Biphasic response of pancreatic beta-cell mass to ablation of tuberous sclerosis complex 2 in mice

Abstract

Recent studies have demonstrated the importance of insulin or insulin-like growth factor 1 (IGF-1) for regulation of pancreatic beta-cell mass. Given the role of tuberous sclerosis complex 2 (TSC2) as an upstream molecule of mTOR (mammalian target of rapamycin), we examined the effect of TSC2 deficiency on beta-cell function. Here, we show that mice deficient in TSC2, specifically in pancreatic beta cells (betaTSC2(-/-) mice), manifest increased IGF-1-dependent phosphorylation of p70 S6 kinase and 4E-BP1 in islets as well as an initial increased islet mass attributable in large part to increases in the sizes of individual beta cells. These mice also exhibit hypoglycemia and hyperinsulinemia at young ages (4 to 28 weeks). After 40 weeks of age, however, the betaTSC2(-/-) mice develop progressive hyperglycemia and hypoinsulinemia accompanied by a reduction in islet mass due predominantly to a decrease in the number of beta cells. These results thus indicate that TSC2 regulates pancreatic beta-cell mass in a biphasic manner.

FIG. 1.

Generation of β-cell-specific TSC2 knockout mice. (A) Structure of targeting vector and Tsc2 alleles. Exons are denoted with filled boxes, some with numbers. Expression cassettes for the diphtheria toxin A chain (DTA) and Neo/HSV-TK (Neo-IRES-TK) are shown as open boxes. Shaded triangles indicate loxP sequences. Dotted lines indicate sites of insertion (targeting vector into wild-type allele) and excision (targeted allele reduced to floxed allele and reduced again to deleted mutant allele). Open triangles indicate primers for genotyping. Positions of probes used for Southern blot analysis are shown as striped boxes below the targeted allele. B; BamHI restriction site. (B) Southern blot analysis of G418-resistant ES clones. BamHI-digested DNAs from two ES clones were analyzed with two probes. Lane 1, nonhomologous recombinant control clone; lane 2, homologous recombinant clone. (C) Southern blot analysis of the floxed Tsc2 allele. BamHI-digested DNA was analyzed. Cre (−), parental homologous recombinant ES clone; Cre (+), FIAU-resistant ES clone with floxed allele. (D) Time course of changes in plasma insulin concentration induced by intraperitoneal administration of glucose (3 mg per gram of body weight) in wild-type (open triangles; n = 3), Ins-Cre (filled squares; n = 3), and TSC2^flox/flox (open circles; n = 4) mice at 8 weeks of age. Data are means ± SEM. (E, F) Immunoblot analysis of TSC2 and either β-actin or α-tubulin (loading controls) in pancreatic islets (E) or in the indicated tissues (F) of 9-week-old control (TSC2^flox/flox) and βTSC2^−/− mice.

FIG. 2.

Effect of β-cell TSC2 ablation on glucose metabolism. Blood glucose concentration (A), plasma insulin concentration (B), and body weight (C) are shown for control (open circles; n = 10) and βTSC2^−/− (filled circles; n = 12) mice at the indicated ages as measured in the randomly fed state. Data are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 for comparison with the corresponding values for control mice.

FIG. 3.

Effect of β-cell-specific ablation of TSC2 on glucose-stimulated insulin secretion in mice at 8 weeks of age. Blood glucose (A) and plasma insulin (B) concentrations during oral glucose tolerance tests are shown for control (open circles; n = 9), βTSC2^+/− (gray circles; n = 5), and βTSC2^−/− (black circles; n = 10) mice. (C) Acute insulin response to intraperitoneal administration of glucose (3 mg per gram of body weight) in control (open circles; n = 4) and βTSC2^−/− (filled circles; n = 4) mice. All data are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 for comparison with the corresponding values for control mice.

FIG. 4.

Effects of β-cell TSC2 ablation on pancreatic islet morphology. (A) Pancreatic sections from 6- and 40-week-old control and βTSC2^−/− mice were stained with hematoxylin-eosin (left panels) or with antibodies to insulin (red) and to glucagon (green) (right panels). Scale bars, 200 μm (left panels) or 50 μm (right panels). (B) Islet density was determined from the number of islets in pancreatic sections divided by the total area of the pancreas in 6- and 40-week-old control and βTSC2^−/− mice. (C) Total β-cell mass was calculated from the area of insulin-positive cells in pancreatic sections divided by the total area of the pancreas in 6- and 40-week-old control and βTSC2^−/− mice. (D) Pancreatic sections from 6- and 40-week-old control and βTSC2^−/− mice were immunostained as for panel A (left panels). Scale bars, 50 μm. The sizes of individual β cells were determined from the total area of insulin-positive cells in pancreatic sections divided by the number of nuclei in insulin-positive cells. (E) The numbers of β cells in 6- and 40-week-old control and βTSC2^−/− mice were determined from the number of β cells in pancreatic sections divided by the total pancreatic area. All quantitative data are means ± SEM (n = 5 and n = 3 for 6- and 40-week-old mice, respectively). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (F) Islets isolated from control and βTSC2^−/− mice at 5 or 35 weeks of age were lysed and subjected to immunoblot analysis with antibodies to the cleaved form of caspase-3 and to β-actin.

FIG. 5.

Effects of β-cell TSC2 ablation on insulin signaling in pancreatic islets. Islets isolated from control and βTSC2^−/− mice of the indicated ages (in weeks [w]) were deprived of serum for 2 h and then incubated in the presence (+) or absence (−) of 100 nM IGF-1 for 1 h. (A, B) The islets were then lysed and incubated with antibodies for immunoblot analysis of phosphorylated (p) or total forms of the indicated proteins.

FIG. 6.

Effects of rapamycin on insulin signaling in pancreatic islets of βTSC2^−/− mice. Intraperitoneal injections of rapamycin were delivered to mice at a concentration of 2 mg/kg every other day from 18 to 40 weeks of age. Blood glucose (A) and plasma insulin (B) concentrations are shown for βTSC2^−/− mice treated with (gray circles; n = 6) and without (closed squares; n = 6) rapamycin. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Pancreatic sections from 40-week-old βTSC2^−/− mice untreated (−) or treated (+) with rapamycin were stained with antibodies to insulin (red) and glucagon (green). Scale bars, 50 μm. (D) Islets isolated from 28-week-old βTSC2^−/− mice untreated (−) or treated (+) with rapamycin were deprived of serum for 2 h and then incubated in the presence of 100 nM IGF-1 for 1 h. The islets were then lysed and subjected to immunoblot analysis with the indicated antibodies.

FIG. 7.

mTORC1 activity in the islets of animal models of obesity and type 2 diabetes. Islets isolated from mice fed with a high-fat diet for 3 weeks (HFD) and 8-week-old db/db mice (db/db) were deprived of serum for 2 h and then incubated in the presence (+) or absence (−) of 100 nM IGF-1 for 1 h. The islets were then lysed and subjected to immunoblot analysis with the indicated antibodies. NC, normal controls; db/m, mice heterozygous for the leptin receptor.