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Endocrinology, doi:10.1210/en.2006-1659
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Endocrinology Vol. 148, No. 8 3887-3899
Copyright © 2007 by The Endocrine Society

Somatostatin Receptor Subtype-2-Deficient Mice with Diet-Induced Obesity Have Hyperglycemia, Nonfasting Hyperglucagonemia, and Decreased Hepatic Glycogen Deposition

Vandana Singh1, Carsten Grötzinger1, Krzysztof W. Nowak, Sylvia Zacharias, Eva Göncz, Gesine Pless, Igor M. Sauer, Ines Eichhorn, Brigitte Pfeiffer-Guglielmi, Bernd Hamprecht, Bertram Wiedenmann, Ursula Plöckinger and Mathias Z. Strowski

Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie (V.S., C.G., S.Z., E.G., I.E., B.W., U.P., M.Z.S.), Interdisziplinäres Stoffwechsel-Centrum: Endokrinologie, Diabetes und Stoffwechsel, and Klinik für Allgemein-, Visceral- und Transplantationschirurgie Campus Virchow-Klinikum (G.P., I.M.S.), Charité-Universitätsmedizin, 13353 Berlin, Germany; Department of Animal Physiology and Biochemistry (K.W.N.), August Cieszkowski University of Agriculture, 60-637 Poznan, Poland; and Interfakultäres Institut für Biochemie (B.P.-G., B.H.), Universität Tübingen, 72076 Tübingen, Germany

Address all correspondence and requests for reprints to: Mathias Z. Strowski, M.D., Medizinische Klinik m. S. Hepatologie, Gastroenterologie and Interdisziplinäres Stoffwechsel-Centrum/Endokrinologie und Diabetes mellitus, 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
 
Hypersecretion of glucagon contributes to abnormally increased hepatic glucose output in type 2 diabetes. Somatostatin (SST) inhibits murine glucagon secretion from isolated pancreatic islets via somatostatin receptor subtype-2 (sst2). Here, we characterize the role of sst2 in controlling glucose homeostasis in mice with diet-induced obesity. Sst2-deficient (sst2–/–) and control mice were fed high-fat diet for 14 wk, and the parameters of glucose homeostasis were monitored. Hepatic glycogen and lipid contents were quantified enzymatically and visualized histomorphologically. Enzymes regulating glycogen and lipid synthesis and breakdown were measured by real-time PCR and/or Western blot. Gluconeogenesis and glycogenolysis were determined from isolated primary hepatocytes and glucagon or insulin secretion from isolated pancreatic islets. Nonfasting glucose, glucagon, and fasting nonesterified fatty acids of sst2–/– mice were increased. Inhibition of glucagon secretion from sst2-deficient pancreatic islets by glucose or somatostatin was impaired. Insulin less potently reduced blood glucose concentration in sst2-deficient mice as compared with wild-type mice. Sst2-deficient mice had decreased nonfasting hepatic glycogen and lipid content. The activity/expression of enzymes controlling hepatic glycogen synthesis of sst2–/– mice was decreased, whereas enzymes facilitating glycogenolysis and lipolysis were increased. Somatostatin and an sst2-selective agonist decreased glucagon-induced glycogenolysis, without influencing de novo glucose production using cultured primary hepatocytes. This study demonstrates that ablation of sst2 leads to hyperglucagonemia. Increased glucagon concentration is associated with impaired glucose control in sst2–/– mice, resulting from decreased hepatic glucose storage, increased glycogen breakdown, and reduced lipid accumulation. Sst2 may constitute a therapeutic target to lower hyperglucagonemia in type 2 diabetes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SOMATOSTATIN (SST) IS AN inhibitory hormone that regulates numerous biological processes. SST exists in two major biologically active isoforms that consist of 14 (SST-14) or 28 (SST-28) amino acids, respectively. SST is synthesized predominantly in the brain, gut, and pancreatic D cells (1). The secretion of SST is regulated by intra- and extrapancreatic (neuro)-mediators and nutrients (1).

SST inhibits the secretion of glucagon and insulin (1, 2). Ontogenetically, SST-producing pancreatic D cells originate from the same Pax4-positive precursor cell as insulin-producing B cells (3). Consistent with this, insulin secretagogues and inhibitors of insulin secretion also stimulate or suppress SST secretion, respectively (4, 5, 6). Similar to insulin, the secretion of SST increases after food ingestion. This prandial SST release is supposed to prevent excessive, postabsorptive insulin and glucagon secretion (7), indicating a role in the control of the postprandial glucose homeostasis (8).

The cellular effects of SST are mediated through a specific interaction with G protein-coupled receptors. Five SST receptor subtypes (sst1–sst5) exist, which have been cloned from human and rodents. Sst isoforms can be distinguished pharmacologically using receptor subtype-selective agonists (1).

In the endocrine rodent pancreas, SST receptors show a cell-type-specific expression pattern, with sst2 predominantly being expressed on A cells and sst5 on B cells. In agreement with the expression pattern, activation of sst2 inhibits glucagon secretion, whereas activation of sst5 mainly suppresses insulin secretion (9, 10).

Physiologically, glucagon secretion increases during fasting and maintains normoglycemia by enhancing hepatic glucose output (11). In healthy individuals, glucagon secretion decreases after food ingestion. In type 2 diabetes, however, the prandial suppression of glucagon secretion is markedly impaired (12, 13). In these patients, not only does glucose fail to inhibit glucagon secretion, but also even a paradoxical increase of circulating glucagon after glucose ingestion has been reported (14). The lack of postprandial suppression of glucagon secretion with resulting hyperglucagonemia contributes to hyperglycemia in type 2 diabetes (15, 16, 17, 18).

It has been demonstrated that immunoneutralization of the endogenous glucagon by glucagon antibodies reduced hyperglycemia in diabetic animals (19). Administration of glucagon receptor (GcgR) antagonists and/or reduction of GcgR expression by antisense oligonucleotides decreased hepatic glucose production (HGP) and lowered glucose concentration (20). Deletion of GcgR reduced blood glucose concentration and improved glucose tolerance (21). Thus, the lowering of circulating glucagon concentration or the blockade of glucagon action may offer a principle to alleviate hyperglycemia in type 2 diabetes.

