Essential role of Pten in body size determination and pancreatic beta-cell homeostasis in vivo

Abstract

PTEN (phosphatase with tensin homology) is a potent negative regulator of phosphoinositide 3-kinase (PI3K)/Akt signaling, an evolutionarily conserved pathway that signals downstream of growth factors, including insulin and insulin-like growth factor 1. In lower organisms, this pathway participates in fuel metabolism and body size regulation and insulin-like proteins are produced primarily by neuronal structures, whereas in mammals, the major source of insulin is the pancreatic beta cells. Recently, rodent insulin transcription was also shown in the brain, particularly the hypothalamus. The specific regulatory elements of the PI3K pathway in these insulin-expressing tissues that contribute to growth and metabolism in higher organisms are unknown. Here, we report PTEN as a critical determinant of body size and glucose metabolism when targeting is driven by the rat insulin promoter in mice. The partial deletion of PTEN in the hypothalamus resulted in significant whole-body growth restriction and increased insulin sensitivity. Efficient PTEN deletion in beta cells led to increased islet mass without compromise of beta-cell function. Parallel enhancement in PI3K signaling was found in PTEN-deficient hypothalamus and beta cells. Together, we have shown that PTEN in insulin-transcribing cells may play an integrative role in regulating growth and metabolism in vivo.

FIG. 1.

Tissue-specific deletion of Pten. (a) Immunohistochemistry showing PTEN deletion in β cells (original magnification, ×25). (b) Western blots (left panels) and quantification of Western blot signal (right panel) showing decreased expression of PTEN in isolated islets and the hypothalamus (Hyp). Residual signal in islets is likely due to the presence of non-β cells. *, P = 0.011; **, P < 0.005. The error bar indicates the standard error of the mean. (c) PCR analysis of Cre-mediated recombination of the Pten locus (Δ4-5, top) in β cells and ear tissue and genotyping for Pten-loxP allele (middle) and Cre (bottom). M, marker; C, control mammary gland tumor; wt, wild type. (d) Expression of PTEN in liver (L), fat (F), and muscle (M) is unchanged. +/+, RIPcre⁺ Pten^+/+ mice; −/−, RIPcre⁺ Pten^fl/fl mice.

FIG. 2.

RIPcre⁺ Pten^fl/fl mice show growth restriction from the early postnatal period onward. (a) Neonatal birth weights of RIPcre⁺ Pten^fl/fl mice are similar to birth weights of littermate controls on postnatal day 1 (n ≥ 3 per genotype). (b) Growth of RIPcre⁺ Pten^fl/fl mice is restricted compared with that of RIPcre⁺ Pten^+/+ and RIPcre⁺ Pten^+/− littermates from 1 week of age onward (n ≥ 14). *, P ≤ 0.005 for comparison between RIPcre⁺ Pten^fl/fl and RIPcre⁺ Pten^+/+ or RIPcre⁺ Pten^+/−; †, P = 0.01 for RIPcre⁺ Pten^fl/fl versus RIPcre⁺ Pten^+/− mice. (c) Growth-restricted RIPcre⁺ Pten^fl/fl mice (right) are proportionately smaller than littermate RIPcre⁺ Pten^+/+ mice (left, 4 weeks old). Error bars indicate standard errors of the means. +/+, RIPcre⁺ Pten^+/+ mice; −/−, RIPcre⁺ Pten^fl/fl mice; +/−, RIPcre⁺ Pten^+/fl mice.

FIG. 3.

Growth restriction in RIPcre⁺ Pten^fl/fl mice is not due to altered food intake or the GH/IGF-1 axis. (a) Food intake levels are similar between RIPcre⁺ Pten^fl/fl and RIPcre⁺ Pten^+/+ mice soon after weaning (left panel) and at 4 to 6 months of age (right panel). Daily food intake is normalized for body weight and expressed as relative food intake (n = 4 per genotype). (b) RT-PCR for various transcripts levels in isolated hypothalamus (n ≥ 8; age, 8 to 12 weeks). #, P = 0.098. (c) Serum IGF-1 levels are similar in RIPcre⁺ Pten^fl/fl and RIPcre⁺ Pten^+/+ or RIPcre⁺ Pten^+/fl mice (n ≥ 3; age, 5 to 8 weeks old). Error bars indicate standard errors of the means. +/+, RIPcre⁺ Pten^+/+ mice; −/−, RIPcre⁺ Pten^fl/fl mice; +/−, RIPcre⁺ Pten^+/fl mice.

FIG. 4.

