addition, there was no effect on insulin secretion in either genotype. These data are fully compatible with the known predominant expression of sst2 on glucagon-producing cells and sst5 on pancreatic B cells (24, 25), and corroborate our previously reported in vitro data in rodents (28). Furthermore, our findings are in agreement with in vitro observations using systems of perfused rat pancreas and isolated rat pancreatic islets in conjunction with an sst2-selective antagonist, DC-41-33, (26). Another study demonstrated that administration of a different sst2-selective antagonist, BIM 23627, increased glucagon and glucose levels in 10-d-old freely moving rats (42). Taken together, these data suggest that the inhibition of glucagon secretion in vitro by sst2 translates in the in vivo situation.

In contrast to these rodent data, using a model of perfused human pancreas, Moldovan et al. (43) found that sst2 suppresses insulin secretion. However, in this study, only a single concentration of an sst2-selective agonist (DC-32-87) has been used. Furthermore, it is not clear whether the effects of DC-32-87, which is less sst2-selective, were mediated via a direct interaction with the receptors on pancreatic B cells. We are unaware of any additional studies demonstrating the ability of DC-32-87 to suppress insulin secretion.

In addition to the experiments on nondiabetic mice, we also characterized the effects of compound 1 in genetically obese ob/ob and db/db mice, which closely resemble human type 2 diabetes. Depending on the age, both mouse models display hyperglycemia, hyperinsulinemia, hyperglucagonemia, hyperphagia, and a marked obesity (31, 32, 33, 34, 35). Hyperglucagonemia, observed in both db/db and ob/ob mice, is due to excessive glucagon secretion; the glucagon to insulin ratio increases with age (31, 32).

Compound 1 reduced glucagon levels by approximately 60–70% and glucose levels maximally by approximately 35–40% compared with untreated controls. This is consistent with the data reported in db/db mice, using glucagon receptor antisense oligonucleotides (17). Liang et al. (17) demonstrated that the reduction of glucagon receptor expression by approximately 80% resulted in a 25% reduction of serum glucose levels. These data indicate that glucagon-dependent glucose production contributes approximately 30% of the glucose increase in the nonfasted state. However, the validation of these data in humans with type 2 diabetes remains to be established. It is noteworthy that in healthy, nondiabetic humans, administration of a glucagon receptor antagonist reduced glucagon-induced hyperglycemia by approximately 30% (44), which correlates well with the data from our study.

Unfortunately, there is limited data available on the in vivo sst2 role in regulating glucose homeostasis in healthy or diabetic animals, simply due to lack of potent sst2-selective agonists. Therefore, it is difficult to compare the in vivo effects of compound 1 with other studies. Octreotide for example, is a clinically used peptidal somatostatin receptor agonist; however, it has only moderate selectivity for sst2. When used in combination with insulin, octreotide reduced the postprandial glycemic peak by decreasing intestinal nutrient absorption and through inhibition of several gluconeogenic hormones, without affecting GH-levels, however (45, 46). Subcutaneous administration of octreotide elicited beneficial effects in a different group of type 2 diabetic patients (47). However, the major obstacle for the use of octreotide for treating type 2 diabetes is the interaction with sst5 and sst3, albeit with a lower affinity (30). Although octreotide reduces glucagon secretion, the predominant effect is the reduction of insulin secretion. This is probably the reason for the deterioration of hyperglycemia frequently observed in type 2 diabetic patients treated with octreotide as monotherapy (20). It seems plausible that agonists with limited selectivity for sst2, which also interact with sst5 and sst3 with a lower affinity, are not sufficient to improve glycemic control, due to a concurrent inhibition of insulin secretion.

Despite the beneficial effects of compound 1 on glucose levels, there are several limitations of the potential therapeutical usefulness of compound 1, which deserve to be pointed out. One of them is a relatively short half life. Compound 1 reduced plasma glucagon levels in a concentration-dependent manner, reaching a maximum after 30 min in both diabetic and nondiabetic animals. Cessation of compound 1 administration led to a normalization of glucagon levels in the nonfasted animals, paralleled by a restoration of hyperglycemia within 45 min (data not shown). These data suggest that compound 1 has a short biological half life and that continuous administration is required to achieve a sufficient glucose control.

An additional limitation of the therapeutical use of the compound 1 in type 2 diabetes is that at its highest tested dose of 0.1 mg/kg, compound 1 diminished glucagon rise during the hyperinsulinemia/starvation-induced hypoglycemia in mice. The decreased rise of glucagon secretion during the hypoglycemia was accompanied by a diminished glucose concentration compared with animals that were not treated with compound 1.