The majority of type 2 diabetic patients are obese. Obesity represents a major risk factor for the development peripheral insulin resistance, a hallmark of type 2 diabetes. In a recent study, we demonstrated that administration of an sst2 agonist into genetically obese mice with type 2 diabetes reduces glucagon secretion and improves glucose control (22). To further understand the mechanisms by which sst2 participates in the control of glucose metabolism, we investigated the consequences of the deletion of the sst2 gene on parameters of glucose homeostasis using a mouse model with high-fat diet (HFD)-induced obesity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All experimental animal procedures were approved by the Institutional Review Board for the Care of Animal Subjects (protocol G 0107/03) and performed in accordance with German legislation on the protection of animals. Principles of laboratory animal care (National Institutes of Health publication no. 85-23, revised 1985) were followed. Animals were individually caged and maintained under controlled conditions of 25 C and 12-h cycle (lights on 0700 h/dark at 1900 h) with food and water available ad libitum. The generation of mice with homozygous deletion of sst2 has been described (23). The mice originally generated on the genetic background 129 SvEv were backcrossed for six generations onto C57BL/6J mice. The wild-type (WT) and sst2-deficient mice used in the study originated from homozygous mating of backcrossed sst2–/– mice to C57BL/6J mice. The resulting progeny was genotyped by a PCR.

Induction of obesity by high-fat diet in mice
Male sst2-deficient mice (10–14 wk of age) and corresponding WT controls were switched from chow diet (4.05 kcal/g, 3.3 kcal/g metabolizable energy, 12.5% kcal from fat; Harlan Teklad, Madison, WI) to HFD [36% fat content (wt/wt), 60 kcal%; (Research Diets Inc., New Brunswick, NJ)] and fed ad libitum for 14 wk. As controls, animals of the same age and body weight (BW) fed chow diet were used. Food intake and changes of BWs were recorded weekly at 1000 h. At the end, all animals were killed between 1000 and 1200 h.

Reagents
Unless otherwise stated, reagents were from Sigma-Aldrich Inc. (St. Louis, MO). Gey’s balanced salt solution, RPMI-1640, DMEM, Williams medium E, PBS, penicillin, streptomycin, and FCS were from Invitrogen Life Technologies, Inc. (Carlsbad, CA), liberase RI from Roche Biochemicals (Indianapolis, IN), and SST-14 from Bachem Inc. (Bubendorf, Switzerland). The sst2-selective nonpeptidal agonist (24) was provided by Dr. Rohrer (Merck Sharp & Dohme, Rahway, NJ).

Antibodies
The secondary antibody (horseradish peroxidase-linked antirabbit IgG), anti-CREB, anti-phospho-CREB, and anti-glycogen synthase kinase-3-ß (Ser9) were from New England Biolabs (Frankfurt, Germany), anti-glycogen synthase (phospho-Ser640) from Abcam Inc. (Cambridge, MA), and anti-glycogen phosphorylase kinase from Abnova (Heidelberg, Germany). The antiserum against the hepatic isoform of glycogen phosphorylase (GP) was generated by vaccinating rabbits with a conjugate consisting of the isozyme-specific heptadecapeptide KISLSKESSNGVNANGK corresponding to the positions 834–850 in the amino acid sequence of the rat liver enzyme and keyhole limpet hemocyanin. Vaccination, determination of the antiserum titer, and demonstration of monospecificity of the antiserum were carried out in analogy to the procedures described for the muscle and brain isoforms of GP (25).

Glucose, insulin, and somatostatin tolerance tests
Glucose tolerance test. Glucose (1.5 g/kg BW or 2 g/kg BW) was injected ip into 8-h fasted animals. Blood was drawn at the indicated time points.

Tolerance tests with insulin and SST. Human insulin (0.5 IU/kg BW) or SST-14 (100 µg/kg BW) was injected ip into 2-h fasted animals. Blood was drawn at the indicated time points.

Determination of metabolic parameters
Blood was collected either by a tail nick or from the retroorbital vein plexus (within 20 sec) using heparin-coated capillaries and transferred immediately into tubes containing 1 µg/ml aprotinin. Blood glucose concentration was determined using a One Touch glucometer (LifeScan, Inc., Milpitas, CA). Plasma concentration of insulin, leptin, and glucagon-like peptide-1 (GLP-1) were measured by ELISA (Alpco, Windham, NH) or by mouse LINCOplex kit (Linco Research Inc., St. Charles, MO), respectively. Glucagon concentration was determined using a rat glucagon RIA (DPC Biermann, Bad Nauheim, Germany). The specificity for glucagon was 95%, the limit of sensitivity 13 pg/ml, and intra- and interassay coefficients of variation less than 6.5 and 12%, respectively. Plasma concentrations of nonesterified fatty acids (NEFA) were quantified by a colorimetric method (half-micro test; Roche Diagnostics, Mannheim, Germany). Plasma levels of triglycerides were quantified by the colorimetric method, using wet reagent diagnostic kits (Roche, Nutley, NJ).

Excision of mouse organs
At the end of the study, epididymal white adipose tissue, livers, interscapular brown adipose tissue, and pancreata were dissected, and the wet weights were measured. Tissues were then quickly frozen in liquid nitrogen and stored at –80 C.

Glucagon and insulin secretion from isolated pancreatic islets
Islets were isolated by injecting liberase RI solution into the main pancreatic duct and processed as described (9). Ten islets per 1 ml Gey’s balanced salt solution were incubated for up to 2 h at 37 C with D-glucose. Medium was then aspirated and analyzed for the concentration of secreted glucagon or insulin.

Hepatic glycogen content
Liver glycogen content was determined as described (26). Livers (150 mg) were treated with 1 ml KOH (30% wt/vol), and heated for 30 min at 95 C. Lysed tissue was cooled to room temperature and dissolved overnight in 95% ethanol at 4 C. Precipitated glycogen was recovered by centrifugation, dissolved in 0.6 M NaOH, heated to 100 C for 2 h, and neutralized with 0.6 M HCl. After centrifugation, 100 µl of the supernatant was analyzed for glycogen content using a glucose hexokinase assay kit (Sigma-Aldrich Inc., St. Louis, MO).

