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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-1578
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 2 673-680
Copyright © 2007 by The Endocrine Society

Characterization of Somatostatin Receptor Subtype-Specific Regulation of Insulin and Glucagon Secretion: An in Vitro Study on Isolated Human Pancreatic Islets

Vandana Singh, Mathias D. Brendel, Sylvia Zacharias, Stefan Mergler, Henning Jahr, Bertram Wiedenmann, Reinhard G. Bretzel, Ursula Plöckinger and Mathias Z. Strowski

Medizinische Klinik mit Schwerpunkt Hepatologie (V.S., S.Z., S.M., B.W., U.P., M.Z.S.), Gastroenterologie & Interdisziplinäres Stoffwechsel-Centrum: Endokrinologie und Diabetes mellitus, Charité—Universitätsmedizin Berlin, Campus Virchow Klinikum, 13353 Berlin, Germany; and Third Medical Department and Policlinic (M.D.B., H.J., R.G.B.), University Hospital, Justus-Liebig University, Rodthohl 6, 35932 Giessen, Germany

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Introduction: Pancreatic A- and B-cells express somatostatin receptors (SSTRs). Five pharmacologically distinct SSTR subtypes are known (SSTR1–SSTR5). In rodents, SSTR2 inhibits glucagon secretion, whereas SSTR5 suppresses the release of insulin. Human pancreatic A- and B-cells express SSTR1–3 and SSTR5; however, their contribution to the regulation of glucagon and insulin secretion is not well known.

Aim of the Study: The goal of this study was to characterize the role of individual SSTR subtypes in regulating human glucagon and insulin secretion in vitro.

Methods: Human pancreatic islets were isolated from healthy donors and incubated with somatostatin, SSTR1–3-selective and SSTR5-selective agonists, or an SSTR2-selective antagonist (DC-41-33). Stimulation of insulin secretion was induced by glucose (10, 20 mM) alone or in combination with 10 nM exendin-4 or 10 mM L-arginine. Glucagon secretion was induced by 20 mM L-arginine. Basal secretion of insulin and glucagon was measured at 2.8 or 3.3 mM glucose.

Results: SSTR1-, SSTR2-, and SSTR5-selective agonists inhibited insulin secretion with the following order of potency: SSTR2 (EC50, 0.08 nM) > SSTR5 (EC50, 5.3 nM) > SSTR1 (EC50, 35 nM). Glucagon secretion was inhibited by SSTR-selective agonists with the following order of potency: SSTR2 (EC50, 0.05 nM) > SSTR1 (EC50, 1.8 nM) > SSTR5 (EC50, 28 nM). DC-41-33 dose-dependently reversed the effects of the SSTR2-selective agonist on insulin and glucagon secretion.

Conclusion: Our study demonstrates that SSTR2-agonist is the most potent inhibitor of insulin and glucagon secretion from isolated human pancreatic islets. Furthermore, we identify SSTR1- and SSTR5-selective agonists as additional inhibitors of insulin and glucagon secretion from human pancreas.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SOMATOSTATIN (SST) ACTS as physiological inhibitor of a variety of biological processes (1, 2, 3, 4). Two major bioactive isoforms of SST, consisting of 14 (SST-14) and 28 amino acids (SST-28), respectively, are synthesized in a tissue-specific manner, predominantly in the brain, gut, and pancreas (2, 3, 4). The secretion of SST increases after food ingestion and inhibits the prandial release of both insulin and glucagon (2, 3, 4, 5).

SST acts on G protein-coupled transmembrane receptors. SST receptors (SSTRs) show a tissue-specific distribution pattern, with the highest levels of expression in the brain, gut, and pancreas (6). Five pharmacologically distinct SST receptor subtypes (SSTR1–SSTR5) exist and bind both major SST-isoforms with a similar affinity.

SSTR-selective agonists and antagonists have been developed to characterize the function of individual SSTR subtypes and to identify potential therapeutical targets. Using SSTR-selective agonists and antagonists, we and others (7, 8) have demonstrated that SSTR2 inhibits glucagon secretion in rodents, whereas SSTR5 confers SST-dependent inhibition of insulin secretion.

