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Expression of Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Substrate-1 in Pancreatic β-Cells and Its Role in Promotion of Insulin Secretion and Protection against Diabetes

Laboratory of Biosignal Sciences (M.K., H.Oh., H.Ok., Y.M., Y.H., H.K., T.M.) and Metabolic Signal Research Center (T.K.), Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371-8512, Japan

Address all correspondence and requests for reprints to: Takashi Matozaki or Hiroshi Ohnishi, Laboratory of Biosignal Sciences, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-Machi, Maebashi, Gunma 371-8512, Japan. E-mail: matozaki{at}showa.gunma-u.ac.jp or ohnishih{at}showa.gunma-u.ac.jp.

	Abstract

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Insulin secretion by β-cells of pancreatic islets is regulated by various soluble factors including glucose and hormones. The importance of direct cell-cell communication among β-cells or between β-cells and other cell types for such regulation has remained unclear, however. Transmembrane proteins Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) and its ligand CD47 interact through their extracellular regions and contribute to intercellular communication. We now show that both SHPS-1 and CD47 are prominently expressed in β-cells of the pancreas. The plasma insulin level in the randomly fed state was markedly reduced in mice that express a mutant form of SHPS-1 lacking most of the cytoplasmic region compared with that in wild-type (WT) mice, although the blood glucose concentrations of the two types of mice were similar. This reduction in the plasma insulin level of SHPS-1 mutant mice was even more pronounced in animals maintained on a high-fat diet. Glucose tolerance was also markedly impaired in SHPS-1 mutant mice on a high-fat diet, whereas both peripheral insulin sensitivity and the insulin content of the pancreas in the mutant animals were similar to those of WT mice. Glucose-stimulated insulin secretion was similar for islets isolated from WT or SHPS-1 mutant mice. The impaired glucose tolerance of SHPS-1 mutant mice was ameliorated by treatment with the 2-adrenergic antagonist yohimbine. These results suggest that SHPS-1 promotes insulin secretion from β-cells and thereby protects against diabetes. Preventing of 2-adrenergic receptor-mediated inhibition of insulin secretion may partly participate in such a function of SHPS-1.

	Introduction

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PANCREATIC β-CELLS ARE essential for glucose homeostasis, with defects in the ability of these cells to secrete adequate amounts of insulin in response to glucose resulting in diabetes mellitus. Secretion of insulin from β-cells is regulated predominantly by the level of glucose in plasma, but it is also modulated by hormones such as glucagon-like peptide-1 (1) and somatostatin (2) as well as by neurotransmitters such as norepinephrine acting at β-adrenergic or 2-adrenergic receptors (3). The effects of such agents on insulin release are mediated in large part by changes in the cytosolic concentrations of Ca²⁺ or cAMP. The roles of protein tyrosine phosphorylation and dephosphorylation in glucose-induced insulin secretion remain unclear, however. In addition to regulation of insulin secretion by soluble factors, direct cell-cell communication among β-cells or between β-cells and other cell types in pancreatic islets is also implicated in this process (4, 5, 6). Indeed, connexin (7), E-cadherin (8), and the EphA-Ephrin-A complex (9), all of which contribute to intercellular interactions, are each thought to participate in such regulation. The molecular basis for regulation of insulin secretion by cell-cell communication remains largely uncharacterized, however.

Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) (10), also known as signal-regulatory protein (SIRP) (11) or BIT (12), is a transmembrane protein whose extracellular region comprises three Ig-like domains and whose cytoplasmic region contains four tyrosine phosphorylation sites that mediate the binding of the protein tyrosine phosphatases SHP-1 and SHP-2. Tyrosine phosphorylation of SHPS-1 is regulated by various growth factors, such as insulin and IGF-I, as well as by integrin-mediated cell adhesion to extracellular matrix proteins (10, 13, 14, 15). SHPS-1 thus functions as a docking protein to recruit and activate SHP-1 or SHP-2 at the cell membrane in response to extracellular stimuli. CD47 is a ligand for the extracellular region of SHPS-1 (16, 17). This protein, which was originally identified in association with _vβ₃ integrin, is also a member of the Ig superfamily, possessing an Ig-V-like extracellular domain, five putative membrane-spanning segments, and a short cytoplasmic tail (18). SHPS-1 is most abundant in neurons, macrophages, and dendritic cells (19, 20, 21, 22), with low or minimal expression of this protein having been detected in other cell types. In contrast, CD47 is expressed in many cell types (18).

