Enhanced beta cell proliferation in mice overexpressing a constitutively active form of Akt and one allele of p21Cip

Abstract

Aims/hypothesis: The ability of pancreatic beta cells to proliferate is critical both for normal tissue maintenance and in conditions where there is an increased demand for insulin. Protein kinase B(Akt) plays a major role in promoting proliferation in many cell types, including the insulin-producing beta cells. We have previously reported that mice overexpressing a constitutively active form of Akt(caAkt (Tg)) show enhanced beta cell proliferation that is associated with increased protein levels of cyclin D1, cyclin D2 and cyclin-dependent kinase inhibitor 1A (p21(Cip)). In the present study, we sought to assess the mechanisms responsible for augmented p21(Cip) levels in caAkt(Tg) mice and test the role of p21(Cip) in the proliferative responses induced by activation of Akt signalling.

Methods: To gain a greater understanding of the relationship between Akt and p21(Cip), we evaluated the mechanisms involved in the modulation of p2(Cip) by Akt and the in vivo role of reduced p21(Cip) in proliferative responses induced by Akt.

Results: Our experiments showed that Akt signalling regulates p21(Cip) transcription and protein stability. caAkt(Tg) /p21(Cip+/-) mice exhibited fasting and fed hypoglycaemia as well as hyperinsulinaemia when compared with caAkt(Tg) mice. Glucose tolerance tests revealed improved glucose tolerance in caAkt(Tg)/p21(Cip+/-) mice compared with caAkt (Tg). These changes resulted from increased proliferation, survival and beta cell mass in caAkt(Tg)/p21(Cip+/-) compared with caAkt(Tg) mice.

Conclusions/interpretation: Our data indicate that increased p21(Cip) levels in caAkt(Tg) mice act as a compensatory brake, protecting beta cells from unrestrained proliferation. These studies imply that p21(Cip) could play important roles in the adaptive responses of beta cells to proliferate in conditions such as in insulin resistance.

Fig. 1

Akt regulates p21^Cip levels by transcriptional regulation and protein stability. a Assessment of p21^Cip mRNA levels in MIN6^GFP and MIN6^caAkt cells using TaqMan RT-PCR. b p21^Cip mRNA stability assays from MIN6 and MIN6^caAkt cells treated with 5 μg/ml actinomycin D in 5.5 mmol/l glucose. Half-lives were calculated using GraphPad Prism Software: MIN6^GFP t_½=6.2 h; MIN6^caAkt t_½=1.1 h; c Immunoblotting for p21^Cip in MIN6^GFP and MIN6^caAkt cells. d p21^Cip protein stability assessed by immunoblotting for p21^Cip and actin in MIN6^GFP and MIN6^caAkt cells cultured with 12.5 μg/ml CHX for 0, 0.5, 1, 2, 4, 6 and 8 h. e Quantification of p21^Cip stability experiments in MIN6^GFP and MIN6^caAkt cells. Protein bands for p21^Cip were quantified and normalised to the levels in cells at time 0. f p21^Cip protein stability assessed by immunoblotting for p21^Cip and tubulin in islets cultured with 12.5 μg/ml CHX for 1 h. Data are presented as mean±SEM of at least three independent experiments (n≥3). *p<0.05. In graphs: black, MIN6^GFP; grey, MIN6^caAkt. WT, wild type

Fig. 2

Altered subcellular localisation and phosphorylation of p21^Cip in cells overexpressing Akt. a Representative immunofluorescence imaging for p21^Cip (red) and nuclear staining with DAPI (blue) in MIN6 and MIN6^caAkt cells. b Quantification of p21^Cip subcellular localisation. c Immunofluorescence imaging of endogenous p21^Cip in pancreatic islet paraffin section from non-transgenic and caAkt mice. Staining of p21^Cip is shown in red and insulin in green. Data are presented as mean±SEM of at least three independent experiments (n≥3). *p<0.05. NT, non-transgenic

Fig. 3

Increased p21^Cip levels bound to CDK4 and CDK2 complex in MIN6 cells and islets with activation of Akt signalling. a,b Assessment of p21^Cip bound to the CDK4 complex in MIN6 (a) and in islets (b). c,d Assessment of p21^Cip bound to the CDK2 complex in MIN6 (c) and in islets (d). Cell lysates from MIN6^GFP and MIN6^caAkt or islet lysates from non-transgenic and caAkt^Tg were immunoprecipitated with CDK4 or CDK2 antibodies followed by immunoblotting for p21^Cip. Quantification of p21^Cip levels bound to CDK4 (a,b) and CDK2 (c,d). In these experiments, CDK4 and CDK2 were immunoprecipitated with their respective antibodies followed by immunoblotting for p21^Cip. Immunoblotting for CDK4 and CDK2 were used as controls. Data are presented as mean±SEM; n≥3; *p<0.05. In graphs, black bars, non-transgenic; grey bars, caAkt^Tg. IP, immunoprecipitated; NT, non-transgenic

Fig. 4

Metabolic assessment of p21^Cip−/− and caAkt^Tg intercross. a Body weight of 3-month-old male mice. Fasting glucose (b) and insulin (c) measurements were performed on overnight-fasted 3–4 month-old male non-transgenic mice or caAkt^Tg containing two alleles (^+/+) or one allele (^+/−) in p21^Cip mice. d,e Random glucose and insulin levels obtained in the same group and age of mice. f Intraperitoneal glucose tolerance tests were performed on the same group of mice. Data are presented as mean±SEM (n=8–10). *p<0.05 compared with p21^Cip+/+; ^†p<0.05 compared with caAkt^Tg/p21^Cip+/+. p21^Cip+/+, grey circle; p21^Cip+/−, grey square; p21^Cip−/−, grey triangle; caAkt^Tg, black triangle; and caAkt^Tg/p21^Cip+/−, black square. NT, non-transgenic

Fig. 5

Determination of pancreatic and islet morphology of caAkt^Tg and p21^Cip intercross. a Representative pancreatic morphology from mice with different genotypes stained for insulin (red) and counterstained with haematoxylin (blue). b Beta cell mass in 4–5-month-old progeny from p21^−/−/caAkt^Tg intercross (n≥3). Data are mean±SEM. *p<0.05. NT, non-transgenic

Fig. 6

Determination of beta cell proliferation and apoptosis among caAkt^Tg and p21^Cip intercrosses. a Proliferation index determined by percentage of Ki67-positive beta cells in p21^Cip and caAkt^Tg intercross (n≥3). TUNEL index determined by percentage of TUNEL-positive beta cells in p21^Cip and caAkt^Tg intercross (n≥3). Data are mean±SEM. *p<0.05. NT, non-transgenic