In agreement with the function, SSTR2 is predominantly expressed in pancreatic A-cells, whereas SSTR5 is found mostly in B-cells in rodents (9, 10). Recently, the presence of all SSTR in pancreatic A- and B-cells of rats and mice has been demonstrated, using different antibodies (11). Striking quantitative differences of SSTR expression pattern between rat and murine islets were observed, implying a species-specific SSTR expression. Using different antibodies, Kumar et al. (12) demonstrated that SSTR1, SSTR2, and SSTR5 are highly expressed in the human endocrine pancreas, whereas both A- and B-cells showed a poor expression of SSTR3 and SSTR4. Reubi et al. (13) found SSTR2 on both A- and B-cells, using a different antibody.

Discrepant observations in the human endocrine pancreas are not only related to the SSTR expression pattern but also related to their function. Using a relative moderate SSTR5-selective agonist, Zambre et al. (14) demonstrated a modest inhibition of insulin secretion from isolated human pancreatic islets, whereas SSTR2-selective agonist was inactive. Atiya et al. (15) reported that SSTR2-selective agonist inhibits insulin secretion from perfused human pancreas, whereas SSTR5- and SSTR3-selective agonists had no effects. Moldovan et al. (16) reported a decrease of insulin secretion from perfused human pancreas using a single dose of an SSTR2-selective agonist DC32–87.

Taken together, the role of individual SSTRs in regulating human insulin secretion is still not sufficiently clear and there are, to our knowledge, no studies that systematically analyze the SSTR subtypes in regulating glucagon secretion in human islets. We therefore aimed to systematically analyze the role of SSTR in regulating human glucagon and insulin secretion using well-characterized SSTR-selective agonists (17), SSTR2-antagonist (19), and isolated pancreatic islets of high quality that are generally used for transplantation procedures in humans.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
SST-14 was from Biotrend Chemikalien GmbH (Cologne, Germany). Exendin-4 (GLP-1 agonist), BSA, D-glucose, and L-arginine were from Sigma-Aldrich Chemie GmbH (Munich, Germany). Tissue culture medium (RPMI 1640, CMRL 1066) and fetal bovine serum were from Invitrogen GmbH (Karlsruhe, Germany). Hank’s balanced salt solution and ficoll-sodium-diatrizoate were from Biochrom AG (Berlin, Germany), and collagenase NB 1 was from Serva Electrophoresis GmbH (Heidelberg, Germany). The human glucagon RIA kit was from DPC Biermann GmbH (Bad Nauheim, Germany) and the human insulin ELISA kit was from DRG Instruments GmbH (Marburg, Germany). The selective agonists for SSTR1–SSTR3 and SSTR5 (Table 1Go) were provided by Dr. Susan P. Rohrer (Merck Research Laboratories, Rahway, NJ) (18). The SSTR2-selective antagonist DC-41-33 (PRL-2903) (19) with the sequence Fpa-c[D-Cys-Pal-D-Trp-Tle-Cys]-Nal-NH2 was provided by Dr. David H. Coy (Tulane University School of Medicine, New Orleans, LA).


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  		TABLE 1. Pharmacological properties of SSTR1–SSTR5-selective agonists (compound 1 – compound 5)

 
Isolation and culture of human pancreatic islets

Human pancreases were surgically harvested from healthy cadavers (Table 2Go), fulfilling criteria of multiorgan donors. Legal consent was obtained either by the organ donor registry or living relatives, in accordance with the Eurotransplant International Foundation. Islet isolation procedure was performed at the Giessen Islet Isolation and Transplant Center, according to institutional standards.


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  		TABLE 2. Characteristics of islet donors with respect to their sex, age, and BMI

 
The pancreases were preserved by hypothermic perfusion with 3000–4000 ml of University of Wisconsin Solution. Each pancreas was intraductally distended with cold (8 C) Hank’s balanced salt solution (1.7 ml/g pancreatic tissue) containing 2000 PZ-U collagenase NB 1 and 40 PZ-U neutral protease (20). Distended organs were placed in a stainless steel chamber. While recirculating the solution, the digestion chamber was set in vertical motion. Digestion temperature was 28–32 C, depending on the amount and dissociation of the tissue. The digested tissue was collected in precooled (4 C) conical centrifugation tubes and, subsequent to washing, was suspended in 400 ml of isoosmolar ficoll-sodium-diatrizoate (density, 1082 g/liter) and loaded into a precooled Cobe 2991 cell separator at 4 C. Islets were separated and purified (21) by automated procedure using a continuous digestion-filtration device (22). Purified islets fractions were washed twice in TCM 199 and finally suspended in CMRL 1066 medium supplemented with 10% fetal bovine serum, 2 mM N-acetyl-L-alanyl-L-glutamine, 1 mM sodium-pyruvate, 10 mM HEPES, 100 IU/ml penicillin, 100 µg/ml streptomycin, 0.01 M hydrocortisone, 0.05 mM dithiothreitol, and 20 µg/ml ciprofloxacine.