SHPS-1 and CD47 appear to constitute a cell-cell communication system that plays an important role in a variety of cell functions. For instance, the binding of CD47 to SHPS-1 contributes to the inhibition of cell migration by cell-cell contact (23). In addition, monoclonal antibodies (mAbs) to CD47 inhibit neutrophil transmigration (24), suggesting that the CD47-SHPS-1 system might mediate bidirectional inhibitory regulation of cell migration. Moreover, the interaction of CD47 on red blood cells with SHPS-1 on macrophages is thought to prevent phagocytosis of the former cells by the latter through SHPS-1-mediated activation of SHP-1 (20, 25, 26). The interaction of SHPS-1 on dendritic cells or Langerhans cells with CD47 on T cells is essential for priming of naive T cells and subsequent differentiation of T cells in the development of experimental autoimmunity (27).

We have now shown that expression of SHPS-1 in pancreatic islets is largely restricted to β-cells. Moreover, within the pancreas, CD47 is expressed preferentially in islets of Langerhans rather than in surrounding acinar cells. We previously generated mice that express a mutant version of SHPS-1 that lacks most of the cytoplasmic region of the protein (26, 28). With the use of these SHPS-1 mutant mice, we have now examined the role of SHPS-1 in regulation of pancreatic β-cells in vivo and in vitro.

	Materials and Methods

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Animals
The generation of mice that express a mutant form of SHPS-1 lacking most of the cytoplasmic region was described previously (26, 28), and the animals were backcrossed onto the C57BL/6N background for nine or 10 generations. Feeding with the high-fat diet (HFD) was initiated at 6 wk of age. Mice were bred and maintained at the Institute of Experimental Animal Research of Gunma University under specific-pathogen-free conditions. They had free access to water and either standard laboratory chow containing 4% fat (CE-2; CLEA Japan, Tokyo, Japan) or a HFD (HFD32; CLEA Japan) containing 32% fat (22.3% of total fat from saturated fat), and they were housed in an air-conditioned room under a 12-h light, 12-h dark cycle. All mice were handled in accordance with the animal care guidelines of Gunma University.

Antibodies and reagents
Hybridoma cells producing rat mAbs to mouse SHPS-1 (p84) or to mouse CD47 (miap301) were kindly provided by C. F. Lagenaur (University of Pittsburgh, Pittsburgh, PA) and P.-A. Oldenborg (Umeå University, Umeå, Sweden), respectively. The mAbs were purified from culture supernatants as described previously (29). Rabbit polyclonal antibodies to SHPS-1 were obtained from Upstate Biotechnology (Lake Placid, NY); guinea pig polyclonal antibodies to insulin, a mouse mAb to glucagon, and rabbit polyclonal antibodies to somatostatin were from Dako (Carpinteria, CA); rabbit polyclonal antibodies to pancreatic polypeptide (PP) were from Chemicon (Temecula, CA); rabbit polyclonal antibodies to SHP-1 or to SHP-2 were from Santa Cruz Biotechnology (Santa Cruz, CA); and a mouse mAb to β-tubulin (TUB 2.1) was from ICN Biomedicals (Aurora, OH). Cyanine 3 (Cy3)-conjugated goat polyclonal antibodies to rabbit or rat IgG were obtained from Jackson ImmunoResearch (West Grove, PA), and Alexa 488-conjugated goat polyclonal antibodies to guinea pig, mouse, or rabbit IgG were from Molecular Probes (Eugene, OR). Yohimbine hydrochloride and clonidine were from Sigma (St. Louis, MO).