Hepatic triglyceride content
The intrahepatic triglyceride content was measured as described (27). Briefly, tissue was weighed and homogenized in a mixture of chloroform/methanol/water for 2 min. The homogenate was centrifuged to separate organic and aqueous phases. The organic phase was evaporated at 70 C and then redissolved with isopropanol. Triglycerides were measured using a colorimetric triglyceride kit (Cypress Diagnostics, Langdorp, Belgium).

Glucose production and glycogenolysis assay in primary human hepatocytes
Experimental protocols were approved by the local ethics committee (protocol 32/2004). Hepatocytes were isolated from patients undergoing liver transplantations due to alcoholic liver cirrhosis using a modified two-step collagenase NB8 (Serva, Heidelberg, Germany) perfusion protocol (28). After purification via Percoll (Biochrom AG, Berlin, Germany) density gradient centrifugation, hepatocytes were suspended in Williams medium E (10% FCS, 50 U/ml penicillin, 50 µg/ml streptomycin), transferred into 2% collagen-coated six-well plates, and allowed to recover for 24 h at 37 C and 5% CO2. The hepatocytes showed approximately 90–95% survival rate as detected by trypan blue exclusion.

To study gluconeogenesis, cells (2 x 106) were washed with PBS and incubated with 10 nM glucagon with or without 100 nM SST-14 or an sst2-selective agonist in 2 ml glucose-free DMEM, containing 0.2% (wt/vol) BSA, antibiotics, 4 mmol/liter pyruvate, and 16 mmol/liter sodium lactate (29).

To study glycogenolysis, after the initial 24 h, the medium was replaced for 20 h with basal medium supplemented with 10 mmol/liter glucose and 10 nmol/liter insulin to build up glycogen reserves (30). Hepatocytes were washed with buffer A (117.6 mmol/liter NaCl, 5.4 mmol/liter KCl, 0.82 mmol/liter Mg2SO4, 1.5 mmol/liter KH2PO4, 20 mmol/liter HEPES, 9 mmol/liter NaHCO3, 0.1% BSA, 2.25 mM CaCl2, pH 7.4), preincubated for 4 h in buffer A, washed, and then treated with glucagon (10 nM) and test agents. The amount of glucose released into buffer A reflected glycogenolysis.

In both assays, aliquots were removed at the indicated time points and centrifuged. Glucose concentration was determined by a glucose (hexokinase) assay kit (Sigma-Aldrich).

Preparation of RNA
RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA). RNA was treated with DNase I (Ambion Inc., Austin, TX) for 60 min at 37 C and purified using PCR clean-up columns (QIAGEN Inc., Valencia, CA).

Quantitative real-time PCR
RNA (1 µg) was reverse transcribed using Superscript first-strand system (Invitrogen). cDNA was denatured (95 C, 5 min), first strand synthesized (42 C, 50 min), and the reaction terminated by heating to 70 C for 15 min. RT-PCR up to 40 cycles followed by the thermal denaturation protocol was performed using 50–100 ng cDNA in quadruplicate on an iCycler PCR machine (Bio-Rad Laboratories, Hercules, CA). Six standards (serial dilutions) and a negative control without template were included in each run. All PCR reagents were from IQ SYBR Green Supermix (Bio-Rad). The expression of each RNA was normalized relative to ß-actin RNA. The sequences of all primers are given in Table 1Go. Real-time PCR analysis of sst1–sst5 in pancreases obtained from sst2–/– and sst2+/+ mice fed HFD were performed using primers and PCR conditions as previously described (31).


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  		TABLE 1. Primer sequences used in the real-time PCR and qualitative RT-PCR

 
Preparation of proteins
Frozen tissue was homogenized with UltraTurrax homogenizer (IKA, Stauffen, Germany) in TNA buffer [50 mmol/liter Tris-Cl (pH 7.0), 150 mmol/liter NaCl, 0.1% sodium azide, 0.1% Nonidet P-40] containing 1x proteinase inhibitor cocktail (Complete; Roche), and 500 µl of a solubilization buffer [50 mmol/liter Tris-Cl (pH 7.0), 150 mmol/liter NaCl, 0.1% sodium azide, 3% Nonidet P-40, 1.5% sodium deoxycholate, 0.3% SDS] was added to the lysates. After solubilization (20 min) and centrifugation (20,000 x g for 45 min at 4 C), supernatants were aspirated and stored at –80 C.

Western blot analysis
Tissue lysates were resuspended in a Laemmli sample buffer (Bio-Rad). Denatured proteins (75 µg per lane) were resolved on 12% SDS-PAGE gels and transferred onto nitrocellulose membranes (0.45 µm) (Bio-Rad) by electroblotting. Membranes were incubated overnight with the primary antibody. Blots were washed, incubated with peroxidase-conjugated secondary antibodies, and processed for enhanced chemiluminescence (ECL detection kit; GE Healthcare Amersham, Piscataway, NJ). Duplicate blots of samples from seven animals of each treatment group were processed.

Equal loading was controlled in Western blotting experiments by incubating the blots of the relevant antibody using anti-ß-actin antibodies as a housekeeping protein. The signal intensity of the bands on x-ray films were quantified using densitometric analysis of film scans. Image acquisition, data processing, and statistics were done using Kodak digital science 1D Image Analysis Software, version 3.02, Microsoft Excel, and GraphPad Prism. The data are expressed in arbitrary intensity units as bar graphs with mean ± SEM.

Immunostaining of tissues
Immunofluorescence and immunohistochemical staining was performed as described (32). For glucagon immunofluorescence, mouse pancreatic tissue sections fixed in 4% paraformaldehyde (PFA) for 20 min were incubated overnight with a polyclonal rabbit antihuman glucagon antibody (A 0565; DakoCytomation, Hamburg, Germany) 1:800 in PBS/0.1% BSA. After three rinses in PBS, slides were incubated for 1 h with a Cy3-labeled goat antirabbit IgG antibody (111-165-045; Dianova, Hamburg, Germany) diluted 1:400 in PBS/0.1% BSA.

For insulin immunohistochemistry, mouse pancreatic tissue sections fixed in 4% PFA for 20 min were incubated overnight with a mouse monoclonal antihuman insulin antibody (MOB234; Biotrend, Köln, Germany) diluted 1:200 in PBS/0.1% BSA. After three rinses in PBS, slides were incubated for 1 h with a peroxidase-labeled goat antimouse IgG antibody (PK-6100; Vector Laboratories, Burlingame, CA). After rinsing and mounting, sections were evaluated and recorded using an Axiophot microscope with Axiocam camera system (Zeiss, Jena, Germany).