Incubation experiments

Islets were allowed to recover for 72–96 h in a humidified atmosphere (5% CO2, 95% air, 37 C). Islets were centrifuged at 800 x g (10 min, room temperature) and resuspended in RPMI 1640 containing 0.1% BSA and 3.3 mM glucose. Islets were handpicked under stereo-microscope and washed twice with serum-free RPMI 1640 and then incubated in the same medium for 1 h at 37 C. Then, islets were washed twice and pretreated in this medium with SST, SSTR-selective agonists, or vehicle for 30 min. Five islets in a volume of 20 µl were transferred into individual wells of a 24-well plate containing 480 µl of RPMI 1640 without additives. The secretion of insulin was stimulated for 1 h using 10 mM or 20 mM glucose, 20 mM glucose/10 nM exendin-4, or a combination of 10 mM glucose/10 mM L-arginine. Unless otherwise stated, glucagon secretion was induced by exposing the islets to 20 mM L-arginine for 1 h. In experiments using the SSTR2-selective antagonist DC-41-33, islets were preincubated for 15 min with the antagonist at the indicated concentrations. Afterward, the SSTR2-selective agonist at the concentration of 10 nM was added to the incubation medium, together with the stimuli of insulin or glucagon secretion. Islets were exposed to the test compounds for 1 h. Medium was aspirated without damaging the islets, centrifuged to remove contaminating cellular debris, and stored at –80 C. Islet purity and viability was monitored by a membrane integrity test (trypan blue exclusion).

Determination of insulin and glucagon concentration

The concentration of secreted insulin from the supernatant and intracellular insulin content, were determined using human insulin ELISA kit. Glucagon concentration in the supernatant and cell lysates was determined using human RIA kit. The intracellular content of insulin and glucagon was measured after an overnight extraction of islets in a mixture containing 95% (vol/vol) ethanol and 5% (vol/vol) of 0.1 N HCl at –20 C.

Data presentation and statistics

Unless otherwise stated, each experiment was performed in sextuplicates-octuplicates, and the data are expressed in percent of maximal hormone secretion. The maximal secretion of insulin was defined as the concentration of secreted insulin in the supernatant, after exposure of islets to a mixture of 20 mM D-glucose with 10 nM exendin-4. In some experiments the maximal insulin secretion was induced by 10 mM glucose, 20 mM glucose alone, or by a combination of 10 mM glucose together with 10 mM arginine, respectively. Unless otherwise stated, the maximal secretion of glucagon was defined as concentration of glucagon released in the supernatant in response to treatment of islets with 20 mM L-arginine.

For the analysis of the concentration-response relationships the data were preprocessed by subtracting the base-line secretion values. The data were then fit by a nonlinear regression analysis (with variable slope), using GraphPad Prism (San Diego, CA). EC50 and maximal response are reported as the mean (±SEM) of individual experiments. Unless otherwise stated all other data are expressed as means ± SEM. Data were tested for the statistical significant differences with Student’s t test or ANOVA, where appropriate. P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Evaluation of insulin responsiveness—quality marker of islet preparation

The quality of pancreatic islets was evaluated by the measurement of insulin secretion of the isolated islets in response to various concentrations of glucose with or without 10 nM exendin-4 (glucagon-like peptide-1 agonist) (Fig. 1AGo). The basal insulin secretion rate was defined as the concentration of the hormone in the supernatant after 1 h incubation of the islets with 2.8 mM D-glucose. Within 1 h (at 2.8 mM D-glucose) islets released approximately 1.33% of the total insulin content (data not shown).