Histology and immunofluorescence analysis of the pancreas
Hematoxylin-eosin staining and immunofluorescence analysis of mouse pancreas were performed as described previously (26). Mice were anesthetized with ether and were then perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer. The pancreas was removed, exposed to the same fixative, embedded in paraffin, sectioned at a thickness of 5 µm, and stained with Mayer’s hematoxylin-eosin as described (26). Immunofluorescence analysis was also performed as described previously (26). Before staining with polyclonal antibodies to SHPS-1, tissue samples were autoclaved in Retrievagen A (pH 6.0) (BD PharMingen, San Diego, CA) at 110 C for 15 min for antigen retrieval. All sections were permeabilized by incubation for 1 h at room temperature in blocking solution (5% goat serum in PBS) supplemented with 0.1% Triton X-100 and were then stained with primary antibodies diluted in the permeabilization solution for 1 h at room temperature or overnight at 4 C. They were then washed with PBS, stained with Cy3- or Alexa 488-conjugated secondary antibodies diluted in the permeabilization solution, and washed again with PBS. Fluorescence or bright-field images were acquired with a BX51 microscope (Olympus, Tokyo, Japan) equipped with epifluorescence optics, a color cooled CCD camera (DP71; Olympus), and DP controller software (Olympus).

Measurement of blood glucose as well as plasma insulin, adiponectin, triglyceride, and nonesterified fatty acids
Blood samples were collected from the tail vein of mice, and blood glucose levels were determined with the use of a Glutest sensor or Glutest Pro R (Sanwa Kagaku, Nagoya, Japan). For measurement of plasma parameters, blood samples were collected from the tail vein into heparinized capillary tubes and centrifuged at 700 x g for 15 min at 4 C. The resulting plasma fractions were stored at –20 C until use. Plasma insulin and adiponectin concentrations were measured with the use of ELISA kits for mouse insulin (LBIS Insulin Mouse S-Type; Shibayagi, Gunma, Japan) or mouse adiponectin (AdipoGen, Seoul, Korea). The plasma levels of triglyceride and nonesterified fatty acids were measured with the use of Triglyceride Test (Wako, Tokyo, Japan) or NEFA C-Test (Wako), respectively.

Glucose and insulin tolerance tests
A glucose tolerance test was performed on mice that had been deprived of food overnight for 18 h as described previously (30). To determine the effect of an 2-adrenergic receptor antagonist on blood glucose and plasma insulin levels after glucose injection, we injected mice ip with yohimbine hydrochloride (10 µmol/kg, dissolved in physiological saline) 5 min before injection of glucose. An insulin tolerance test was performed between 1500 and 1800 h on mice fed normal chow freely or on HFD-fed mice deprived of food for 4 h before the test. Blood samples were collected from the tail vein at various times after ip injection of human insulin (Humulin R; Eli Lilly, Indianapolis, IN) at a dose of 0.75 U/kg.

Measurement of pancreatic insulin content
The excised pancreas was cut into small pieces, homogenized in ice-cold 75% ethanol containing 0.18 M HCl, and maintained overnight at 4 C. The homogenate was centrifuged at 15,000 x g for 10 min at 4 C, and the resulting supernatant was stored at –20 C until assayed for insulin as described above.

Isolation of pancreatic islets and assay of insulin secretion
Islets were isolated by collagenase digestion of the pancreas as described previously (31). They were suspended in ice-cold Krebs-Ringer solution containing 11.2 mM glucose and then incubated for 1 h at 37 C. Islets were manually selected with the use of a dissection microscope and a pipette, transferred to ice-cold Krebs-Ringer solution containing 2.8 mM glucose, and incubated for 15 min at 37 C. At the end of this preincubation period, the islets were exposed for 1 h or 15 min at 37 C to low (2.8 mM) or high (16.8 or 25 mM) concentrations of glucose in the same solution. They were then collected by centrifugation, and the supernatant was assayed for insulin as described above. To test the effect of an 2-adrenergic agonist on insulin secretion, we included clonidine (3 ng/ml) in both the preincubation and incubation mixtures.

