Muscle-specific Pten deletion protects against insulin resistance and diabetes

Abstract

Pten (phosphatase with tensin homology), a dual-specificity phosphatase, is a negative regulator of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Pten regulates a vast array of biological functions including growth, metabolism, and longevity. Although the PI3K/Akt pathway is a key determinant of the insulin-dependent increase in glucose uptake into muscle and adipose cells, the contribution of this pathway in muscle to whole-body glucose homeostasis is unclear. Here we show that muscle-specific deletion of Pten protected mice from insulin resistance and diabetes caused by high-fat feeding. Deletion of muscle Pten resulted in enhanced insulin-stimulated 2-deoxyglucose uptake and Akt phosphorylation in soleus but, surprisingly, not in extensor digitorum longus muscle compared to littermate controls upon high-fat feeding, and these mice were spared from developing hyperinsulinemia and islet hyperplasia. Muscle Pten may be a potential target for treatment or prevention of insulin resistance and diabetes.

FIG. 1.

Targeted deletion of Pten in muscle and general characterization of mckPten^+/+ and mckPten^−/− mice. (A) PCR analysis of Cre-mediated recombination of the Pten locus (Δ4-5, top) and genotyping for the Pten-loxP allele (middle) and cre (bottom) as described previously (2). Results obtained from genomic DNAs of soleus (S) and EDL (E) muscles and tail (T) are shown. (B) Absence of Pten expression in muscle. Western blots of expression of Pten in livers (L), epididymal fat (F), brains (B), and thigh muscles (M) of 6-month-old mckPten^+/+ and mckPten^−/− mice are shown (top). Soleus (S) and EDL (E) muscle types show the same degree of Pten deletion (bottom). (C) Immunohistochemistry of Pten staining in soleus muscles of 6-month-old mckPten^+/+ and mckPten^−/− mice (magnification, ×25). (D) Similar weights of soleus and EDL muscles from 6-month-old mckPten^+/+ (closed bars) and mckPten^−/− (open bars) mice on a high-fat diet (n = 8 to 10 muscles per group; P = NS). (E) Similar weight gains by mckPten^+/+ (closed symbols) and mckPten^−/− (open symbols) mice on a chow (circles) or high-fat (squares) diet (n = 10 to 15 mice per group; P = NS). (F) Similar epididymal fat pad weights from 6-month-old mckPten^+/+ and mckPten^−/− mice after a chow or high-fat diet (P = NS). Error bars indicate standard errors of the means.

FIG. 2.

Protection from insulin resistance and diabetes in high-fat-fed mckPten^−/− mice. (A) Fasting glucose levels of mckPten^+/+ and mckPten^−/− mice on a chow or high-fat diet. *, P < 0.05, comparing mckPten^+/+ and mckPten^−/− mice on a high-fat diet. (B) Insulin tolerance tests were performed on 6-month-old mckPten^+/+ and mckPten^−/− mice as described in Materials and Methods. *, P < 0.05, comparing mckPten^+/+ and mckPten^−/− mice on a high-fat diet. (C) Glucose tolerance tests were performed on 6-month-old mckPten^+/+ and mckPten^−/− mice on a chow or high-fat diet as described in Materials and Methods. * and **, P < 0.01 and P < 0.1, respectively, comparing mckPten^+/+ and mckPten^−/− mice on a high-fat diet. All results are expressed as means ± standard errors of the means from at least 10 animals of each genotype.

FIG. 3.

Enhanced insulin-stimulated 2-deoxyglucose uptake into isolated soleus muscles of high-fat-fed mckPten^−/− mice. (A and B) Soleus or (C) EDL muscles isolated from 6-month-old mice fed (A) chow or (B and C) high fat were incubated with or without 2 mU of insulin per ml for 30 min. 2-Deoxyglucose uptake was measured over 20 min. Results are the means ± standard errors from four independent experiments. All values are normalized relative to the lowest 2-deoxyglucose uptake value in order to express all values as greater than one.

FIG. 4.

Enhanced insulin-stimulated Akt phosphorylation in isolated soleus muscles of high-fat-fed mckPten^−/− mice. Soleus muscles isolated from 6-month-old mice fed (A) chow or (B) high fat were incubated with or without 2 mU of insulin per ml for 10 min. (C) Lysates (30 μg) were resolved by SDS-10% PAGE and immunoblotted with anti-phospho-T308 Akt or anti-pan-Akt antibody. Representative immunoblots are shown. Immunoblots were scanned within the linear range and quantified with NIH Image J software. The quantified values represent the means ± standard errors from five independent experiments. All values are normalized relative to basal Akt phosphorylation in mckPten^+/+ mice.

FIG. 5.

Increased glycogen as measured by PAS staining in soleus muscles of mckPten^−/− mice compared to mckPten^+/+ mice. There is no difference in PAS staining in EDL muscles between the two genotypes. Overnight-fasted mice (high-fat fed, 6 months old) were injected with glucose (1 g/kg) and insulin (1.5 U/kg) and sacrificed at 3 h postinjection.

FIG. 6.

Continued protection from insulin resistance and diabetes in older mckPten^−/− mice on prolonged high-fat feeding. (A) Insulin tolerance tests were performed on 15-month-old mckPten^+/− and mckPten^−/− mice on a chow or high-fat diet as described in Materials and Methods. (B) Glucose tolerance tests were performed on 15-month-old mckPten^+/− and mckPten^−/− mice on a chow or high-fat diet as described in Materials and Methods. * and **, P < 0.01 and P < 0.1, respectively, comparing mckPten^+/− and mckPten^−/− mice on a high-fat diet. All results are expressed as means ± standard errors of the means from at least 10 animals of each genotype.

FIG.7.

Insulin-stimulated 2-deoxyglucose uptake into isolated soleus muscle and Akt phosphorylation in prolonged high-fat feeding. (A) Soleus (left panel) or EDL (right panel) muscles isolated from 15-month-old mice fed a high-fat diet were incubated with or without 2 mU of insulin per ml for 30 min. 2-Deoxyglucose uptake was determined over 20 min. Results are the means ± standard errors from five independent experiments. (B) Net insulin effect on 2-deoxyglucose uptake in both muscles. (C) Soleus (left panel) or EDL (right panel) muscles isolated from 15-month-old mice fed a high-fat diet were incubated with or without 2 mU of insulin per ml for 10 min. Lysates (30 μg) were resolved by SDS-10% PAGE and immunoblotted with anti-phospho-T308 Akt-antibody. Representative immunoblots are shown (upper panels). Immunoblots were scanned within the linear range and quantified with the NIH image computer software. The quantified values (lower panels) represent the means ± standard errors from five experiments. All values are expressed relative to basal Akt phosphorylation in mckPten^+/− mice, which was assigned a value of 1. (D) Lysates (25 μg) of isolated muscles were resolved by SDS-10% PAGE and immunoblotted with anti-Akt. A representative immunoblot is shown.

FIG. 8.

Protection from hyperinsulinemia and islet hyperplasia in high-fat-fed mckPten^−/− mice. (A) Fasting plasma insulin levels. After an overnight fast, plasma insulin levels in venous blood samples from 6-month-old mckPten^+/+ and mckPten^−/− mice fed a chow (left two columns) or high-fat diet (right two columns) were determined. Dashed lines represent arbitrary cutoff levels for comparative analysis. (B) Sparing of islet hyperplasia in mckPten^−/− mice fed a high-fat diet. Representative hematoxylin- and eosin-stained histological sections of pancreata from 6-month-old mckPten^+/+ and mckPten^−/− mice fed a chow or high-fat diet are shown.