Figure 1
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  		FIG. 1. Evaluation of the responsiveness of isolated human pancreatic islets exposed to various known stimuli of insulin secretion. Islets were derived from donors indicated in Fig. 2Go. A, Dose-dependent effects of glucose (2.8 mM and 20 mM) in combination with exendin-4 (10 nM), a glucagon-like peptide-1-agonist. The data represent the amount of secreted insulin (ng/ml) per five islets after 1 h incubation period. B, Effects of L-arginine (10 mM, 20 mM) with or without potassium chloride (20 mM) at three different concentrations of glucose (2.8 mM, 10 mM, and 20 mM). Basal insulin secretion was measured at 2.8 mM glucose. Data were obtained from six to eight samples. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. corresponding controls (1-h insulin secretion at 2.8 mM glucose).

 
At 20 mM glucose, the secretion rate of insulin increased from 2.1 ± 0.3 to 14.2 ± 4.9 ng/ml·h, P < 0.01 (n = 6) (Fig. 1AGo). At 2.8 mM D-glucose, exendin-4 (at the known most effective stimulatory concentration of 10 nM) failed to increase insulin secretion (2.8 mM glucose: 2.1 ± 0.3 ng/ml·h, 2.8 mM glucose + 10 nM exendin-4: 2.9 ± 1.7 ng/ml·h; P > 0.05; n = 6) (Fig. 1AGo). In contrast, exendin-4 potentiated 20 mM glucose-induced insulin secretion (35.2 ± 5.7 ng/ml, P < 0.001; n = 7 vs. 20 mM glucose, and P < 0.001 vs. 2.8 mM glucose) (Fig. 1AGo).

To further characterize the quality of islets, we evaluated the insulin responsiveness of the isolated human islets exposed to 10 or 20 mM glucose, either alone or in combination with L-arginine and/or KCl (Fig. 1BGo). Glucose (10 and 20 mM) potently stimulated insulin secretion (Fig. 1BGo). A marked increase of insulin secretion could be detected when 10 mM L-arginine or 20 mM L-arginine/20 mM KCl were added to 2.8 mM glucose (Fig. 1BGo). The maximal response (~50-fold increase over basal) was observed after exposure of islets to 20 mM arginine/20 mM KCl at 20 mM glucose (Fig. 1BGo).

Taken together, isolated human pancreatic islets showed a physiological response to various known stimuli of insulin secretion. A combination of 20 mM glucose with 10 nM exendin-4 proved to be more potent stimulus of insulin secretion compared with 20 mM glucose alone.

Effects of SST and SSTR-selective agonists on glucose/exendin-4–stimulated insulin secretion

Because basal secretion of insulin was not affected by SST-14 (data not shown), we characterized the effects of SST-14, or SSTR1-, 2-, 3-, and 5-selective agonists on 20 mM glucose/10 nM exendin-4-stimulated insulin secretion (Fig. 2Go). SST-14 potently inhibited insulin secretion with an EC50 of 0.09 nM (Fig. 2AGo and Table 3Go). In contrast, SSTR1 displayed a moderate inhibitory potency (EC50, 35 nM) (Fig. 2BGo and Table 3Go). SSTR2 and SSTR5 were more potent inhibitors of insulin secretion (SSTR2: EC50, 0.08 nM; SSTR5: EC50, 5.3 nM) (Fig. 2Go, C and D, and Table 3Go). SSTR3 failed to influence insulin secretion (data not shown).


Figure 2
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  		FIG. 2. Effects of SST-14 and SSTR-selective agonists on insulin secretion from isolated human pancreatic islets. Islets were incubated for 1 h with SST-14 [donors: 46-yr-old male, body mass index (BMI) of 27.8 kg/m2; and 55-yr-old female, BMI of 22.8], and nonpeptidal agonists selective for SSTR1 (donor: 46-yr-old male, BMI of 27.8), SSTR2 (donors: 46-yr-old male, BMI of 27.8; and 60-yr-old female, BMI of 27.7), and SSTR5 (donor: 54-yr-old female, BMI of 22.7) in the presence of 20 mM D-glucose/10 nM exendin-4. The concentration of released insulin in the medium was determined. The data are expressed in percent of maximal secretion (±SEM), defined as secreted insulin in the presence of 20 mM D-glucose/10 nM exendin-4. Basal secretion was determined at 2.8 mM glucose (n = 6–8).

 

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  		TABLE 3. Summary of the effects of SST-14 and SSTR1-, SSTR2-, and SSTR5-selective agonists on insulin and glucagon secretion from isolated human pancreatic islets

 
Thus, SSTR2-agonist is the most potent inhibitor of insulin secretion, followed by SSTR5- and SSTR1-selective agonist.

