Creation of transgenic mice overexpressing SOCS3 in skeletal muscle.

<p>(<b>A</b>) The SOCS3 transgene construct. Transgenic mice (SOCS3) were crossed with MCK-Cre mice to generate mice over-expressing SOCS3 specifically in skeletal muscle (SOCS3/MCK). (<b>B</b>) SOCS3 mRNA is increased in SOCS3/MCK mice compared to SOCS3 mice. SOCS3 mRNA in various types of skeletal muscle was measured by real-time RT PCR. Gas: gastrocnemius; Sol: soleus; EDL: extensor digitorum longus; TA: tibialis anterior. n = 4. (C) The tag protein GFP and the transgene-encoded protein SOCS3-HA are expressed in gastrocnemius skeletal muscle. The proteins were detected by immunoblotting using specific antibodies.</p

Mice with skeletal muscle over-expression of SOCS3 have suppressed basal AMPK signaling.

<p>(<b>A</b>) α2AMPK activity, (<b>B</b>) AMPK signaling, (<b>C</b>) expression of genes involved in fatty acid oxidation, (<b>D</b>) β-hydroxyacyl-CoA dehydrogenase activity (HADH), (<b>E</b>) citrate synthase activity (CS), and (<b>F</b>) COXI DNA content in skeletal muscle of SOCS3/MCK and control mice on a chow diet. Soleus, EDL or gastrocnemius muscles from 5-month-old chow-fed male mice were evaluated for AMPK activity using an immune complex assay. Phosphorylation and total protein levels of AMPK and ACC were measured by immunoblotting. Gene expression was measured by Real-time RT-PCR and corrected with cyclophilin levels. HADA and CS activities were determined spectrophotometrically using skeletal muscle (gastrocnemius) homogenates. Mitochondrial DNA content was measured in muscle (gastrocnemius) DNA samples by Real-time PCR using primer/probe sets corresponding to mitochondrial DNA-encoded cytochrome c oxidase subunit I (COXI) gene and normalized with nuclear DNA-encoded gene UCP2. Data are expressed as Mean ± SE, n = 6–8. *p<0.05 vs. control SOCS3 mice in (<b>A</b>), (<b>C</b>), (<b>D</b>), and (<b>F</b>); p = 0.05 vs. control SOCS3 mice in (<b>E</b>).</p

SOCS3 mRNA is elevated in skeletal muscle of diet-induced obese (A), <i>ob/ob</i> (B), TNFα-injected (C) and lipid-infused (D) mice.

<p>Total RNA was isolated from gastrocnemius and SOCS3 RNA levels were measured by quantitative real-time RT-PCR. (<b>E</b>). Over-expressing SOCS3 in C2C12 myotubes inhibits insulin-stimulated glucose uptake. C2C12 myotubes were treated with or without 100 nM insulin for 20 min. Data are expressed as mean ± SE, n = 6–8. *p<0.05. A.U.: arbitrary units.</p

Skeletal muscle SOCS3 over-expression impairs systemic insulin sensitivity.

<p>(<b>A</b>) Blood glucose levels (left panel) and blood insulin levels (right panel) in MCK/SOCS3 and control mice fed a low fat chow diet. (<b>B</b>): ITTs (left) and GTTs (right) in MCK/SOCS3 and control mice. ITTs and GTTs were performed on mice fed a chow diet as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047493#s4" target="_blank">Materials and Methods</a>. (<b>C</b>) Glucose infusion rate, insulin-stimulated glucose turnover, hepatic glucose production under clamp, and insulin-stimulated glucose uptake in gastrocnemius/soleus muscle and epididymal fat. Hyperinsulinemic-euglycemic clamps were conducted in MCK/SOCS3 and control mice fed a chow diet as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047493#s4" target="_blank">Materials and Methods</a>. Data are expressed as mean ± SE; n = 5, *p<0.05 vs. control SOCS3 mice.</p

Skeletal muscle SOCS3 over-expression blocks insulin signaling in skeletal muscle.

