Pten (phosphatase and tensin homologue gene) haploinsufficiency promotes insulin hypersensitivity

Abstract

Aims/hypothesis: Insulin controls glucose metabolism via multiple signalling pathways, including the phosphatidylinositol 3-kinase (PI3K) pathway in muscle and adipose tissue. The protein/lipid phosphatase Pten (phosphatase and tensin homologue deleted on chromosome 10) attenuates PI3K signalling by dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate generated by PI3K. The current study was aimed at investigating the effect of haploinsufficiency for Pten on insulin-stimulated glucose uptake.

Materials and methods: Insulin sensitivity in Pten heterozygous (Pten(+/-)) mice was investigated in i.p. insulin challenge and glucose tolerance tests. Glucose uptake was monitored in vitro in primary cultures of myocytes from Pten(+/-) mice, and in vivo by positron emission tomography. The phosphorylation status of protein kinase B (PKB/Akt), a downstream signalling protein in the PI3K pathway, and glycogen synthase kinase 3beta (GSK3beta), a substrate of PKB/Akt, was determined by western immunoblotting.

Results: Following i.p. insulin challenge, blood glucose levels in Pten(+/-) mice remained depressed for up to 120 min, whereas glucose levels in wild-type mice began to recover after approximately 30 min. After glucose challenge, blood glucose returned to normal about twice as rapidly in Pten(+/-) mice. Enhanced glucose uptake was observed both in Pten(+/-) myocytes and in skeletal muscle of Pten(+/-) mice by PET. PKB and GSK3beta phosphorylation was enhanced and prolonged in Pten(+/-) myocytes.

Conclusions/interpretation: Pten is a key negative regulator of insulin-stimulated glucose uptake in vitro and in vivo. The partial reduction of Pten due to Pten haploinsufficiency is enough to elicit enhanced insulin sensitivity and glucose tolerance in Pten(+/-) mice.

Fig. 1

Insulin hypersensitivity in Pten^+/−mice. a Insulin challenge test. Mice were fasted for 15 h prior to i.p. injection of insulin (0.6 mU/g body weight). Blood glucose levels were determined at the indicated times. Means±SEM for six wild-type mice (white circles) and six Pten^+/− mice (black circles). b Glucose tolerance test. Mice were fasted for 15 h prior to i.p. injection of glucose at 2 mg/g body weight. Means±SEM for six wild-type mice (white circles) and seven Pten^+/− mice (black circles)

Fig. 2

Pancreatic islet morphology and immunohistochemistry for insulin (green) and glucagon (red) in wild-type (a) and Pten^+/− (b) mice. Pancreatic sections were immunostained as described in Materials and methods and visualised by fluorescence microscopy. Original magnification ×40

Fig. 3

Insulin-stimulated uptake of ¹⁸FDG in wild-type and Pten^+/− mice. Mice were fasted for 15 h prior to i.p. injection of 0.6 mU insulin/g body weight. Twenty minutes after insulin injection, mice were injected with ¹⁸FDG via a tail vein, and whole-body distribution of ¹⁸FDG was monitored by microPET for 60 min. The experiment was repeated three times with similar results. Typical results are shown. a–f Imaging of ¹⁸FDG distribution in coronal section; wild-type mouse is on the left and Pten^+/− mouse is on the right (a 5 min; b 15 min; c 25 min; d 35 min; e 45 min; f 55 min). Hindlimb muscles were highlighted as regions of interest, and ¹⁸FDG activity in each region of interest quantified across coronal planes at each time-point, as described in Materials and methods. g Graphical representation of relative ¹⁸FDG activity. Data are shown as proportion of ¹⁸FDG activity in hindlimbs relative to activity in whole body for wild-type mice (circles) and Pten^+/− mice (triangles) (mean±SD; n = 3 per point)

Fig. 4

a 2-Deoxy[³H]glucose uptake in wild-type and Pten^+/− myocytes. Myocytes were serum-starved for 12 h prior to incubation with 10 μmol/l 2-deoxy[³H]glucose (0.037 MBq/μmol/l) for the indicated times with 0 or 1 μmol/l insulin. Following incubation cells were lysed and aliquots of lysates were taken for determination of radioactivity by scintillation counting. Means±SD for three separate determinations. White circles, wild-type cells 0 μmol/l insulin; black circles, wild-type cells 1 μmol/l insulin; white triangles, Pten^+/− cells 0 μmol/l insulin; black triangles, Pten^+/− cells 1 μmol/l insulin. b Cell-surface GLUT1 and GLUT4 levels. Levels of cell-surface GLUT1 and GLUT4 proteins in wild-type (WT) and Pten^+/− cells were determined as described in Materials and methods

Fig. 5

Phosphorylation of PKB and GSK3β. Myocytes were serum-starved for 12 h prior to incubation with 1 μmol/l insulin for the indicated times. Cell lysates were prepared, and proteins were separated by PAGE followed by western immunoblot analysis for vinculin, total PKB, phospho-PKB (p-PKB S473) and phospho-GSK3β (p-GSK3β) as indicated. This experiment was repeated three times with typical results shown. WT, wild-type