SST and an SSTR2-selective agonist potently inhibit glucose and glucose/arginine-induced insulin secretion from isolated human pancreatic islets

To further characterize the effects of the two most potent inhibitors of insulin secretion, we tested the effects of SST-14 and a SSTR2-selective agonist on 10 mM glucose (Fig. 3Go, A and B) and 10 mM glucose/10 mM arginine-induced insulin secretion (Fig. 3Go, C and D). SST-14 and SSTR2-selective agonist inhibited 10 mM glucose-stimulated insulin secretion with an EC50 of 1.6 nM and 0.33 nM, respectively (Fig. 3Go, A and B). SST-14 and SSTR2-selective agonist inhibited 10 mM glucose/10 mM arginine-stimulated insulin secretion with an EC50 of 6.2 nM and 0.52 nM, respectively (Fig. 3Go, C and D).


Figure 3
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  		FIG. 3. Effects of SST-14 and an SSTR2-selective agonist on 10 mM glucose (A and B) and 10 mM glucose/10 mM arginine-induced insulin secretion (C and D) from isolated human pancreatic islets. Islets were derived from 55-yr-old female donor with a BMI of 27.7 kg/m2 (A and B) or 51-yr-old female with a BMI of 25.8 (C and D). Islets were incubated for 1 h with 10 mM glucose or 10 mM glucose/10 mM arginine to stimulate insulin secretion. The effects of either SST-14 or an SSTR2-selective nonpeptidal agonist (at the indicated concentrations) on stimulated insulin secretion from pancreatic islets were tested. After 1 h of incubation with test agents, the amount of released insulin in the medium was measured. The data are expressed in percent of maximal secretion (±SEM), defined as the amount of secreted insulin from islets incubated with 10 mM glucose (A and B) or 10 mM glucose/10 mM arginine. Basal secretion was determined at 2.8 mM glucose (n = 6–8).

 
SSTR2-selective antagonist reverses SSTR2-induced inhibition of glucose-stimulated insulin secretion from isolated pancreatic islets

To confirm further the involvement of SSTR2 in regulating insulin secretion, islets were incubated with an SSTR2-selective agonist (10–7 M) and an SSTR2-selective antagonist (DC-41-33). DC-41-33 reversed the SSTR2-agonist–induced inhibition of glucose-stimulated stimulated insulin secretion with an EC50 of 0.19 nM (Fig. 4Go).


Figure 4
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  		FIG. 4. Dose-dependent reversal of SSTR2-agonist-induced insulin suppression by the SSTR2 antagonist, DC-41-33. Islets were derived from a 43-yr-old female donor (BMI of 24.5). Islets were preincubated with the SSTR2-selective antagonist, DC-41-33 (at the indicated concentrations for 15 min). Subsequently an SSTR2-selective agonist (10–7 M) together with 20 mM glucose was added to the islets. The incubation with test agents was continued for 1 h and the amount of insulin released in the medium was measured. Each data point represents mean (percent reversal of SSTR-2-induced suppression of insulin secretion, corresponding to antagonist activity) ± SEM obtained from 6–8 wells. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. SSTR2-agonist-induced inhibition of insulin secretion.

 
Evaluation of glucagon responsiveness—quality marker of islet preparation

The ability of glucose to suppress glucagon secretion was tested to functionally evaluate the quality of the islet preparations. At 3.3 mM, glucose (defined as basal), the secretion rate of glucagon was 62.6 ± 4.7 pg/ml per 1 h (Fig. 5AGo), which represents approximately 3.34% of the total intra-islet glucagon content. The secretion rate decreased at 10 mM glucose (39.66 ± 11.5pg/ml; P < 0.01 vs. basal, n = 6) or 20 mM glucose (46.0 ± 3.6 pg/ml; P < 0.01 vs. basal, n = 6), respectively. Next, we tested L-arginine (10 and 20 mM) or KCl (10 and 20 mM), either alone or in combination with each other. KCl (20 mM) or L-arginine (20 mM) stimulated glucagon secretion by approximately 8-fold (P < 0.01; n = 6) or approximately 35-fold (P < 0.001; n = 6), compared with basal secretion (Fig. 5BGo). The secretion rate was highest following treatment with 10 mM L-arginine/10 mM KCl (Fig. 5BGo).