Cell culture and immunoblot analysis
MIN-6 mouse insulinoma cells were kindly provided by T. Takeuchi (Gunma University, Gunma, Japan). Immunoblot analysis was performed as described previously (32). MIN-6 cells were cultured under a humidified atmosphere of 5% CO₂ at 37 C in DMEM (Sigma) supplemented with 10% fetal bovine serum (Sigma) and 100 µM β-mercaptoethanol. Isolated pancreatic islets or MIN-6 cells were washed with ice-cold PBS and then lysed on ice in SDS-PAGE sample buffer [62.5 mM Tris-HCl (pH 7.6), 10% glycerol, 2% sodium dodecyl sulfate, 0.005% bromophenol blue]. The lysates were subjected to immunoblot analysis as described previously (32).

Statistical analysis
Data are presented as means ± SEM. Statistical analysis was performed by Student’s t test or Welch’s t test with the use of StatView 5.0 software (SAS Institute, Cary, NC). A P value of <0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of SHPS-1 and CD47 in pancreatic islets
We first examined the expression and localization of SHPS-1 in pancreatic islets. Immunohistofluorescence analysis of the pancreas of 10-wk-old mice revealed that SHPS-1 immunoreactivity was localized predominantly to islets of Langerhans, in particular to β-cells identified by insulin immunoreactivity (Fig. 1A). SHPS-1 was thus virtually undetectable in -cells (identified by glucagon immunoreactivity), -cells (somatostatin immunoreactivity), and PP cells (PP immunoreactivity) of islets as well as in surrounding acinar cells and duct cells (Fig. 1A). In addition, SHPS-1 immunoreactivity was markedly concentrated in β-cells at sites of cell-cell adhesion (Fig. 1B). Immunoblot analysis also showed that SHPS-1, which migrated as a broad band at a position corresponding to a molecular size of 120 kDa, was expressed in isolated islets as well as in insulin-secreting mouse MIN-6 cells (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Immunoreactivity for the SHPS-1 ligand CD47 was also predominant in pancreatic islets, being largely undetectable in surrounding acinar cells (Fig. 2A). However, the distribution of CD47 overlapped not only with that of insulin but also with that of glucagon, somatostatin, or PP (Fig. 2A). Immunoreactivity for CD47 was also markedly concentrated at sites of adhesion between adjacent endocrine cells (Fig. 2B).

View larger version (37K): [in this window] [in a new window]   FIG. 1. Expression of SHPS-1 in pancreatic islets. A, Pancreatic sections prepared from 10-wk-old male mice were double stained with either polyclonal antibodies (upper two rows) or a mAb (lower two rows) to SHPS-1 (red) as well as with antibodies to insulin (top row, green), to glucagon (second row, green), to somatostatin (third row, green), or to PP (bottom row, green). Merged images are also shown. Scale bar, 100 µm. B, Higher-magnification image of a pancreatic islet stained with polyclonal antibodies to SHPS-1. Scale bar, 20 µm.

View larger version (40K): [in this window] [in a new window]   FIG. 2. Expression of CD47 in pancreatic islets. A, Pancreatic sections prepared from 10-wk-old male mice were double stained with a mAb to CD47 (left panels, red) as well as with antibodies to insulin (top row, green), to glucagon (second row, green), to somatostatin (third row, green), or to PP (bottom row, green). Merged images are also shown. Scale bar, 100 µm. B, Higher-magnification image of a pancreatic islet stained with a mAb to CD47. Scale bar, 20 µm.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Expression of SHPS-1 in pancreatic islets as well as levels of blood glucose and plasma insulin in WT and SHPS-1 mutant mice. A, Pancreatic sections from a 31-wk-old male WT mouse (upper left panel) or a 32-wk-old male SHPS-1 mutant mouse (upper right panel) were double stained with polyclonal antibodies (pAbs) to SHPS-1 (red) and to insulin (green in insets). Pancreatic sections from 39-wk-old female WT (lower left panel) or SHPS-1 mutant (lower right panel) mice were also stained with the p84 mAb to SHPS-1 (red). Scale bars, 100 µm. B, Levels of blood glucose in WT and SHPS-1 mutant mice fed a normal chow were measured at the indicated ages. Data are means ± SEM of seven to 15 mice per group. C, Levels of blood glucose in WT and SHPS-1 mutant mice fed a HFD from 6 wk of age were measured at an age of 33–35 wk. Data are means ± SEM of five mice per group. D, Levels of plasma insulin in WT and SHPS-1 mutant mice fed a normal diet were measured at the indicated ages. Data are means ± SEM of seven to 14 mice per group. E, Levels of plasma insulin in WT and SHPS-1 mutant mice fed a HFD from 6 wk of age were measured at 30 wk of age. Data are means ± SEM of six to 10 mice per group. *, P < 0.05 vs. corresponding value for WT mice.