Visualization of hepatic glycogen
Glycogen periodic acid Schiff staining was performed using solutions and protocol from a Sigma-Aldrich kit (395b-1KT). After fixation in Carnoy solution, sections were incubated in Schiff’s reagent and 96% ethanol. Sections were mounted using Faramount (Dako, Hamburg, Germany).

Visualization of hepatic lipid content
Lipids were detected by a Sudan stain procedure. Tissue sections were fixed in 4% PFA (20 min), washed, and incubated in 50% ethanol (2 min), Sudan IV solution (2C282; Waldeck, Münster, Germany) (15 min), 50% ethanol (three times for 2 min each), and H2O (twice for 5 min each). Sections were counterstained with hemalum, rinsed, and mounted using glycerol gelatin (Merck, Darmstadt, Germany).

Statistical analysis
The results presented in figures and tables are representative for at least three experiments with comparable results. Unless otherwise stated, the data were analyzed by two-way ANOVA with Bonferroni post tests or Student’s t test. Values of P < 0.05, P < 0.01, and P < 0.001 were considered as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SST receptor subtype-2-deficient mice with diet-induced obesity have impaired glucose control
After 14 wk of HFD feeding, the BWs of mice of both genotypes were comparable (Table 2Go). Sst2+/+ mice gained 8.03 ± 3.59 g, whereas BW of sst2–/– mice increased by 9.22 ± 5.6 g (P > 0.05; n = 12–13). The liver weight of both genotypes was similar (Table 2Go). In contrast, the wet weight of epididymal white adipose tissue as well as interscapular brown adipose tissue of sst2-deficient mice was lower compared with the WT controls (Table 2Go), despite comparable food intake (data not shown).


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  		TABLE 2. Characterization of sst2–/– and WT mice (sst2+/+ ) fed HFD for 14 wk

 
Already 4 wk after onset of HFD feeding, blood glucose concentrations were higher in sst2-deficient mice than in WT animals (Fig. 1Go, A and B).


Figure 1
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  		FIG. 1. A and B, Changes of nonfasting (A) and overnight fasting (B) blood glucose levels of WT (sst2+/+) and sst2-deficient (sst2–/–) mice during the feeding with HFD (60 kcal%). At the indicated time points, blood from individually housed animals was taken by a tail incision and blood glucose concentrations were measured. C, The ip glucose tolerance test (1.5 g glucose/kg BW) in WT and sst2–/– animals, after an overnight fasting period. D, Insulin tolerance test (0.5 IU human insulin/kg BW, ip) in sst2+/+ and sst2–/– animals fed HFD after 2 h of food deprivation. The results are expressed in percentage of change compared with basal glucose concentration. E, The ip glucose tolerance test (2 g glucose/kg BW) in sst2+/+ and sst2–/– animals fed chow diet. ***, P < 0.001; **, P < 0.01; *, P < 0.05 using two-way ANOVA test.

 
During the glucose tolerance test (1.5 g/kg BW), blood glucose concentrations at 40 and 60 min were higher in sst2-deficient mice than in ST animals (Fig. 1CGo). Exogenous insulin (0.5 IU/kg BW) lowered blood glucose concentration less efficiently in sst2-deficient mice than in WT animals (Fig. 1DGo).

In contrast to animals fed HFD, animals of both genotypes fed the chow diet showed similar responses to glucose challenge (2 g/kg BW) as detected by glucose tolerance test (Fig. 1EGo). The concentrations of fasting and ambient blood glucose, plasma insulin, and glucagon in animals of both genotypes that were fed normocaloric chow diet were similar (Table 3Go). Detailed analysis of several organ weights and basic metabolic parameters of animals of both genotypes fed HFD or chow diet are shown in Tables 2Go and 3Go.


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  		TABLE 3. Characterization of age-matched sst2–/– and WT mice (sst2+/+) fed chow diet

 
It is important to note that sst2-deficient mice showed higher expression levels of sst5 and sst1 in the pancreas, whereas the expression of sst4 was decreased, as detected by the real-time PCR (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).

Together, these data indicate that sst2-deficient mice fed HFD have impaired glucose tolerance.

SST receptor subtype-2-deficient mice with diet-induced obesity have increased plasma concentration of nonfasting glucagon and fasting NEFA
Nonfasting plasma glucagon concentration and fasting NEFA of sst2–/– mice fed HFD were higher compared with control mice (Table 2Go). In contrast, NEFA plasma concentrations of animals fed chow diet were comparable (Table 3Go).

Sst2–/– mice fed HFD had comparable concentrations of fasting glucagon and nonfasting NEFA to WT mice (Table 2Go). Plasma concentrations of insulin, GLP-1, and leptin were similar in both genotypes (Table 2Go).

Taken together, sst2–/– mice with diet-induced obesity have higher nonfasting plasma glucagon levels and increased fasting levels of NEFA than their WT littermates.

Deletion of SST receptor subtype-2 in mice with diet-induced obesity leads to impaired inhibition of glucagon secretion by exogenous SST
We investigated how exogenous SST-14 influences glucose, glucagon, and insulin concentrations in WT and sst2–/– mice. Sst2–/– mice injected with SST-14 (100 µg/kg BW) had increased glucose concentration, whereas glucose concentration in sst2+/+ mice remained nearly constant (Fig. 2AGo).


Figure 2
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  		FIG. 2. SST tolerance test in 2-h fasted WT and sst2–/– animals. Fasted animals were injected with SST-14 (100 µg/kg BW, ip) or vehicle, and blood glucose (A), plasma glucagon (B), and insulin (C) levels were determined at the indicated time points. Each data point represents mean ± SEM from n = 5–7 animals per genotype. **, P < 0.01; *, P < 0.05 using two-way ANOVA test.

 
Consistent with the glucagonostatic activity of SST, glucagon concentration in WT controls decreased; however, it failed to decrease in sst2–/– mice injected with SST-14 (Fig. 2BGo). In contrast, plasma insulin concentrations were comparable in both genotypes after administration of exogenous SST-14 (Fig. 2CGo).