Figure 5
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  		FIG. 5. Characterization of the responsiveness of isolated human pancreatic islets exposed to known regulators of glucagon secretion. Islets were derived from donors indicated in Figs. 6Go and 7Go. A, Inhibitory effects of glucose (3.3, 10, and 20 mM). B, Effects of L-arginine (10, 20 mM) with or without potassium chloride (10, 20 mM) at 3.3 mM glucose. The data represent the amount of secreted glucagon (pg/ml) per five 5 islets after 1-h incubation period (n = 6–8). **, P < 0.01; ***, P < 0.001 vs. corresponding controls (1-h glucagon secretion at 3.3 mM glucose).

 
In summary, 20 mM L-arginine (at 3.3. mM glucose) proved to be a reliable stimulus of glucagon secretion and showed less stimulatory effect on insulin secretion compared with mixture of L-arginine/KCl (Fig. 1BGo). This tempted us to use L-arginine for further experiments.

Effect of SST and SSTR-selective agonists on arginine-stimulated glucagon secretion

SST-14 failed to influence basal glucagon secretion (data not shown); however, it potently inhibited 20 mM L-arginine-stimulated glucagon secretion (EC50, 0.08 nM) (Fig. 6AGo and Table 3Go). SSTR1-selective agonist less potently suppressed glucagon secretion (EC50, 1.8 nM) (Fig. 6BGo) compared with SSTR2-selective agonist (EC50, 0.05 nM) (Fig. 6CGo and Table 3Go). The SSTR5-selective agonist was much less effective (EC50, 28 nM) (Fig. 6DGo and Table 3Go), whereas the SSTR3-selective agonist was inactive (data not shown). Thus, the rank of potency at inhibiting L-arginine-stimulated glucagon secretion was: SSTR2 > SSTR1 > SSTR5.


Figure 6
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  		FIG. 6. Effects of SST-14 and SSTR-selective agonists on arginine-induced glucagon secretion from isolated human pancreatic islets. Islets were incubated for 1 h with SST-14 (donors: 55-yr-old female, BMI of 22.8 kg/m2); and 39-yr-old male, BMI of 23.2), or nonpeptidal agonists, selective for: SSTR1 (donor: 39-yr-old male, BMI of 23.2), SSTR2 (donors: 46-yr-old male, BMI of 27.8; 39-yr-old male, BMI of 23.2; and 60-yr-old female, BMI of 27.7), and SSTR5 (donor: 54-yr-old female, BMI of 22.7). Glucagon secretion in all experiments was stimulated by 20 mM L-arginine. The concentration of released glucagon in the incubation medium was determined. The data are expressed in percent of maximal secretion (±SEM), defined as secreted glucagon in the presence of 20 mM L-arginine. Basal secretion was determined at 3.3 mM glucose (n = 6–8).

 
SSTR2-selective antagonist reverses SSTR2-induced inhibition of arginine-stimulated glucagon secretion from isolated pancreatic islets

To confirm the role of SSTR2 in regulating glucagon secretion, pancreatic islets were incubated with the SSTR2-selective agonist (10–7 M) and DC-41-33. DC-41-33 reversed the SSTR2-selective agonist-induced inhibition of arginine-stimulated glucagon secretion with an EC50 of 0.13 nM (Fig. 7Go).


Figure 7
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  		FIG. 7. Dose-dependent reversal of SSTR2-agonist-induced glucagon suppression by the SSTR2 antagonist, DC-41-33. Islets were derived from a 43-yr-old female donor (BMI of 24.5 kg/m2). Islets were preincubated with the SSTR2-selective antagonist, DC-41-33 (at the indicated concentrations for 15 min). Subsequently an SSTR2-selective agonist (10–7 M) together with 20 mM arginine was added to the islets. The incubation with test agents was continued for 1 h and the amount of glucagon released in the medium was measured. Each data point represents mean (percent reversal of SSTR-2-induced suppression of glucagon secretion, corresponding to antagonist activity) ± SEM obtained from six to eight wells. *, P < 0.05; **, P < 0.01 vs. SSTR2-agonist-induced inhibition of glucagon secretion.