Given that feeding mice a HFD increases insulin secretion, most likely as a result of the insulin resistance induced by the large increase in adipose tissue mass (33), we next examined the levels of blood glucose and plasma insulin in WT and SHPS-1 mutant mice that had been maintained on a HFD between 6 and 30–35 wk of age. Blood glucose concentration did not differ between WT and SHPS-1 mutant mice (male or female) on the HFD (Fig. 3C). The plasma insulin level in WT mice on the HFD was greatly increased compared with that in those fed normal chow, whereas the insulin level of both male and female SHPS-1 mutant mice on the HFD was markedly decreased compared with that in WT mice on the same diet (Fig. 3E).

Impaired glucose tolerance in HFD-fed SHPS-1 mutant mice
To investigate further glucose metabolism and insulin secretion in SHPS-1 mutant mice, we evaluated glucose disposal by ip injection of glucose (1 g/kg body weight) in animals that had been deprived of food overnight. The changes in blood glucose concentration in response to the glucose challenge did not differ significantly between WT and SHPS-1 mutant mice maintained on normal chow at 33–37 wk of age (Fig. 4A). In contrast, blood glucose levels at 30 or 60 min after glucose loading were significantly higher in SHPS-1 mutant mice fed a HFD than in WT controls (Fig. 4B). Whereas the levels of plasma insulin after the overnight fast were indistinguishable between WT and SHPS-1 mutant mice on the HFD, those after glucose loading were lower in SHPS-1 mutant mice than in WT mice, although the differences were not statistically significant (Fig. 4B). These results suggested that glucose tolerance is impaired in SHPS-1 mutant mice, possibly as a result of a reduced insulin secretory response to glucose.

View larger version (23K): [in this window] [in a new window]   FIG. 4. Glucose tolerance tests in WT and SHPS-1 mutant mice. A, Levels of blood glucose in WT and SHPS-1 mutant mice (33–37 wk of age, male and female) maintained on normal chow were determined after an overnight fast and ip injection of glucose (1 g/kg). Data are means ± SEM from nine or 10 animals per group. B, Levels of blood glucose (upper panels) and plasma insulin (lower panels) in WT and SHPS-1 mutant mice (43–47 wk of age, male and female) maintained on a HFD were determined after an overnight fast and injection of glucose as in A. Data are means ± SEM from four or five animals per group. *, P < 0.05 vs. corresponding value for WT mice.

View larger version (15K): [in this window] [in a new window]   FIG. 5. Insulin tolerance tests and pancreatic insulin content in WT and SHPS-1 mutant mice. A, Levels of blood glucose in female WT and SHPS-1 mutant mice maintained on normal chow (left panel; 37–39 wk of age, n = 9) or on a HFD (right panel; 33–36 wk of age, n = 5) were determined after ip injection of insulin (0.75 U/kg). Mice on the HFD were deprived of food for 4 h before insulin injection. B, Insulin content of the pancreas was determined for WT and SHPS-1 mutant mice maintained on normal chow (38–42 weeks of age, male and female, n = 5 or 6) or on a HFD (50–53 wk of age, male and female, n = 2–5). All data are means ± SEM, with the exception of those for pancreatic insulin content of male SHPS-1 mutant mice fed the HFD (mean of values for two animals).