The lower doses of compound 1 (0.001 and 0.01 mg/kg) failed to reach a statistical significance, despite a trend toward reduction of circulating glucagon levels (Table 1Go). Despite its inability to lower circulating glucagon, the intermediate dose of compound 1 (0.01 mg/kg) significantly decreased blood glucose concentration. The lack of correlation between the reduction of glucagon and glucose concentration (at 0.01 mg/kg) suggests that a small (nonsignificant) reduction of glucagon may cause a profound decline of hepatic glucose output. An alternative explanation could be that hyperinsulinemia at euglycemic or hypoglycemic condition is able to increase the activity of the sympathoadrenergic system and enhance the secretion of cortisol (48, 49). Activation of the sympathoadrenergic system and increased concentration of cortisol contribute to an increased hepatic glucose production and output. It is known that sst2 can inhibit the secretion of epinephrine, norepinephrine, and ACTH (50, 51). The consequence of this effect is the reduction of the hepatic glucose output (52). Thus, it is possible that compound 1, at the intermediate dose, reduced blood glucose levels through a suppression of the sympathoadrenergic system and or ACTH-cortisol axis, but not through suppression of glucagon secretion. However, this issue is beyond the scope of the current manuscript.

The results from the hyperinsulinemia/starvation-induced hypoglycemia suggest that, in situations demanding glucagon hypersecretion (e.g. life-threatening hypoglycemia), the suppression of A cell secretion by sst2-selective agonists may provide a limitation of their therapeutic usefulness.

An important observation made in dogs was that the time course of glucose and glucagon levels does not match entirely. Although glucose concentration reached or even exceeded the starting glucose concentrations, the levels of glucagon still remained suppressed. A possible explanation of this discrepancy is that compound 1 causes a moderate decrease of insulin concentration, although this effect failed to reach statistical significance. The fall of insulin levels after administration of compound 1 appears to be a secondary event, which probably results from the decrease of blood glucose levels (53). This notion is supported by several earlier reports which demonstrated that a small, noninsulin-dependent and mild decrease of glucose levels in dogs (e.g. after an overnight fasting) provides a very sensitive signal that leads to an increased secretion of glucagon and decreased secretion of insulin (53, 54).

It is important to note that somatostatin is a potent inhibitor of GH secretion and, therefore, we extended our study by investigation of the effects of sst2-selective agonist on GH behavior in vivo. In rodents, at least three different somatostatin receptor subtypes control the secretion of GH, including sst1, sst2, and sst5. Due to considerable temporal oscillatory fluctuations of GH secretion, already low GH levels in both animal models of type 2 diabetes, and limited blood volume in mice, the measurement of GH in mouse plasma is a very difficult task. Therefore, we chose to characterize the effects of compound 1 on GH levels in beagles that had been fasting overnight, because fasting favors the stimulation of GH secretion. Despite the increased fasting levels of GH secretion, compound 1, at all three doses tested, failed to reduce circulating GH. Thus, the effects of compound 1 on blood glucose levels cannot be explained by the reduction of GH concentration.

However, our interpretation of GH data in beagles has to be cautious because limited data are available on somatostatin receptors regulating GH secretion in dogs. Two different somatostatin agonists were recently used in dogs, to evaluate their potential to decrease IGF-I secretion in vivo (55). Octreotide, an agonist that binds with the highest affinity to sst2, followed by sst5, and sst3 (at pharmacological doses) failed to reduce IGF-I levels, whereas the novel pan-agonist SOM230, which binds with similar affinity to sst1, sst2, sst3, and sst5, decreased IGF-I secretion. Unfortunately, the authors did not investigate the effects of the somatostatin agonists on GH secretion. Although no direct evidence for the role of somatostatin receptors in regulating the GH-IGF-I axis has been provided in this study, we speculate that different somatostatin receptor subtypes regulate GH secretion in dogs and rats.

In summary, we demonstrate that glucagon significantly contributes to hyperglycemia in rodent animal models of type 2 diabetes, consistent with the "bihormonal theory" first proposed by Unger and colleagues (14, 15, 56). Furthermore, we identify an sst2-selective, nonpeptide agonist as an effective suppressor of glucagon secretion, which translates into a fall in glucose levels in the animal models of type 2 diabetes.

The results of our study indicate that suppression of glucagon secretion by a highly sst2-selective, nonpeptide somatostatin agonist may provide a novel therapeutic approach for the treatment of type 2 diabetes. Because sst2 agonists do not interfere with insulin release, the judicious use of this class of compounds may prove effective in reducing insulin requirements early in type 2 diabetes, which would reduce the need for insulin secretagogues. Lastly, this class of small nonpeptide compounds provides the basis for the development of orally active agents for the treatment of type 2 diabetes.


   Acknowledgments
 
We thank Dr. D. E. Moller, Dr. B. B. Zhang, and Dr. U. Kintscher for their helpful comments during the preparation of this manuscript. We thank Ms Elizabeth Zach for the careful edit of the manuscript.


   Footnotes
 
The project was supported by a grant from the Deutsche Forschungsgemeinschaft (Str558/4-1 to M.Z.S.).

Disclosure: D.E.C., E.T.B., L.Y., T.M.J., S.P.R., A.A.P., R.G.S., and J.M.S. are stock holders at the Merck Sharp Dome.

First Published Online July, 20, 2006

Abbreviations: BW, Body weight; FBS, fetal bovine serum; GBSS, Gey’s balanced salt solution; IRG, immunoreactive glucagon; sst1–5, cloned somatostatin receptor subtypes 1–5; sst2–/–, homozygous somatostatin receptor subtype 2-gene deficiency; WT, wild type.

Received March 1, 2006.

Accepted for publication July 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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