 
Taken together, SSTR2-selective agonist is the most effective inhibitor of glucagon secretion followed by SSTR1 and SSTR5-selective agonist.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Despite discrepant observations concerning the SSTR expression pattern in the endocrine pancreas (9, 10, 11), the function of SSTR in regulating glucagon and insulin secretion in rodents is well documented (7, 8, 23). In contrast, conclusive studies that address the in vitro regulation of glucagon or insulin secretion in humans are either lacking or provide contradictory results (14, 15, 16). The discrepancies may originate due to different islet preparation techniques; the use of variable biological systems (e.g. islet perfusion, static incubation, perfusion of the whole pancreas) or different ligands.

The present study was designed to provide unequivocal evidence for the role of individual SSTRs in regulating glucagon and insulin secretion from isolated human pancreatic islets. To overcome some of these stated potential limitations, we used highly selective and well-characterized SSTR-agonists (7, 17, 18, 23, 24, 25) (Table 1Go).

A substantial advantage in comparison to previous studies is the parallel investigation of glucagon and insulin secretion using high-quality islets, derived from a reference laboratory, which routinely supplies islets for transplantation procedures. Additional significant methodological improvement was the functional assessment of the quality of islet preparations. Islets were routinely prescreened for their ability to adequately secrete insulin or glucagon in response to different physiological stimuli.

For example, exendin-4 increased insulin secretion only at high glucose concentrations, which is in accordance with the literature (26, 27). These data indicate a high quality of the islets preparations and optimal cultivation conditions.

Noteworthy, SSTR-selective agonists used in our study have been well characterized either in heterologous expression systems or, more importantly, on tissues (pancreatic islets) derived from animals carrying a genetic deletion of the individual SSTR (7, 23). Thus, the use of agonists that have been validated in respect to their SSTR selectivity provides a substantial advantage compared with other studies.

Our observations revealed that SSTR2 is the most potent inhibitor of insulin secretion from isolated human pancreatic islets. SSTR2-selective agonist potently reduced insulin stimulated either by high glucose alone or in combination with exendin-4 or L-arginine. Additional evidence for the role of SSTR2 as an inhibitory SSTR receptor subtype regulating insulin secretion was confirmed by the use of the SSTR2-selective antagonist DC-41-33.

Altogether, these results are quite unexpected, because they do not correspond to the expression pattern of SSTRs on B-cells (SSTR2 is expressed in approximately 46% of B-cells, SSTR5 in 100%, SSTR1 in 87%) (12). Although our findings do not entirely match the expression pattern of SSTR they are in agreement with two previous studies on perfused human pancreas, describing SSTR2 as an inhibitor of insulin secretion (15, 28). However, in these studies less SSTR2-selective agonists have been tested. Because these studies have been performed on perfused pancreas it was not possible to rule out that the effects of SSTR2-agonists on insulin secretion could be mediated through extra-islet or other factors, because acinar cells, blood vessel cells also express SSTR2.

Another study on perfused human pancreas model (16) has reported that the SSTR2 selective agonist DC32–87 (5 ng/ml) inhibited insulin secretion; however, the dose-response relationship was not investigated. In subsequent studies, however, the selectivity of DC32–87 has been questioned (3). In a different study SSTR2-selective agonists failed to suppress glucose-stimulated insulin secretion in isolated human islets (14). In contrast, moderate SSTR5-selective agonists were effective at lowering insulin secretion, with the EC50 of 0.2 and 1.0 nM (14) that are comparable to our data. However, the limitations of the study by Zambre et al. were a moderate selectivity and the lack of evaluation of other SSTR-selective agonists (29, 30).

In contrast to these reports, an SSTR5-selective agonist (5 ng/ml) failed to influence glucose-induced insulin secretion from perfused human pancreas (16). Because in this experiment the islets were repeatedly used, the authors concluded that loss of responsiveness to SSTR5-selective agonist may have occurred due to decreased viability of the perfused tissue, which is highly sensitive to environmental conditions (temperature, lack of oxygen, etc.). Thus, a clear conclusion regarding the role of SSTR5 in regulating insulin secretion in humans has not been drawn.

Because in the past only SSTR2- and SSTR5-selective agonists have been tested, some of which display a relative moderate selectivity, references to the effect of other SSTR in regulating insulin secretion are lacking.