Insulin secretion from isolated pancreatic islets of SHPS-1 mutant mice
We next examined glucose-stimulated insulin secretion by isolated pancreatic islets of WT and SHPS-1 mutant mice fed with normal chow. Insulin secretion in the presence of low or high concentrations of glucose did not differ significantly between islets from the two types of mice (Fig. 6A). Glucose-stimulated insulin secretion consists of two phases, a rapid first phase and long-lasting second phase (34). Thus, we further compared the insulin secretion from isolated islets between WT and SHPS-1 mutant mice at the early phase of insulin secretion. However, insulin secretion during the first 15 min of incubation did not differ between islets from the two types of mice (supplemental Fig. 4). We next examined insulin secretion from islets of WT and SHPS-1 mutant mice fed with HFD. Insulin secretion was markedly greater for islets from both WT and SHPS-1 mutant mice maintained on a HFD than for those from both types of mice fed normal chow (Fig. 6B), consistent with previous observations (35, 36). However, insulin secretion in the presence of low or high concentrations of glucose did not differ between islets from the two types of mice fed the HFD (Fig. 6B). We also examined the effects of forskolin (an activator of adenylyl cyclase), 12-O-tetradecanoylphorbol 13-acetate (an activator of protein kinase C), or a high K⁺ concentration on insulin secretion from isolated islets. However, no marked differences in the insulin secretory responses to these stimulants were apparent between WT and SHPS-1 mutant mice (data not shown).

View larger version (9K): [in this window] [in a new window]   FIG. 6. Glucose-stimulated insulin secretion from isolated pancreatic islets of WT and SHPS-1 mutant mice. Pancreatic islets from WT or SHPS-1 mutant mice fed normal chow (male, 24–25 wk of age, n = 4 or 5) (A), or a HFD (female, 45–54 wk of age, n = 7) (B) were exposed to the indicated concentrations of glucose for 1 h, after which the amount of insulin secreted into the medium was determined. All data are means ± SEM.

View larger version (15K): [in this window] [in a new window]   FIG. 7. Effects of yohimbine and clonidine on insulin secretion. A and B, WT and SHPS-1 mutant mice (40–50 wk of age, female, n = 9) maintained on a HFD were injected ip with yohimbine (10 µmol/kg) 5 min before ip injection of glucose for a glucose tolerance test. The levels of blood glucose (A) and plasma insulin (B) were determined at the indicated times after glucose injection. C, Pancreatic islets from WT or SHPS-1 mutant mice (male, 28–30 wk of age, n = 5) fed normal chow were exposed to the indicated concentrations of glucose in the absence or presence of clonidine (3 ng/ml) for determination of insulin secretion. All data are means ± SEM.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have shown here that SHPS-1 expression is largely restricted to β-cells in the pancreas, being virtually undetectable in other endocrine cells of islets or in acinar cells. Moreover, the SHPS-1 ligand CD47 is also expressed prominently in β-cells, although this protein was also detected in other endocrine cells including -cells, -cells, and PP cells. Both SHPS-1 and CD47 appear to be localized in β-cells at sites of cell-cell adhesion.

Indeed, the plasma level of insulin was markedly decreased in SHPS-1 mutant mice in the randomly fed state. In addition, SHPS-1 mutant mice maintained on a HFD manifested impaired glucose tolerance and a reduced insulin secretory response to glucose loading. These observations suggest that insulin secretion from β-cells is impaired in SHPS-1 mutant mice, resulting in a reduced plasma insulin concentration in the randomly fed state or reduced glucose tolerance in the HFD-fed condition. SHPS-1 thus appears to be important for normal insulin secretion and hence may protect against the development of type 2 diabetes. SHPS-1 undergoes tyrosine phosphorylation in response to cell stimulation by insulin or IGF-I (10, 15, 39) and has thus been thought to contribute to the regulation of cell metabolism by insulin. However, the ability of injected insulin to lower blood glucose concentration was not impaired in SHPS-1 mutant mice, suggesting that the impaired glucose tolerance in these mice is not attributable to reduced insulin sensitivity. In addition, both insulin and IGF-I are important regulators of the proliferation of β-cells (40, 41). However, the insulin content of the pancreas was also similar in WT and SHPS-1 mutant mice, suggesting that SHPS-1 may not participate in regulation of β-cell proliferation.

