The Effect of Global SSTR5 Gene Ablation on the Endocrine Pancreas and Glucose Regulation in Aging Mice1

doi:10.1016/j.jss.2005.05.024

Journal of Surgical Research

Volume 129, Issue 1, November 2005, Pages 64-72

https://doi.org/10.1016/j.jss.2005.05.024 Get rights and content

Introduction

The purpose of this study was to examine the effect of global gene ablation of SSTR5 on the endocrine pancreas, insulin secretion, and glucose tolerance in aging mice, as SSTR5 is a primary regulator of insulin secretion in the mouse pancreas.

Methods

Global SSTR5^−/− mice were generated and genotypes were verified using Southern blot and RT-PCR. Glucose tolerance and in vivo insulin secretion in SSTR5^−/− and WT mice were examined using intraperitoneal glucose tolerance test (IPGTT;1.2–2.0 mg/kg) at 3 and 12 months of age (n = 8 per group). Basal and glucose-stimulated insulin secretion in vitro was studied using the isolated perfused mouse pancreas model at 3 and 12 months. Pancreata were removed and levels of insulin, glucagon, somatostatin, and SSTR1 were studied using immunohistochemical analysis along with H&E staining of the pancreata.

Results

Genotyping verified the absence of SSTR5 in SSTR5^−/− mice. IPGTT demonstrated that 3-month-old SSTR5^−/− mice were glucose intolerant despite similar insulin secretion both in vivo and in vitro and enlarged islets. At 12 months of age, SSTR5^−/− mice had basal hypoglycemia and improved glucose intolerance associated with hyperinsulinemia in vivo and in vitro and enlarged islets. SSTR5^−/− mice had increased insulin clearance at 3 and 12 months of age. SSTR1 expression was significantly increased in islets at 3 months of age, but was nearly absent in islets at 12 months of age, as was somatostatin staining in SSTR5^−/− mice.

Conclusions

These results suggest that both SSTR5 and SSTR1 play a pivotal role in insulin secretion and glucose regulation in mice and that their regulatory effects are age-related.

Introduction

Somatostatin (SST) is an inhibitory peptide initially discovered in the brain but was later found in a variety of tissues and organs [1, 2]. SST-14 is the primary form found throughout the body, whereas SST-28 is mainly located in the gut mucosa [3]. Studies have shown it to have an inhibitory function on cellular signaling and insulin secretion. The inhibitory effects of SST peptide are mediated by a family of seven transmembrane G-protein-coupled receptors (SSTRs) that inhibit cAMP, K⁺ and Ca²⁺ channels, and activate protein phosphatases [4]. Five SST receptors (SSTR1-5) have been isolated and cloned from multiple mammalian species. They are located on five different chromosomes and code for a protein with molecular weight from 45 to 65 kDa [5, 6, 7, 8].

In humans, SSTR1 and 2 were found in the pituitary, small intestine, heart, and spleen with SSTR2 predominating in the pancreas, pituitary, and stomach. SSTR3 and SSTR4 were similarly expressed in the pituitary, heart, liver, spleen, stomach, small intestine, and kidney, whereas SSTR5 was found in high concentrations in the pituitary. In the pituitary, various groups also have shown that SSTR2 and SSTR5 play key roles in the inhibition of growth hormone secretion. Shimon et al. were able to demonstrate that combining SSTR2 and SSTR5 specific agonists had an additive effect on growth hormone suppression from human pituitary adenoma cultures [9]. Analysis of SSTR1-5 using reverse transcriptase polymerase chain reaction (RT-PCR) detected the presence of SSTR2 and not SSTR5 on human islets [10]. However, recent studies by Kumar et al. showed a majority of cells expressing SSTR1, 2, and 5; with SSTR3 and 4 present, but in reduced quantities. On the α cell, the predominant receptor was SSTR2. The β cell had high numbers of SSTR1 and SSTR5 while the δ cell had predominantly SSTR5 [11].

However, the distribution of SSTRs in mice is controversial. The recent cloning of five somatostatin receptor subtypes (SSTR) has lent great excitement to studies related to somatostatin and its receptors. In the rat, SSTRs1 through 5 were found in the brain and pituitary. In the rat brain, however, there also is a different SSTR expression pattern between the cortex and the cerebellum [12]. SSTR2, 3, and 5 also were found in the stomach and pancreas. Infusion of the SSTR5 agonist into the rat pancreata resulted in a significant inhibition of glucose-stimulated insulin secretion. An earlier report from our laboratory using in vitro islet culture technique showed that SSTR5 is the dominant receptor in regulating insulin release in the mouse pancreas [13]. These studies suggest that the inhibition of insulin secretion in mice was mediated by SSTR5 [14].