In our experiments, the SSTR1-selective agonist displayed a weaker potency to inhibit insulin secretion, compared with SSTR2- and SSTR5-selective agonists, despite being highly expressed on pancreatic B-cells (12). Hypothetically, this may be due to a less efficient coupling to G-proteins, a lack of efficacy of the SSTR1 compound and/or possible involvement of SSTR1 in totally different biological processes. So far, the hypothesis of low efficacy can be ruled out because SSTR1 has been proved to be a potent regulator of other physiological processes (31).

Thus, SSTR2 and SSTR5 are the predominant two receptor subtypes involved in the regulation of insulin secretion in a system of isolated human pancreatic islets in vitro. It is important to note that the role of SSTR5 at inhibiting insulin secretion is in agreement with the data reported in rodents using the same agonists as in our current studies (7, 23).

We and others (7, 8) have previously demonstrated that SSTR2 is the main receptor involved in the regulation of murine glucagon secretion in vitro and these data were recently confirmed by a different study using DC-41-33. In contrast to rodents (7, 8, 23, 32, 33), it is not known which SSTR regulates glucagon secretion in humans. Based on the expression pattern (~89% of human A-cells express SSTR2) (12), we predicted that SSTR2 is a potent regulator of glucagon secretion in humans. Consistent with the expectation, the SSTR2-selective agonist was highly effective at inhibiting glucagon secretion. The role of SSTR2 at inhibiting glucagon secretion was strengthened by the use of an antagonist for this receptor subtype (DC-41-33).

In agreement with the lower expression levels of SSTR5 on A-cells (12), the effects of the SSTR5-selective agonist were markedly less potent, indicating a positive correlation between the SSTR expression and function. Noteworthy, in rodents SSTR5 does not play a major role in regulating glucagon secretion (7).

Surprisingly, the SSTR1-selective agonist inhibited glucagon secretion. Thus, the inhibitory potency of the SSTR1-selective agonist appears not to correspond with the low expression level this SST receptor subtype in human pancreatic A-cells.

Taken together, similar to rodents (7, 8), SSTR2-agonist appears to be the most effective at suppressing glucagon secretion, followed by SSTR1- and SSTR5-selective agonists. Thus, the high expression of SSTR2 on pancreatic A-cells in humans correlates with the SSTR2-function as an effective inhibitor of glucagon secretion.

In summary, our study demonstrates that SSTR2 potently regulates both insulin and glucagon secretion from isolated human pancreatic islets. In addition, SSTR5 appears to be an additional receptor subtype playing a role in regulating human insulin secretion and a less potent inhibitor of glucagon secretion. SSTR1-selective agonist is more potent at inhibiting glucagon secretion compared with its effects on insulin secretion. The function of SSTR2 in regulating the secretion of both pancreatic hormones is of clinical importance, because SST agonists, such as octreotide, which interact with at least three different SSTRs (SSTR2, SSTR5, and SSTR3) are used in the therapy of hypersecretory disorders. Because increased glucagon secretion markedly contributes to hyperglycemia in type 2 diabetes, agents that reduce glucagon secretion appear to be attractive therapeutics. The current data possibly provide an explanation for the lack of beneficial effects with octreotide in type 2 diabetes. This agent suppresses both insulin and glucagon secretion. Octreotide has the highest affinity to SSTR2. Our study demonstrates that SSTR2 potently inhibits both insulin and glucagon secretion; therefore, it is plausible that octreotide suppresses the secretion of both hormones due to the interaction with SSTR2. Therefore, a further search for other selective suppressors of glucagon secretion is necessary to address this attractive approach in the therapy of hyperglucagonemia in type 2 diabetes or glucagon-secreting neuroendocrine tumors.


   Acknowledgments
 
We acknowledge Dr. David H. Coy, Ph.D. (Tulane University School of Medicine, New Orleans, LA), for providing the SSTR2-selective antagonist DC-41-33 (PRL-2903) and Elizabeth Zach for a careful reading and editing of the manuscript.


   Footnotes
 
This work was supported by the Deutsche Forschungsgemeinschaft (Str558/4-1, 4-2), Deutsche Diabetes-Gesellschaft (to M.Z.S.), and the Sonnenfeld-Stiftung.

First Published Online November 14, 2006

Abbreviations: BMI, Body mass index; SST, somatostatin; SSTR, SST receptor.

Received July 20, 2006.

Accepted November 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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