Insulin secretion induced by a high concentration of glucose from isolated islets did not differ between WT or SHPS-1 mutant mice maintained either on normal chow or a HFD. Moreover, insulin secretion in response to forskolin, a phorbol ester, or a high K⁺ concentration was not impaired in islets isolated from SHPS-1 mutant mice. Thus, despite the preferential expression of SHPS-1 in β-cells of islets, stimulus-secretion coupling for glucose-stimulated insulin release appears to be intact in islets of SHPS-1 mutant mice. In contrast, administration of yohimbine partly restored normal glucose tolerance in SHPS-1 mutant mice maintained on a HFD. Consistent with this finding, the difference in glucose loading-induced insulin secretion between WT and SHPS-1 mutant mice was diminished by administration of yohimbine. These results suggest that the impaired glucose tolerance and insulin secretion apparent in HFD-fed SHPS-1 mutant mice are attributable, at least in part, to enhanced inhibition of insulin secretion by 2-adrenergic receptor signaling.

The inhibition of insulin secretion by 2-adrenergic receptors is thought to be mediated physiologically by the binding of norepinephrine, which is released from sympathetic nerve terminals located in and around islets in the pancreas (42, 43), to such receptors on β-cells (3). However, the extent of inhibition of glucose-stimulated insulin secretion by the 2-adrenergic agonist clonidine was similar for islets isolated from WT or SHPS-1 mutant mice. This finding suggests that SHPS-1 on β-cells does not regulate 2-adrenergic receptor-mediated signaling in these cells present in isolated islets. Both SHPS-1 and its ligand CD47 are abundant in the nervous system (16, 19). Moreover, electron microscopic analysis of the pancreas has revealed that the axonal terminals of peripheral nerves are often localized near or in direct contact with the endocrine cells of pancreatic islets (43, 44). Furthermore, both SHPS-1 and CD47 are present at ribbon synapses in the retina (45). It is thus possible that the interaction of SHPS-1 on β-cells with CD47 on axonal terminals (or that of CD47 on β-cells with SHPS-1 on axonal terminals, or both alternatives) is important for efficient antagonism by SHPS-1 of the inhibition of insulin secretion by norepinephrine acting at 2-adrenergic receptors on β-cells.

Sympathetic nerve activation also promotes glucagon and somatostatin secretion from pancreatic islets (3). Whereas glucagon increases the blood concentration of glucose (46), somatostatin inhibits insulin secretion (2). Enhanced activation of sympathetic nerves in SHPS-1 mutant mice might increase the secretion of both of these hormones, inhibit insulin secretion, and impair glucose tolerance. Indeed, SHPS-1 expressed in the central nervous system is implicated in regulation of the activity of autonomic nerves (47). Thus, regardless of the predominant expression of SHPS-1 in β-cells themselves, neuronal SHPS-1 might be important for regulation of insulin secretion through activation or inactivation of the sympathetic nervous system.

In conclusion, our results suggest that SHPS-1, which is highly expressed in pancreatic β-cells, promotes insulin secretion and hence protects against diabetes. SHPS-1 is thus a potential therapeutic target for diabetes.

	Acknowledgments

 
We thank C. F. Lagenaur and P.-A. Oldenborg for hybridoma cells, T. Takeuchi and S. Torii for MIN-6 cells, K. Tomizawa and Y. Niwayama for technical assistance, and I. Kojima and A. Hara for reagents and helpful discussion.

	Footnotes

 
This study was supported by a Grant-in-Aid for Scientific Research on Priority Areas Cancer, a Grant-in-Aid for Scientific Research (B) and (C), and a grant of the Global COE Program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

First Published Online July 17, 2008

Abbreviations: Cy3, Cyanine 3; HFD, high-fat diet; mAb, monoclonal antibody; PP, pancreatic polypeptide; SHPS-1, Src homology 2 domain-containing protein tyrosine phosphatase substrate-1; WT, wild type.

Received February 19, 2008.

Accepted for publication July 3, 2008.

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