<p>5-month old mice on chow diet were intraperitoneally injected with 10 units/kg body weight of human insulin or saline. 10 min after injection, gastrocnemius muscle strips were dissected and saved for immunoprecipitation and immunoblotting analysis of insulin signaling molecules as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047493#s4" target="_blank">Materials and Methods</a>.</p

MnTBAP treatment reduces caloric intake in mice fed a HFD.

<p><b>(A)</b> Caloric intake was assessed at various time points of MnTBAP treatment in mice previously fed a LFD or HFD for 5 months. <b>(B)</b> Correlation between the change in body weight and the change in caloric intake from pre-treatment to post-treatment. *, Denotes statistically significant difference from HFD-Vehicle. ⁺, Denotes statistically significant difference from respective LFD mice. P-value for post-hoc analysis for LFD-Vehicle vs. HFD-Vehicle mice: Day 9, P = 0.057; Day 17, P = 0.085. N = 6–8 mice per group.</p

MnTBAP treatment improves insulin-assisted glucose tolerance (IAGT) in mice fed a HFD.

<p>Mice were simultaneously injected with 2.0 U/Kg insulin and 2.0 g/Kg glucose and blood glucose values were assessed at baseline and 20, 40, and 60 min following the injection (Panels A) and the area under the IAGT curve was calculated (Panels B). *, Denotes statistically significant difference from HFD-Vehicle. ⁺, Denotes statistically significant difference from LFD-Vehicle mice. N = 7–14 mice per group.</p

MnTBAP treatment increase insulin-stimulated PKB phosphorylation and content in muscles from mice fed a HFD.

<p>Mice were fed a LFD or HFD for 5 months and then treated with or without MnTBAP (10 mg/kg) daily for 5 weeks. At 15 min following an intraperitoneal injection of insulin (2 U/Kg), quadriceps muscles were excised and rapidly frozen in liquid nitrogen for subsequent Western blot analyses. <b>(A)</b> Representative PKB immunoblots for pThr³⁰⁸ and pSer⁴⁷³ PKB as well as total PKB-α and PKB-β isoforms. GAPDH was used as a loading control. Quantification of <b>(B)</b> pThr³⁰⁸ PKB, <b>(C)</b> pSer³⁰⁸ PKB, <b>(D)</b> PKB-α and <b>(E)</b> PKB-β content, each normalized to GAPDH. *, Denotes statistically significant difference from HFD-Vehicle. N = 5–6 mice per group.</p

SnMP improves MnTBAP’s ability to reduce blood glucose levels during IAGT tests.

<p>Mice were fed a LFD or HFD for 5 months and then treated with MnTBAP (10 mg/kg) alone or in combination with SnMP (20 mg/Kg) daily for 5 weeks. Mice were simultaneously injected with 2.0 U/Kg insulin and 2.0 g/Kg glucose and blood glucose values were assessed at baseline and 20, 40, and 60 min following the injection (Panels A) and the area under the IAGT curve was calculated (Panels B). *, Denotes statistically significant difference from HFD-Vehicle. ⁺, Denotes statistically significant difference from LFD-Vehicle mice. ^#, Denotes statistically significant difference from HFD-MnTBAP mice. N = 5 mice per group.</p

MnTBAP treatment alters insulin-stimulated PKB phosphorylation but not content in EWAT from mice fed a HFD.

<p>Mice were fed a LFD or HFD for 5 months and then treated with or without MnTBAP (10 mg/kg) daily for 5 weeks. At 15 min following an intraperitoneal injection of insulin (2 U/Kg), EWAT was excised and rapidly frozen in liquid nitrogen for subsequent Western blot experiments. <b>(A)</b> Representative PKB immunoblots for pThr³⁰⁸ and pSer⁴⁷³ PKB as well as total PKB-α and PKB-β isoforms. α-tubulin was used as a loading control. Quantification of <b>(B)</b> pThr³⁰⁸ PKB, <b>(C)</b> pSer³⁰⁸ PKB, <b>(D)</b> PKB-α and <b>(E)</b> PKB-β content, each normalized to α-tubulin. *, Denotes statistically significant difference from HFD-Vehicle. ⁺, Denotes statistically significant difference from LFD-MnTBAP. N = 5–6 mice per group.</p