Furthermore, the distribution of SSTRs in vivo has been found in both cell-subtype and tissue-specific patterns [15]. SSTRs are expressed in different levels in different organs at different ages with sexual dimorphisms [16, 17]. The concept that more than one receptor may serve the same function or work together on the same cell has been hinted at by Rocheville et al. who showed that SSTR1 and SSTR5 were able to form heterodimers. Subsequent studies also have shown that other family members were able to form dimmer/heterodimers as a mechanism to modulate their functions [18, 19, 20, 21].

It has been demonstrated previously that the inhibitory effect of SST on insulin secretion in the endocrine pancreas is mediated via SSTR5 expressed on β and δ cells [13, 22, 23, 24]. The objective of this study was to examine the specific role of SSTR5 in regulating insulin secretion and glucose tolerance and its association with other SSTR family members during mouse development.

Section snippets

Mouse Colony

The mouse SSTR5 gene was cloned and the DNA sequences have been published previously [25]. Global SSTR5 gene-ablated mice were generated and a colony have been maintained in our laboratory [22, 23, 24]. Mice were weaned 3 weeks after birth. Genomic DNA was prepared from each mouse and PCR assays were used for genotyping [22]. (The animals were cared for in compliance with the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources, Commission

Early Growth Retardation in SSTR5^−/− Mice

The body weight of male and female mice at 3 to 24 months of age was measured and compared to those of age-matched, WT controls (n = 20 for each group). As shown in Fig. 1A, SSTR5 gene ablation resulted in a significant decrease in body weight of female mice. Compared to that of age-matched WT controls, the reduction of body weight in female mice was statistically significant at 3, 6, 12, and 24 months of age (P < 0.05). Results showed that in WT controls, the body weight of female mice

Discussion

The islets of Langerhans are composed of four major endocrine cell types: α, β, δ, and pancreatic polypeptide (PP) cells, which secrete glucagon, insulin, SST, and pancreatic polypeptide, respectively. They comprise 10, 70, 5, and 15% of the islet cells, respectively. The appearance, intra-islet location, and the amount of these cell types are age- and species-dependent [26]. Using in vivo microscopy to examine microcirculation in islet cells of rats and mice, it was shown that cell-cell

Acknowledgments

We would like to thank our colleagues working in Dr. Brunicardi’s laboratory for their support and technical assistance. Gratitude also is extended to Katie Elsbury for her editorial assistance throughout this project.

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¹: This project was supported in part by Dr. F. Charles Brunicardi’s NIH grant NIDDK R01-DK46441.

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Metabolism/nutritionThe Effect of Global SSTR5 Gene Ablation on the Endocrine Pancreas and Glucose Regulation in Aging Mice11

Introduction

Methods

Results

Conclusions

Introduction

Section snippets

Mouse Colony

Early Growth Retardation in SSTR5−/− Mice

Discussion

Acknowledgments

J. Biol. Chem.

Neuroscience

Surgery

Biochem. Biophys. Res. Comm.

Life Sci.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Surgery

J. Surg. Res.

Exp. Toxicol. Pathol.

Am. J. Surg.

Pancreas

J. Gastrointest. Surg.

FEBS Lett.

Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone

Science

N. Engl. J. Med

Multiple forms of immunoreactive somatostatinComparison of distribution in neural and nonneural tissues and portal plasma of the rat

Endocrinology

Molecular biology of somatostatin receptors

Endocrinol Rev

Cloning and functional characterization of a family of human and mouse somatostatin receptors expressed in brain, gastrointestinal tract, and kidney

Proc. Natl. Acad. Sci. USA

Characterization of cloned somatostatin receptors SSTR4 and SSTR5

Mol. Pharmacol.

Clone somatostatin receptorsIdentification of subtype-selective peptides and demonstration of high affinity binding of linear peptides

Mol. Pharmacol.

Metabolism/nutrition
The Effect of Global SSTR5 Gene Ablation on the Endocrine Pancreas and Glucose Regulation in Aging Mice¹1

Early Growth Retardation in SSTR5^−/− Mice