Investigation of pancreatic sections of both genotypes by immunofluorescence and immunohistochemistry and analysis of intra-islet contents of isolated pancreatic islets revealed comparable glucagon and insulin contents as well as similar pancreas morphology (supplemental Figs. 2Go and 3Go).


Figure 3
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  		FIG. 3. Changes of plasma glucagon (A) and insulin (B) levels in WT and sst2–/– animals after administration of 1.5 g glucose/kg BW (ip). Changes of glucagon (C) and insulin (D) secretion from pancreatic islets isolated from WT and sst2–/– animals after exposure to different concentrations of glucose. The results are expressed as percent change vs. 3.3 mmol/liter (defined as 100%) glucose. Each data point represents mean ± SEM from four independent experiments performed in quadruplicate. **, P < 0.01 vs. the value in sst2–/– islets using two-way ANOVA test.

 
Together, these results are consistent with sst2 as the main mediator of SST-dependent inhibition of glucagon secretion in rodents.

Deletion of SST receptor subtype-2 in mice with diet-induced obesity leads to impaired inhibition of glucagon secretion by glucose
In addition, we investigated the effects of exogenous glucose on glucagon and insulin secretion in vivo and in vitro. Plasma glucagon concentration of sst2–/– mice injected with glucose (1.5 g/kg BW) was slightly higher (at the 15-min time point, P > 0.05) than in WT mice (Fig. 3AGo), whereas insulin concentration was comparable between both genotypes (Fig. 3BGo).

In vitro, 20 mmol/liter glucose suppressed glucagon secretion by 58% from islets isolated from sst2+/+ mice and by only 23% from islets lacking sst2 (P < 0.01 vs. sst2+/+ islets) (Fig. 3CGo). In contrast, no difference was observed when islets were exposed to lower glucose concentrations (Fig. 3CGo). The glucose-induced stimulation of insulin secretion from isolated pancreatic islets derived from both genotypes was similar (Fig. 3DGo).

Together, these data indicate that sst2 mediates the inhibition of glucagon secretion after exposure to higher glucose concentration.

Increased plasma glucagon levels in SST receptor subtype-2-deficient mice correlate with reduced hepatic glycogen content and lower activity of enzymes facilitating glycogen synthesis
Liver is the primary target tissue for glucagon action. Glucagon decreases hepatic glucose utilization and storage as glycogen. Because deletion of sst2 resulted in increased glucagon concentration, we measured hepatic glycogen content and the activity/expression of enzymes regulating glycogen metabolism.

Sst2-deficient mice had lower hepatic glycogen content compared with sst2+/+ mice (Fig. 4Go, A and B). RNA levels of glucokinase (GK) were decreased in livers obtained from sst2–/– mice (Fig. 5AGo). The expression of the GK gene is stimulated by the transcription factor sterol-regulated element binding protein (SREBP). In agreement with the lower hepatic expression of GK, livers isolated from sst2–/– mice had lower SREBP RNA compared with WT mice (Fig. 5BGo).


Figure 4
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  		FIG. 4. Hepatic glycogen content. A, Quantitative determination of glycogen content (µg/mg of wet tissue) in livers isolated from nonfasted WT and sst2–/– animals; B, histochemical detection of hepatic glycogen using McMannus’ periodic acid Schiff staining after 14 wk feeding of animals with HFD: top, representative cryosection of livers obtained from sst2+/+ mice; bottom, representative cryosection of livers obtained from sst2–/– mice. Images were taken at x400 magnification using a Zeiss Axiophot microscope. *, P < 0.05 using Student’s t test.

 

Figure 5
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  		FIG. 5. Expression and phosphorylation of enzymes regulating hepatic glycogen synthesis. A and B, Real-time analysis of the mRNA levels of hepatic GK (A) and sSREBP (B) in nonfasted WT and sst2–/– animals. C, Phosphorylation of GS in livers isolated from nonfasted WT (lanes 1–5) and sst2–/– animals (lanes 6–10) after 14 wk of HFD feeding: top, quantitative analysis; bottom, representative Western blots. D, Phosphorylation of hepatic GSK-3 in WT (lanes 1–4) and sst2–/– animals (lanes 5–9) after 14 wk of HFD feeding: top, quantitative analysis; bottom, representative Western blots. **, P < 0.01; *, P < 0.05 using Student’s t test.

 
Glycogen synthase (GS) is the rate-limiting enzyme in the synthesis of hepatic glycogen, which is active in the nonphosphorylated state. GS is activated by GS kinase-3 (GSK-3), which is active in the phosphorylated state. Consistent with the reduced glycogen content, phosphorylation of hepatic GS of sst2-deficient mice was increased, whereas GSK-3 was decreased (Fig. 5Go, C and D).

Together, the reduced glycogen content of sst2-deficient mice correlates with reduced activity and expression of hepatic enzymes facilitating glycogen synthesis.

SST receptor subtype-2-deficient mice have increased expression and activity of enzymes stimulating glycogenolysis und gluconeogenesis
Hepatic GP initiates the catalytic breakdown of glycogen. Expression of GP in livers of sst2–/– mice and the protein content were higher compared with sst2+/+ mice (Fig. 6Go, A and B). In agreement with this, the phosphorylation of GP kinase, an enzyme that activates GP, was increased in livers from sst2–/– mice (Fig. 6CGo).


Figure 6
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  		FIG. 6. Changes of the mRNA expression and protein phosphorylation of enzymes regulating hepatic glycogen breakdown. A, Quantification of the mRNA levels by real-time analysis of GP in livers isolated from nonfasted WT and sst2–/– animals after 14 wk of HFD feeding. B, Detection of GP protein in livers of sst2+/+ (lanes 1–3) and sst2–/– animals (lanes 4–8) by Western blot analysis, using rabbit antiserum raised against the hepatic isoform of GP. Note a specific signal of 97 kDa recognized by the antiserum (1:500 dilution). The upper panel shows the results of quantitative analysis of Western blots. C, Phosphorylation of the hepatic phosphorylase kinase in nonfasted WT (lanes 1–3) and sst2–/– animals (lanes 4–7): top, quantitative analysis of signal intensities in Western blots; bottom, representative Western blots. ***, P < 0.001; **, P < 0.01 using Student’s t test.

