Insulin hypersensitivity and resistance to streptozotocin-induced diabetes in mice lacking PTEN in adipose tissue

Abstract

In adipose tissue, insulin controls glucose and lipid metabolism through the intracellular mediators phosphatidylinositol 3-kinase and serine-threonine kinase AKT. Phosphatase and a tensin homolog deleted from chromosome 10 (PTEN), a negative regulator of the phosphatidylinositol 3-kinase/AKT pathway, is hypothesized to inhibit the metabolic effects of insulin. Here we report the generation of mice lacking PTEN in adipose tissue. Loss of Pten results in improved systemic glucose tolerance and insulin sensitivity, associated with decreased fasting insulin levels, increased recruitment of the glucose transporter isoform 4 to the cell surface in adipose tissue, and decreased serum resistin levels. Mutant animals also exhibit increased insulin signaling and AMP kinase activity in the liver. Pten mutant mice are resistant to developing streptozotocin-induced diabetes. Adipose-specific Pten deletion, however, does not alter adiposity or plasma fatty acids. Our results demonstrate that in vivo PTEN is a potent negative regulator of insulin signaling and insulin sensitivity in adipose tissue. Furthermore, PTEN may be a promising target for nutritional and/or pharmacological interventions aimed at reversing insulin resistance.

FIG. 1.

Targeted deletion of Pten is adipose tissue specific. (A) PCR analysis performed on tissues from a Pten^loxP/loxP aP2-Cre^+/− mouse. LoxP alleles (1,100 bp) flanking exon 5 of the Pten locus were detected in all tissues except WAT and BAT. The Δ5 band (450 bp) appeared only in WAT, BAT, and skin, which has an adipose component. (B) Western blot analysis of protein extracts from mesenteric fat pads. Blots were hybridized with anti-PTEN (54 kDa, top panel) and anti-actin (40 kDa, bottom panel) antibodies. (C) Immunohistochemical (IHC) staining for PTEN in paraffin-embedded WAT (right panels). Arrows indicate blood vessels. Loss of PTEN status did not disturb the gross morphology of WAT as seen by hematoxylin and eosin (H&E) staining (left panels). Scale bar = 50 μm. Sk muscle, skeletal muscle; Con, control; Mut, mutant; α-PTEN, PTEN antibody.

FIG. 2.

Pten deletion in adipose tissue led to an increase in AKT signaling. (A) Western blot analyses of mesenteric fat pad protein extracts. Blots were probed with anti-P-AKT, IR-β, P-GSK3β, P-FOXO1, and P-FOXO3. (B) Immunofluorescent staining of control (Con) and mutant (Mut) WAT sections with P-AKT antibody (α-pAKT). Scale bar = 50 μm.

FIG. 3.

Pten mutant mice retained normal body weight and fat mass. (A) Body weights of control (Con) and mutant (Mut) mice at 3 weeks, 1 month, and 3 months of age. (B) Fat pad morphology and fat mass. (Left panel) Gross fat pad dissection; (right panel) percent total body fat content, as assessed by NMR spectroscopy (9 control mice and 11 mutant mice). (C) Mean plasma leptin (left panel) and adiponectin (right panel) levels for 12 mice. (D) Plasma triglyceride (left panel) (16 control mice and 18 mutant mice) and NEFA (right panel) (13 control mice and 12 mutant mice) concentrations. Data are presented as means ± SEM.

FIG. 4.

Metabolic indices and glucose and insulin tolerance tests. (A) Intraperitoneal GTT indicated that Pten mutant mice (Mut) (n = 12) were more tolerant of glucose challenge than controls (Con) (n = 11). ITT revealed that mutant mice (n = 13) were more sensitive to insulin challenge than controls (n = 12) and became seriously hypoglycemic (arrows). Data are normalized as percentages of initial blood glucose levels in fasting mice. (B) Blood glucose concentrations in fasting mice (12 control mice; 12 mutant mice) and plasma insulin levels and glucose/insulin ratios in fasting mice (there were 14 mice of each genotype for each assay). Data are presented as means ± SEM. *, P ≤ 0.05.

FIG. 5.

Pten deletion in aP2-expressing cells decreased resistin in adipose tissue and increased insulin and AMP kinase signaling in liver. (A, top panel) Serum resistin concentrations were significantly lower in Pten mutants (Mut) (n = 7) than in controls (Con) (n = 9). (A, bottom left panels) Western blot analysis of mesenteric fat pad protein extracts. Blots were probed with antiresistin (12.5 kDa, top panel) and antiactin (40 kDa, bottom panel) antibodies. (A, bottom right panel) Densitometry data of resistin Western blot analysis. Resistin levels in control samples were considered to be 1 (three mice). (B) Western analysis of insulin signaling and AMP kinase activity in liver protein extracts. WT, wild type.

FIG. 6.

Pten deletion in adipose tissue alters GLUT4 membrane localization in both WAT and skeleton muscles. (Top panels) Western blots of GLUT4 subcellular fractions (total protein, LDM, and PM; 80 μg of protein/lane; run on the same gel) from adipose tissue (A) and skeletal muscle (B). (Bottom three panels) Densitometry data of GLUT4 levels from Western analysis in WAT (A) and skeletal muscle (B). The relative density of control samples at the basal stage is considered to be 1 in all cases. Three mice were used. WT, wild type; Mut, mutant mice; con, control mice.

FIG. 7.

Pten deletion protects mutants from developing STZ-induced diabetes. (A, left panels) Insulin levels in fasting mice. WT, wild-type mice. (A, right panels) Glucose/insulin ratios in control (Con) and mutant (MUT) mice. (B) Hematoxylin and eosin (H&E) staining of paraffin-embedded pancreas (top panels). After the STZ treatment, islets of Langerhans cells are damaged (arrows) and insulin immunoreactivity is markedly reduced (bottom panels; red stain). (C) Blood glucose concentrations of fasting mice (left panel) measured 14 days after STZ treatment demonstrated hyperglycemia in controls and normal glycemia in Pten mutants. The GTT (right panel) measured glucose 14 days after STZ treatment. Scale bar = 50 μm. Each value represents a mean ± SEM. *, P < 0.05; **, P < 0.001, compared with values for control mice. For all experiments, 13 control and 4 mutant mice were used.