Defective insulin secretion in pancreatic beta cells lacking type 1 IGF receptor - PubMed (original) (raw)

. 2002 Oct;110(7):1011-9.

doi: 10.1172/JCI15276.

Tadahiro Kitamura, Jun Nakae, Katerina Politi, Yoshiaki Kido, Peter E Fisher, Manrico Morroni, Saverio Cinti, Morris F White, Pedro L Herrera, Domenico Accili, Argiris Efstratiadis

Affiliations

Defective insulin secretion in pancreatic beta cells lacking type 1 IGF receptor

Shouhong Xuan et al. J Clin Invest. 2002 Oct.

Abstract

Defective insulin secretion is a feature of type 2 diabetes that results from inadequate compensatory increase of beta cell mass and impaired glucose-dependent insulin release. beta cell proliferation and secretion are thought to be regulated by signaling through receptor tyrosine kinases. In this regard, we sought to examine the potential proliferative and/or antiapoptotic role of IGFs in beta cells by tissue-specific conditional mutagenesis ablating type 1 IGF receptor (IGF1R) signaling. Unexpectedly, lack of functional IGF1R did not affect beta cell mass, but resulted in age-dependent impairment of glucose tolerance, associated with a decrease of glucose- and arginine-dependent insulin release. These observations reveal a requirement of IGF1R-mediated signaling for insulin secretion.

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Figures

Figure 1

Figure 1

Generation of a conditional IGF1R knockout in β cells. (a) A diagram of the Igf1r locus around exon 3 (filled square) is shown, followed by diagrams of the null (Igf1r+/–) (41), floxed (Igf1rlox) (42), and recombined floxed (Igf1rΔlox) (42) alleles. The positions of the P1, P2, and P3 primers (arrows) used for genotyping are indicated. (b) PCR analyses using DNA from islets and control tissues. The lanes labeled – and + correspond to the negative and positive controls. Primers P1 and P2 yield a product of approximately 120 bp in WT and null Igf1r alleles, and 220 bp in the Igf1rlox allele. In the Igf1rΔlox allele, excision of exon 3 enables amplification to occur between primers P3 and P2, yielding 320 bp. In the lanes to the right of the markers, DNA from different tissues of Igf1rΔlox/– mice was used. Br, brain; Li, liver; and Ki, kidney. (c) Western blot analysis of IGF1R expression. We obtained protein extracts from wild-type mouse embryonic fibroblasts or from fibroblasts derived from Igf1r–/– mice as positive and negative controls (first two lanes on the left). We purified islets from wild-type (third lane from the left) and Igf1rΔlox/– mice (right lane). Following detection of IGF1R, the blot was stripped and reprobed with anti-tubulin antiserum. The position of the various bands is indicated on the left of the autoradiogram.

Figure 2

Figure 2

Intraperitoneal glucose tolerance tests in mice lacking IGF1R in β cells. Tests were performed in overnight-fasted mice as indicated in Methods. The difference between Igf1rΔlox/– and Igf1rΔlox/+ is significant at all time points except 0. The difference between Igf1rΔlox/– and Igf1rlox/– is statistically significant at times 90 and 120. The difference between Igf1rΔlox/+ and Igf1rlox/– is statistically significant at times 30, 60, and 90 (P < 0.05 by ANOVA). Bars represent SEM.

Figure 3

Figure 3

Islet morphology in mice lacking IGF1R in β cells. Examples of pancreatic sections from P30 mice of the indicated genotypes that were stained with anti-insulin and anti-glucagon Ab’s are shown.

Figure 4

Figure 4

Analysis of Glut-2 expression. (a) Real-time PCR amplification. mRNA was isolated from Igf1rΔlox/–, Igf1rlox/–, and Igf1rΔlox/+ mice (n = 3 each; equal amounts were pooled). Amplification was performed using a one-step RT-PCR reaction. The amount of RNA in the reaction was normalized using β-actin RNA as a control. *P < 0.05 by ANOVA. (b) Immunohistochemistry with anti-GLUT-2 antiserum. Representative sections of Igf1rΔlox/+ and wild-type mice are shown.

Figure 5

Figure 5

Ki67, histone H3, and BrdU labeling of β cells. Adjacent 4-μm-thick pancreatic section from mice at E18.5, P10, P30, and P120 were costained with anti-insulin Ab’s (to identify β cells) and anti-Ki67, or anti-histone H3 or anti-BrdU Ab’s (to provide an index of proliferation). Positive cells were visually scored, and the number of Ki67-positive cells was plotted as a function of the number of histone H3–positive (upper panel) or BrdU-positive cells (lower panel). Regression analysis was performed using the Statsview software. _r_2 = 0.9 for the H3/Ki67 and 0.9 for the BrdU/Ki67 correlation (P < 0.0001).

Figure 6

Figure 6

Developmental analysis of Ki67 labeling. A Ki67 labeling index was determined in mice of different ages by multiplying the mean number of Ki67/insulin double-positive cells per section by β cell mass (μg). The values at E18.5 were normalized to 100%. None of the differences observed between the different genotypes are statistically significant. Two mice for each genotype were used at each time point, except for Igf1rlox/+ and Igf1rΔlox/+ at P10, where one mouse was used. Three to five sections per pancreas were scored as described in Methods.

Figure 7

Figure 7

Insulin secretion and content in purified islets. Insulin release was determined in response to varying glucose (a) and arginine (b) concentrations. For each glucose concentration, ten islets derived from two or three mice of each genotype were used. Three separate experiments were performed and the results were averaged. (c) Insulin content in islets of the indicated genotypes was measured by high-salt buffer extraction followed by radioimmunoassay.

Figure 8

Figure 8

Electron microscopy of β cells. Representative images of β cells in wild-type and mutant mice are shown at ×11,300 magnification.

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