Beta-Cell hyperplasia induced by hepatic insulin resistance: role of a liver-pancreas endocrine axis through insulin receptor A isoform - PubMed (original) (raw)
Increase of IR-A isoform in pancreatic islets: β-cell lines expressing IR-A (Rec A), but not IR-B (Rec B), induce proliferation in response to insulin or IGF-1. A: mRNA levels of insulin receptor and the insulin receptor isoforms distribution were analyzed by real-time quantitative PCR in 6-month-old control and iLIRKO mice as described in
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. Values are expressed as mean ± SEM; n = 4 of each genotype, (*P < 0.05; **P < 0.005; ***P < 0.001 iLIRKO vs. control). B: Pancreatic islet proliferation was assessed by BrdU incorporation in 6-month-old control and inducible liver-specific insulin receptor knockout (iLIRKO) mice. The islets were isolated as described and seeded in 96-well plates for 16 h in RPMI medium. After that, the medium was removed and fresh medium with the stimuli (10 nmol/l insulin, 
; 10 nmol/l IGF-1, ▤) and BrdU was added for 24 h. Finally, the BrdU incorporation was measured as indicated by the manufacturer. Values are expressed as mean ± SEM; n = 4 of each genotype. Data were subjected to ANOVA with Bonferroni post-test (*P < 0.05; IGF-1 vs. basal in iLIRKO mice). C: IR expression was analyzed by Western blot and RT-PCR in IRLoxP, IRKO, Rec A, and Rec B β-cells. Arrow at the RT-PCR panels indicates the IR-A and IR-B. A representative experiment out of four is shown. D: Functional assessment of insulin receptor reconstitution was carried out by immunoprecipitation of insulin– or IGF-1–stimulated β-cells with antibodies against IR or IGF-1R and subsequent Western blot against phospho-tyrosine residues. E: IRLoxP, IRKO, Rec A, and Rec B β-cells were cultured to 80% confluence and then serum and glucose starved for 4–6 h. Glucose uptake induced by insulin () or IGF-1 (▤) was measured as described in
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. Data were subjected to ANOVA with Bonferroni post-test (*P < 0.05, IRKO and Rec A vs. IRLoxP in basal conditions; #P < 0.05, IGF-1 vs. basal of each cell line). F: IRLoxP, IRKO, Rec A, and Rec B β-cells were cultured to 50% confluence in 10% FBS-DMEM overnight. After that, 10 nmol/l insulin (), 10 nmol/l IGF-1 (▤), or both (■) were added to the wells in serum-starved 5 mmol/l glucose DMEM. After 24 h, the medium was withdrawn and the four cell lines were stained with violet crystal as described in
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. Statistical significance was carried out by Student's t test by comparison of basal conditions with insulin-stimulated conditions of each cell line (*P < 0.05) or basal conditions with IGF-1–stimulated conditions of each cell line (#P < 0.05). G: IRLoxP, IRKO, Rec A, and Rec B β-cells were cultured to 50% confluence in 10% FBS-DMEM overnight. After that, 10 nmol/l insulin (), 10 nmol/l IGF-1 (▤), or both (■) were added to the wells in serum-starved 5 mmol/l glucose DMEM for 24 h. Thymidine incorporation was measured in these conditions as described in
research design and methods
. Statistical significance was carried out by Student's t test by comparison of basal conditions with insulin-stimulated conditions of each cell line (*P < 0.05) or basal conditions with IGF-1–stimulated conditions of each cell line (#P < 0.05).