Glucose metabolism and insulin secretion. (a) Fasting blood glucose levels in RIPcre⁺ Pten^fl/fl mice are lower than those in littermate RIPcre⁺ Pten^+/+ controls (n ≥ 8). *, P ≤ 0.005. (b) Systemic-fasting serum insulin levels are lower in RIPcre⁺ Pten^fl/fl mice (n ≥ 6). *, P ≤ 0.05. (c) Insulin tolerance tests demonstrate greater insulin sensitivity in RIPcre⁺ Pten^fl/fl mice. The hypoglycemic response to an i.p. injection of human regular insulin at a dose of 0.5 mU/g of body weight is expressed as a percentage of baseline blood glucose (n ≥ 3; age, 8 to 10 weeks). *, P < 0.05. (d) RIPcre⁺ Pten^fl/fl mice have lower glucose excursions after i.p. glucose tolerance tests compared with that for RIPcre⁺ Pten^+/+ and RIPcre⁺ Pten^+/fl mice (n ≥ 4; age, 2 to 4 months). *, P < 0.005 for comparison of RIPcre⁺ Pten^fl/fl versus RIPcre⁺ Pten^+/+ or RIPcre⁺ Pten^+/fl; **, P = 0.011 for RIPcre⁺ Pten^+/fl versus RIPcre⁺ Pten^+/+ and P = 0.042 for RIPcre⁺ Pten^+/fl versus RIPcre⁺ Pten^fl/fl. (e) In vivo glucose-stimulated insulin secretion after i.p. glucose injection at 2 and 30 min is preserved. P was not significant. (f) Insulin secretion in isolated perfused pancreas. The left panel shows the response to glucose at the molar concentrations indicated. The right panel shows the response to arginine and 16.7 mM glucose. P was not significant for all time points. Error bars indicate standard errors of the means. +/+, RIPcre⁺ Pten^+/+ mice; −/−, RIPcre⁺ Pten^fl/fl mice; +/−, RIPcre⁺ Pten^+/fl mice.

FIG. 5.

Islet morphology and function. (a) Increased total islet area in RIPcre⁺ Pten^fl/fl mice compared with that in littermate RIPcre⁺ Pten^+/+ mice shown by immunohistochemistry of representative pancreatic sections stained for synaptophysin (left panels; original magnification, ×4) and expressed as a percentage of total pancreatic area (right panel, synaptophysin stained area divided by total pancreatic area) (n ≥ 7). *, P = 0.015; **, P = 0.028. (b) β-Cell size is increased in islets of RIPcre⁺ Pten^fl/fl mice compared with that in the islets of RIPcre⁺ Pten^+/+ littermates, costained for insulin and DAPI (left panels; original magnification, ×40) and expressed as a ratio of insulin-stained area to number of nuclei within insulin-stained area (right panel) (n ≥ 7). *, P ≤ 0.005. (c) Total pancreatic insulin content is increased in RIPcre⁺ Pten^fl/fl mice compared to that in RIPcre⁺ Pten^+/+ mice, (n ≥ 3; age, 8 to 12 weeks). *, P = 0.029. (d) Islet architecture is preserved without evidence of tumorigenesis. Insulin and glucagon staining show normal β- and α-cell distributions (original magnification, ×20), basement membranes are intact as shown by laminin staining (original magnification, ×10), and β-catenin localizes to the cell membrane (original magnification, ×20), demonstrating intact cell-to-cell adhesion. Error bars indicate standard errors of the means. +/+, RIPcre⁺ Pten^+/+ mice; −/−, RIPcre⁺ Pten^fl/fl mice.

FIG. 6.

β-Cell proliferation and apoptosis. (a) RIPcre⁺ Pten^fl/fl mice tend to have a higher percentage of Ki67-positive cells than do their RIPcre⁺ Pten^+/+ littermates (n = 8 per genotype). (b) Effect of MLDS on blood glucose levels showing protection against MLDS-induced diabetes in RIPcre⁺ Pten^fl/fl mice (n = 2 to 5; age, 7 to 9 weeks). con, control; STZ, streptozotocin. (c) Representative islets of MLDS-treated RIPcre⁺ Pten^fl/fl and RIPcre⁺ Pten^+/+ mice stained for TUNEL, showing fewer TUNEL-positive nuclei in RIPcre⁺ Pten^fl/fl mice. +/+, RIPcre⁺ Pten^+/+ mice; −/−, RIPcre⁺ Pten^fl/fl mice.

FIG. 7.

Effect of Pten deletion on the insulin/IGF-1 signaling pathway. (a) Immunohistochemistry of pancreatic sections shows that RIPcre⁺ Pten^fl/fl mice have increased phospho-AKT, cytoplasmic localization of FoxO-1, increased nuclear PDX-1, and increased GLUT2. (b) Western blots (left panel) and quantification (right panel) of isolated islets showing that absence of PTEN in β cells leads to increased phospho-AKT, FoxO-1, and GSK3β as well as enhanced PDX-1, GLUT2, and IRS-2 levels. **, P < 0.005. (c) Western blots (left panel) and quantification (right panel) from isolated hypothalamus showing that partial deletion of PTEN leads to enhanced phospho-AKT, similar levels of phospho- and total FoxO-1, and elevated GLUT2 and IRS-2 expression in RIPcre⁺ Pten^fl/fl mice. *, P < 0.05; **, P < 0.005. Error bars indicate standard errors of the means. p, phospho; +/+, RIPcre⁺ Pten^+/+ mice; −/−, RIPcre⁺ Pten^fl/fl mice.