Muscle total mTOR protein (A) and mTOR pS2448 (B) in GHRKO (KO) and normal (N) mice fed <i>ad libitum</i> (AL) or 30% calorie restriction (CR) for one year were determined using western blotting.

<p>Bars represent 6–8 animals per phenotype/diet group.</p

Summarizes the effects of CR, or lack thereof, in wildtype and GHRKO mice under insulin-stimulated conditions.

<p>Solid grey proteins represent stimulatory activation, while solid black proteins are inhibitory.</p

Metabolic parameters of male GHRKO (KO) and normal (N) mice fed <i>ad libitum</i> (AL) or subjected to 30% calorie restriction (CR) for one year.

<p>Panel (A) shows time-course changes in body weight over a one year period. After one year of CR, all groups were fasted overnight and bodyweight (B), peripheral glucose (C), insulin (D), and calculated homeostatic model of assessment (HOMA, E) were determined. Values with unlike superscripts/letters are significantly different (P<0.05).</p

Muscle total IRS-1 protein (A) and IRS-1 pS307 inhibitory phosphorylation (B) in GHRKO (KO) and normal (N) mice fed <i>ad libitum</i> (AL) or 30% calorie restriction (CR) for one year was determined using ELISA.

<p>Bars represent 6–8 animals per phenotype/diet group. Values with unlike superscripts/letters are significantly different (P<0.05).</p

Muscle insulin signaling cascade total proteins and phospho-proteins in response to insulin stimulation (10 IU/kg) versus saline treated controls in GHRKO (KO) and normal (N) mice fed <i>ad libitum</i> (AL) or 30% calorie restriction (CR) for one year.

<p>Levels of total insulin receptor (IR; A), IR pY1158 (B), AKT1 (D), and AKT1 pS473 (E) were determined using ELISA. Liver homogenates were immunoprecipitated (IP) with anti-p85, separated using SDS-PAGE and the level of non-specific Tyr phosphorylation was determined at approximately 180 kDa (corresponding to IRS proteins) using anti-pY99 (C). Total AKT2 protein (F) and AKT2 pS473/474 (G) was determined from liver protein homogenates first subjected to IP using anti-AKT2. Total GLUT4 protein (H) was determined in muscle homogenates using anti-GLUT4. Panel (I) summarizes key insulin-stimulated phospho-proteins compared to insulin-stimulated N AL mice. Bands are representative blots from 4–6 male mice per phenotype/diet/treatment group. Values with unlike superscripts/letters are significantly different (P<0.05).</p

Liver PI3K subunit abundance in GHRKO (KO) and normal (N) mice fed <i>ad libitum</i> (AL) or 30% calorie restriction (CR) for one year.

<p>Liver protein isolates were immunoprecipitated (IP) with anti-pan-p85. Total p85α (A), 55α (B) and 50α (C) subunits were separated using SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-pan-p85. Bands are representative blots from 4–6 male mice per phenotype/diet/treatment group. Values with unlike superscripts/letters are significantly different (P<0.05).</p

Liver insulin signaling cascade total proteins and phospho-proteins in response to insulin stimulation (10 IU/kg) versus saline treated controls in GHRKO (KO) and normal (N) mice fed <i>ad libitum</i> (AL) or 30% calorie restricted (CR) for one year.

<p>ELISA with antibodies directed towards total insulin receptor (IR; A), IR pY1158 (B), AKT1 (D), and AKT1 pS473 (E) were performed. Liver homogenates were immunoprecipitated (IP) with anti-p85, separated using SDS-PAGE and the level of non-specific Tyr phosphorylation was determined at approximately 180 kDa using anti-pY99 (C). Total AKT2 protein (F) and AKT2 pS473/474 (G) was determined from liver protein homogenates first subjected to IP using anti-AKT2. Panel (H) summarizes key insulin-stimulated phospho-proteins compared to insulin-stimulated N AL mice. Bands are representative blots from 4–6 male mice per phenotype/diet/treatment group. Values with unlike superscripts/letters are significantly different (P<0.05).</p