 
The expression of phosphoenolpyruvate carboxykinase (PEPCK), which increases de novo glucose production, was increased in livers lacking sst2 (Fig. 7AGo).


Figure 7
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  		FIG. 7. A, Real-time analysis of the expression of the gluconeogenic PEPCK in livers of nonfasted WT and sst2–/– animals after 14 wk of HFD feeding. B, Expression of CREB at the protein level: top, quantitative analysis of total CREB signals detected by Western blots; bottom, representative Western blot of total hepatic CREB; lanes 1–4, Livers from sst2+/+ mice; lanes 5–8, livers from sst2–/– mice. C, Quantitative analysis of phosphorylated CREB by Western blots (top); bottom, representative Western blots; lanes 1–4, livers from sst2+/+ mice; lanes 5–8, livers from sst2–/– mice. D, Expression of G6Pase in livers isolated from sst2+/+ and sst2–/– animals. *, P < 0.05; **, P < 0.01 by Student’s t test.

 
PEPCK gene is transcriptionally regulated by the cAMP response element B (CREB). In agreement with this, livers with sst2-null mutation had increased expression and phosphorylation (activity) of CREB (Fig. 7Go, B and C). The expression of glucose-6-phosphatase (G6Pase) was slightly (P > 0.05) increased in livers obtained from sst2–/– mice (Fig. 7DGo).

These data suggest that sst2-null mice have increased activity and expression of enzymes enhancing hepatic glycogenolysis and gluconeogenesis. Both mechanisms are able to contribute to hyperglycemia in sst2–/– mice.

Activation of SST receptor subtype-2 inhibits glucagon-stimulated glycogenolysis
SST was shown to influence HGP (33, 34). Sst2 is expressed in murine livers, human hepatocytes, and HepG2 hepatoma cells as detected by RT-PCR (supplemental Fig. 4) (35). Therefore, the deletion of sst2 may cause a change of HGP and glycogenolysis, independent of glucagon. To prove this hypothesis, we tested the effects of SST-14 and an sst2-selective agonist on de novo HGP and glycogenolysis in isolated human hepatocytes.

First, we investigated the expression profile of several genes normally expressed in hepatocytes after the maximal cultivation period of 44 h. The rationale for the evaluation of the gene expression is the possibility of hepatocytes to dedifferentiate into fibroblasts after longer cultivation periods. After 44 h, cultivated hepatocytes expressed glucose transporter 2 (GLUT-2), GK, fatty acid synthase, acetyl-CoA-carboxylase, G6Pase, but not GLUT-1, as detected by RT-PCR (data not shown). The same expression profile was detected in freshly isolated hepatocytes (data not shown).

Using isolated primary hepatocytes, we observed a time-dependent increase of basal HGP (Fig. 8AGo). Glucagon (10 nM) stimulated HGP by about 3-fold; however, neither SST-14 nor the sst2 agonist reduced basal or glucagon-stimulated HGP (Fig. 8BGo). In contrast, glucagon-induced glycogenolysis (Fig. 8CGo) was significantly reduced by SST-14 and sst2 agonist (Fig. 8DGo).


Figure 8
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  		FIG. 8. A, Time-dependent basal hepatic glucose production (µg/ml per 106 cells) in isolated primary human hepatocytes; B, effects of SST-14 (100 nM) or an sst2-selective agonist (100 nM) with or without glucagon (10 nM) on hepatic glucose production in isolated primary human hepatocytes; C, time-dependent stimulation of glycogenolysis by glucagon in isolated human hepatocytes; D, effects of SST-14 (100 nM) or an sst2-selective agonist (100 nM) on glucagon-induced (10 nM) glycogenolysis in isolated primary human hepatocytes. Glycogen synthesis was induced by 24-h incubation of hepatocytes with 10 mmol/liter insulin and 10 mmol/liter glucose. Glycogenolysis was measured as the amount of glucose released into incubation buffer. The results are expressed as percentage of basal glucose production (defined as 100%). *, P < 0.05; **, P < 0.01; ***, P < 0.001 using two-way ANOVA or Student’s t test.

 
Thus, reduced hepatic glycogen deposition in sst2–/– mice may result from hyperglucagonemia and impaired hepatic action of SST.

Livers of SST receptor subtype-2-deficient mice have decreased hepatic triglyceride content
Feeding with HFD for 14 wk led to hepatic lipid accumulation. Consistent with the lipolytic effects of glucagon, sst2-deficient mice had decreased hepatic triglyceride content as detected biochemically (Fig. 9AGo) and by histochemistry (Fig. 9BGo). The expression of lipoprotein lipase (LPL) in livers isolated from sst2-deficient was increased compared with WT mice (Fig. 9CGo).


Figure 9
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  		FIG. 9. A, Quantitative determination of triglyceride content in livers isolated from nonfasted WT and sst2–/– animals. B, Histochemical detection of hepatic triglyceride content (x400 magnification) in cryosections obtained from livers of sst2+/+ and sst2–/– animals: top, representative cryosection of livers obtained from sst2+/+ mice; bottom, representative cryosection of livers obtained from sst2–/– mice. C, Regulation of LPL mRNA expression in livers isolated from sst2+/+ and sst2–/– animals by real-time analysis. **, P < 0.01 by Student’s t test.

 
These findings are consistent with lipolytic activity of glucagon.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major finding of our study is that the deletion of sst2 in mice with diet-induced obesity leads to impaired suppression of glucagon secretion by glucose and SST. Consequently, sst2-deficient mice have increased postprandial plasma glucagon concentration. Impaired glucose tolerance in these mice results from decreased hepatic glucose utilization and storage as glycogen. Lower hepatic glycogen and lipid content of sst2–/– mice are associated with reduced activity and expression of enzymes controlling glycogen synthesis, whereas glycogenolytic and lipolytic enzymes are increased. In addition, the inability of SST to inhibit glycogenolysis, due to the absence of sst2 in the liver, can also contribute to the increased hepatic glucose output.

It is important to note that sst2-deficient mice fed standard laboratory chow diet (12.5 kcal% from fat) do not show any abnormalities of glucose homeostasis or BW and body composition compared with WT control mice (data not shown), indicating that stress factors (e.g. feeding HFD) are necessary to develop the above described major phenotypical abnormalities. Because HFD can contribute to peripheral insulin resistance and/or impaired function of pancreatic B cells (due to lipotoxicity), we assume that these mechanisms are responsible for the development of impaired glucose control in our animals.

