Insulin receptors in beta-cells are critical for islet compensatory growth response to insulin resistance

Abstract

Insulin and insulin-like growth factor 1 (IGF1) are ubiquitous growth factors that regulate proliferation in most mammalian tissues including pancreatic islets. To explore the specificity of insulin receptors in compensatory beta-cell growth, we examined two models of insulin resistance. In the first model, we used liver-specific insulin receptor knockout (LIRKO) mice, which exhibit hyperinsulinemia without developing diabetes due to a compensatory increase in beta-cell mass. LIRKO mice, also lacking functional insulin receptors in beta-cells (beta IRKO/LIRKO), exhibited severe glucose intolerance but failed to develop compensatory islet hyperplasia, together leading to early death. In the second model, we examined the relative significance of insulin versus IGF1 receptors in islet growth by feeding high-fat diets to beta IRKO and beta-cell-specific IGF1 receptor knockout (beta IGFRKO) mice. Although both groups on the high-fat diet developed insulin resistance, beta IRKO, but not beta IGFRKO, mice exhibited poor islet growth consistent with insulin-stimulated phosphorylation, nuclear exclusion of FoxO1, and reduced expression of Pdx-1. Together these data provide direct genetic evidence that insulin/FoxO1/Pdx-1 signaling is one pathway that is crucial for islet compensatory growth response to insulin resistance.

Fig. 1.

βIRKO/LIRKO mice display early hyperinsulinemia, hyperglycemia, glucose intolerance, loss of acute phase insulin release, and insulin resistance. (a–e) Serum insulin (a) levels and blood glucose (b) were measured 7, 14, 17, 21, 28, and 42 days after birth. ∗, P < 0.01, βIRKO/LIRKO vs. control, βIRKO, or LIRKO; †, P < 0.05, βIRKO/LIRKO vs. LIRKO. Insulin is plotted on log scale on y axis. (c) Acute (first)-phase insulin secretion following an intraperitoneal (i.p.) injection of glucose (3 g/kg body weight). Data are expressed as percentage increase at 2 and 5 min relative to time 0. ∗, P < 0.01, vs. 0 or 5 min; n = 8–10. (d) Blood glucose after i.p. injection of glucose (2 g/kg body weight). ∗, P < 0.001 βIRKO vs. control; †, P < 0.05, βIRKO vs. control, or LIRKO vs. βIRKO/LIRKO; n = 8. (e) Blood glucose after i.p. injection of insulin (Humulin, 1 unit/kg body weight). ∗, P < 0.01 βIRKO/LIRKO or LIRKO vs. control and βIRKO; n = 8. Experiments described in c–e are from male mice ages 4–5 weeks..

Fig. 2.

Absent compensatory islet hyperplasia in βIRKO/LIRKO mice. (a) Immunostaining of pancreas sections for non-β-cells by using a mixture of antibodies to glucagon, somatostatin, and pancreatic polypeptide as described in Methods. Representative sections from 2- and 6-week-old mice. (Magnification: ×10.) (Scale bar: 50 μm.) (b) β-cell mass at 2 (in micrograms) and 6 (in milligrams) weeks assessed by morphometric analysis as described in Methods. ∗, P < 0.05 LIRKO vs. all other groups. (c) After overnight culture, size-matched islets were incubated in different concentrations of glucose for 45 min. Then the medium was removed and assayed for insulin. Islet insulin content was assayed by acid ethanol extraction as described in Methods. ∗, P < 0.05, 5.5 mM vs. 11 mM glucose; †, P < 0.05, βIRKO/LIRKO or βIRKO vs. control or LIRKO; +, P < 0.05, βIRKO/LIRKO vs. control or βIRKO or LIRKO. (d) Representative pancreas sections from 4-week-old male mice immunostained for insulin by using DAB as a substrate as described in Methods. (Magnification: ×40.) (Scale bar: 50 μm.)

Fig. 3.

LIRKO mice show massive islet hyperplasia secondary to enhanced β-cell replication. (a) Morphometric analyses of β-cell mass in 10- to 12-month-old control, βIRKO, and LIRKO mice as described in Methods. ∗, P < 0.05 control vs. βIRKO or LIRKO; †, P < 0.01, βIRKO vs. LIRKO (n = 4–6). βIRKO/LIRKO mice were dead by ≈8 weeks. Analyzed by single observer blinded to genotypes. (b) BrdU+ β-cells in 10-month-old control, βIRKO, and LIRKO mice. ∗, P < 0.05 control vs. βIRKO or LIRKO; †, P < 0.01, βIRKO vs. LIRKO (n = 4–6). (c) Representative islets from three male LIRKOs immunostained for insulin (red), BrdU (green), and nuclear stain DAPI (blue). Arrowheads point to BrdU+ cells. (Magnification: ×63.) (Scale bar: 100 μM.) (d) Alterations in blood glucose, serum insulin, and BrdU+ β-cells in 6-week- and 6-, 10-, and 20-month-old control and LIRKO male mice. ∗, P < 0.05 LIRKO vs. control; n = 4–7. Serum insulin is plotted on log scale. BrdU immunostaining was performed as described in Methods.

Fig. 4.

βIRKO, but not βIGFRKO, mice fail to manifest compensatory islet hyperplasia in response to high-fat diet-induced insulin resistance due to nuclear restriction of FoxO1. (a) Insulin tolerance tests in control, βIRKO, and βIGFRKO mice on chow or high-fat diets at the end of 20 weeks. ∗, P < 0.05 high-fat diet vs. chow (n = 9–16). (b) Glucose tolerance tests in control, βIRKO, and βIGFRKO mice on chow and high-fat diets at the end of 20 weeks. ∗, P < 0.05 high-fat diet vs. chow; †, P < 0.01, βIRKO (high-fat) vs. control (high-fat) or βIGFRKO (high-fat). βIRKO mice on chow diet are significantly different from control group on chow diet at 15, 30, 60, and 120 min. βIGFRKO mice on chow diet are significantly different from control mice on chow diet at 15 and 30 min. βIRKO mice on chow diet are significantly different from βIGFRKO mice on chow diet at 30, 60, and 120 min (n = 9–16). (c) Representative pancreas sections from control, βIRKO, and βIGFRKO mice at the end of 20 weeks on chow or high-fat diets immunostained with mixture of antibodies against non-β-cell hormones as described in Methods. (Magnification: ×20.) (Scale bar: 100 μM.) (d) Morphometric analyses of β-cell mass in control, βIRKO, and βIGFRKO mice on chow or high-fat diets as described in Methods. ∗, P < 0.05 chow vs. high-fat diet in each group; +, P < 0.05 βIRKO (chow) vs. control (chow); †, P < 0.01, βIRKO (high-fat) vs. control (high-fat) or βIGFRKO (high-fat) (n = 5–7). Analyzed by a single observer blinded to genotypes. (e) Representative pancreas sections of control, βIGFRKO, and βIRKO mice on high-fat diet immunostained with anti-FoxO1 antibody (dark brown) and nuclear stain DAPI (blue) (Upper) or for FoxO1 only (Lower) by using immunofluorescent antibody as described in Methods (n = 3–4 per group). (Magnification: ×63.) (f) Representative Western blot of control β-cell lines, β-cells with a 95% knockdown of insulin receptors, or β-cells with a ≈90% knockdown of IGF1 receptors. Expression of insulin receptors (IR) and IGF1 receptors (IGF1R) was examined in whole-cell lysates, and expression of FoxO1 and PDX-1 protein was examined in cytosolic and nuclear fractions as described in Methods.