Food intake and glucose stimulate the secretion of SST and inhibit the secretion of glucagon. It has been postulated that SST confers the inhibition of glucagon secretion by glucose (36) Because the deletion of sst2 leads to impaired inhibition of glucagon secretion by glucose in our study, we suggest that sst2 is required for the prandial inhibition of glucagon secretion.

There are several evidences from our study supporting this notion. Using isolated pancreatic islets, we detected that glucose at all tested doses inhibits glucagon secretion from WT islets, although the effect was markedly impaired using islets lacking sst2.

Administration of exogenous SST-14 failed to influence glucose concentration in WT mice, which is in agreement with the glucagono- and insulinostatic activity of SST. However, sst2-deficient mice had increased blood glucose concentration in response to the treatment with SST-14. This effect was due to the lack of inhibition of glucagon secretion by SST-14 in sst2-deficient mice, which is in agreement with the results of previous studies (9, 10).

The impaired suppression of glucagon secretion in sst2-deficient mice is comparable to the situation in type 2 diabetes (12, 13, 14). Up to 70% of patients with type 2 diabetes have increased glucagon secretion, which significantly contributes to postprandial hyperglycemia. Reasons for the lack of postprandial glucagon suppression in type 2 diabetes are still unclear. However, it is possible that changes in the expression or function of sst2 may contribute to impaired inhibition of glucagon secretion in type 2 diabetes, but this has yet to be demonstrated.

Sst2-deficient mice had lower hepatic glycogen content, which is compatible with the results of a study in which the deletion of GcgR led to increased hepatic glycogen content (21). However, GcgR antisense oligonculeotides (ASO)-treated db/db mice had, unexpectedly, decreased hepatic glycogen content (20). The discrepant observation may be explained either by the incomplete reduction of GcgR expression by ASO treatment or through markedly increased insulin and GLP-1 levels in db/db mice (20). In contrast to db/db mice, sst2–/– mice had normal circulating plasma GLP-1 and insulin. GLP-1 is a potent stimulus of insulin secretion (37), and insulin facilitates hepatic glucose uptake as well as glycogen synthesis (38, 39). Hyperinsulinemia could therefore overcome the consequences of the blockade of glucagon action by GcgR ASO; however, to date, these questions have not yet been fully answered.

Reducing the expression of the hepatic GcgR in db/db mice by 83% using GcgR ASO resulted in an improved glucose disappearance rate during an insulin tolerance test and improvement of lipid control (20). The important role of glucagon is again confirmed by our own experiments, which demonstrate impaired glucose control in sst2-deficient mice with hyperglucagonemia. This observation can be explained by glucagon-dependent inhibition of hepatic glucose uptake with the concomitantly reduced glycogen content in sst2-deficient animals, which is supported by the results of a study showing an improved glucose control and insulin sensitivity in GcgR null mice (21).

SST has been shown to partially inhibit glucagon-stimulated HGP (33, 34). Hepatocytes are known to express SST receptors; however, it is unknown which of the five sst isoforms expressed in the liver inhibits HGP. The results of our experiments using isolated primary hepatocytes indicate that sst2 inhibits glycogenolysis, without affecting de novo glucose synthesis. Therefore, both hyperglucagonemia and decreased effects of SST in sst2-deficient mice may contribute to increased hepatic glycogenolysis. The clinical relevance of this hepatic action of sst2 remains to be established.

The results obtained in isolated hepatocytes should be interpreted cautiously, because we have used human hepatocytes in our study. We have performed studies on isolated hepatocytes obtained from humans due to the limited amount of high-quality murine hepatocyte preparations. Murine hepatocytes are very difficult to isolate; the rate of apoptosis and immediate damage and poor survival and dedifferentiation into fibroblasts unfortunately exceeded 25%. In addition, murine hepatocytes were considerably contaminated with fibroblasts. The necessity to cultivate high-quality hepatocytes to study glycogenolysis and gluconeogenesis for up to 48 h (starting from overnight recovery and lasting for up to an additional 24 h), limited the use of isolated murine hepatocytes.

Although we did not directly demonstrate the diminished rate of hepatic glucose uptake and glycogen synthesis, there are several lines of indirect evidence supporting this notion.

GK stimulates the hepatic glucose uptake. The expression and activity of GK in livers correlates with the hepatic glucose uptake. In livers of sst2–/– mice, GK expression was decreased, suggesting reduced uptake of glucose by hepatocytes.

The hepatic glycogen metabolism is controlled by the coordinated action of GS and GP (40, 41, 42). Through this cAMP/protein kinase A (PKA)-dependent pathway, glucagon is able to lower the expression of GS and to stimulate that of GP (40, 41, 42). Glucagon promotes phosphorylation (lowers activity) of GS through activation (dephosphorylation) of GSK-3 (41, 42). Consistent with this notion, the phosphorylation of GSK-3 of sst2–/– mice was lower and of GS higher than in WT mice.

The expression of GP and phosphorylation of GP-activating phosphorylase kinase (PhK) of sst2-null mice were increased. Both enzymes are known to stimulate the glycogen breakdown, thereby increasing hepatic glucose content.

Thus, both decreased expression/activity of enzymes stimulating glycogen synthesis and increased activity of glycogenolytic enzymes may contribute to decreased storage of glucagon in the liver of sst2–/– mice.

Glucagon increases the rate of de novo glucose production, which requires the activation of PEPCK. Glucagon is able to increase the expression and phosphorylation of CREB through cAMP/PKA, which then leads to increased PEPCK transcription (43, 44, 45). PEPCK catalyzes one of the rate-limiting steps of gluconeogenesis, the conversion of oxaloacetate to phosphoenolpyruvate (46). Moreover, glucagon increases the activity of G6Pase, which catalyzes the final step of gluconeogenesis, the production of glucose from glucose-6-phosphate. Consistent with these effects of glucagon on the gluconeogenic enzymes, expression and phosphorylation of CREB and PEPCK in sst2-null mice were increased. The expression of G6Pase, however, was not significantly increased.

Hepatic steatosis is frequently associated with obesity and type 2 diabetes (47). Administration of glucagon has been demonstrated to alleviate fatty liver diseases (48). Decreased hepatic triglyceride content of GcgR-ASO-treated db/db mice (20) provides additional support for the lipolytic action of glucagon. This is stressed by the decreased hepatic triglyceride content of sst2-null mice, compared with WT controls. Hepatic lipolysis is activated in the cAMP/PKA/CREB pathway (49), whereas overexpression of SREBP increases hepatic lipid accumulation (50). Consistent with the ability of glucagon to decrease SREBP gene expression in isolated hepatocytes (51), we detected low CREB and SREBP expression in sst2-null mice. Increased expression of LPL suggests that the decreased hepatic lipid content is due to increased lipolysis.

The increased rate of lipolysis is in line with increased levels of NEFA in sst2–/– mice. NEFA can reduce insulin sensitivity (52). Thus, the decreased lowering of blood glucose concentration in sst2-deficient mice in response to exogenous insulin may be due to increased circulating NEFA concentration. However, future studies in sst2-null mice are required to validate this hypothesis and to delineate why fasting NEFA are increased and how they affect the peripheral insulin sensitivity in sst2–/– mice.

It is important to note that the role of the sst2 described here in mice with diet-induced obesity cannot be extrapolated onto another species, which is a limitation of the study. Despite numerous studies characterizing the expression and function of sst, differences between species were reported. There is some controversy on the sst expression in various species, and the results of morphological and functional studies focusing on SST receptor subtypes suggest that considerable species-specific expression and function exist.

In rodents, sst2 is predominantly expressed in pancreatic A cells, whereas sst5 is found mostly in B cells (53, 54). Recently, the presence of all SST receptor subtypes has been demonstrated in pancreatic A and B cells of rats and mice, using different antibodies (55). Ludvigsen et al. (55) reported in their study that murine pancreatic B cells express a high density of sst1, sst2, and sst5, whereas glucagon-immunoreactive cells expressed high levels of sst2 and sst5. In contrast to rat islets, Ludvigsen et al. (55) found only a sparse expression of sst3 in murine islets. Using different antibodies, Kumar et al. (56) demonstrated that sst1, sst2, and sst5 are highly expressed in the human endocrine pancreas, whereas both A and B cells showed a poor expression of sst3 and sst4. For example, sst2 was expressed in approximately 46% of B cells, sst5 was found in 100%, and sst1 in 87% of insulin-immunoreactive cells. Approximately 89% of human A cells expressed sst2, whereas sst5 and sst1 colocalized with 35 and 26% of A cells, respectively (56). In another study, Reubi et al. (57) reported the expression of sst2 on both A and B cells, using a different antibody. Discrepant observations in the human endocrine pancreas are related not only to the sst expression pattern but also to their function. Using a relatively moderate sst5-selective agonist, Zambre et al. (58) demonstrated a modest inhibition of insulin secretion from isolated human pancreatic islets, whereas sst2-selective agonist was inactive. Atiya et al. (59) reported that sst2-selective agonist inhibits insulin secretion from perfused human pancreas, whereas sst5- and sst3-selective agonists had no effects. Moldovan et al. (60) reported a decrease of insulin secretion from perfused human pancreas using a single dose of an sst2-selective agonist, DC32–87. We have recently demonstrated that both insulin and glucagon secretion are mainly regulated via sst2, using sst-selective agonists and an sst2-selective antagonist (61).

Thus, based on the previous results, the role and expression of individual SST receptor subtypes shows a species-specific pattern. At least for the sst2, there is strong evidence that this isoform regulates glucagon secretion in both humans (in vitro) and in rodents (in vivo and in vitro). However, these data are based mostly on the use of sst-selective agonist. The convincing evidence describing the function of sst can be derived from studies in animals lacking or overexpressing the sst of interest. Our data thus provide strong evidence that sst2 has an in vivo role as an inhibitor of glucagon secretion, which has consequences in the regulation of glucose household. The limitation of the study is that the data obtained in rodents cannot be extrapolated to another species due to the above mentioned limitations. Thus, additional studies are required that may use, hopefully soon, clinically available highly potent and selective sst2 agonists in humans to answer this questions.

In summary, our study demonstrates that sst2 confers the SST- and glucose-induced suppression of glucagon secretion. In the absence of sst2, mice develop nonfasting hyperglucagonemia. Hyperglucagonemia is associated with decreased glycogen content, resulting from decreased glycogen synthesis and increased glycogenolysis. Reduced hepatic glucose utilization, decreased hepatic triglyceride storage, and increased lipolysis may contribute to impaired glucose control in sst2-deficient mice. Ablation of sst2 decreases the inhibition of glycogenolysis by SST in a mouse model of diet-induced obesity. Obesity represents a major risk factor to develop type 2 diabetes, and the majority of type 2 diabetic patients are obese. The clinical relevance of the sst2 function in the context of type 2 diabetes and diet-induced obesity in humans remains to be established.


   Acknowledgments
 
We thank Ms. Elizabeth Zach for the careful editing of the manuscript.


   Footnotes
 
M.Z.S. was supported by a research grant from the Deutsche Forschungsgemeinschaft (STR558/4-1 and 4-2) and the Sonnenfeld-Stiftung.

Disclosure Statement: All authors have nothing to disclose.

First Published Online May 24, 2007

1 V.S. and C.G. contributed equally to the paper. Back

Abbreviations: ASO, Antisense oligonucleotides; BW, body weight; CREB, cAMP response element B; GcgR, glucagon receptor; GK, glucokinase; GLP-1, glucagon-like peptide-1; GLUT-1, glucose transporter 1; GP, glycogen phosphorylase; G6Pase, glucose-6-phosphatase; GS, glycogen synthase; GSK-3, GS kinase-3; HFD, high-fat diet; HGP, hepatic glucose production; LPL, lipoprotein lipase; NEFA, nonesterified fatty acids; PEPCK, phosphoenolpyruvate carboxykinase; PFA, paraformaldehyde; PKA protein kinase A; SREBP, sterol-regulated element-binding protein; SST, somatostatin; sst2, somatostatin receptor subtype-2; sst2–/–, homozygous deletion of sst2; WT/sst2+/+, wild type.

Received December 11, 2006.

Accepted for publication May 11, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
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