Role of G_i/o proteins, Gβγ dimers and PI3K in Akt phosphorylation in response to ghrelin.

<p>A, Time-course of the effect of ghrelin on Akt HM (S473) and A-loop (T308) phosphorylation. B, Ghrelin-induced Akt phosphorylation in the absence or presence of PTX (100 ng/mL, 12 h), PI3K inhibitor wortmannin (1 µM, 30 min) and βγ sequester β-ARK-CT. In A and B, serum-starved HEK-GHSR1a cells were treated with ghrelin (100 nM) at 37°C for the indicated times. Cells were lysed and analyzed by SDS-PAGE using specific antibodies. Akt phosphorylation was quantified by densitometry and expressed as a percentage of the maximal phosphorylation obtained for each residue (mean±S.E. of three independent experiments). Blots are representative of three independent experiments.</p

c-Src is required for phosphorylation of Akt in response to ghrelin.

<p>A, Effect of siRNA depletion of c-Src on ghrelin-induced Akt phosphorylation. HEK-GHSR1a cells transfected with c-Src siRNA were serum-starved for 12 h and then stimulated with ghrelin (100 nM) at 37°C. After stimulation, equal amounts of protein in each sample were used to assess the expression of c-Src (left panel) and Akt phosphorylation (right panel) by western blotting. Expression of c-Src was quantified by densitometry and expressed as percentages of the level of c-Src in control siRNA-transfected cells (mean±S.E.). Akt phosphorylation was quantified by densitometry and expressed as a percentage of the maximal phosphorylation at HM (S473) and A-loop (T308) after ghrelin addition to control siRNA-transfected cells (mean±S.E.). B, Effect of ghrelin on Y phosphorylation of Akt and interaction between Akt and c-Src. Cells were incubated with ghrelin (100 nM, 5 min) at 37°C, lysed and immunoprecipitated (IP) with antibodies to Akt, and then analyzed by western blotting with pAkt HM(S473), pY, pSrc (Y416) antibodies. C, Effect of ghrelin on Y phosphorylation of Akt in the presence of c-Src siRNA. HEK-GHSR1a cells transfected with c-Src siRNA were serum-starved for 12 h and then stimulated with ghrelin (100 nM, 5 min) at 37°C. Equal amounts of protein in each sample were used to assess the expression of c-Src [upper panel; values shown (mean±S.E.) are percentages of the level of c-Src in control siRNA-transfected cells]. Cells were lysed and immunoprecipitated (IP) with antibodies to Akt, and then analyzed by western blotting with with pAkt HM(S473), pY, pSrc (Y416) antibodies. Immunoblots are representative of three independent experiments.</p

Mean differences of cognitive scales after 1 year of follow-up between control and intervention group and stratified by nutritional status.

<p>Mean differences of cognitive scales after 1 year of follow-up between control and intervention group and stratified by nutritional status.</p

Omega3_cognitionUntitled Item

Dataset for the placebo-controlled double-blind randomized trial of a supplementation rich in omega-3 PUFA in patients aged 75 or more years. <div><br></div><div>PLEASE BEFORE USING THIS DATA SET CONTACT TO MAIRA BES-RASTROLLO ([email protected])</div

Adjusted^* mean differences of cognitive scales after 1 year of follow-up between control and intervention group.

<p>Adjusted^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193568#t004fn001" target="_blank">*</a>} mean differences of cognitive scales after 1 year of follow-up between control and intervention group.</p

Flow chart of participants.

<p>Flow chart of participants.</p

mRNA expression levels measured by real-time PCR for ghrelin in <i>ad libitum</i> fed animals (A) or 36-hour fasted animals (B).

<p>Ghrelin protein levels and representative Western blot from the mucosa from <i>ad libitum</i> fed animals (C) or 36-hours fasted animals (D). Animals that received different in vivo treatments (control/rimonabant) and surgical procedures (vagotomy/sham-operated). C: control, R: rimonabant, V: vagotomy; V+R: vagotomy+rimonabant. CB1 receptor mRNA levels in the gastric mucosae of <i>ad libitum</i> fed animals (E) or 36-hour fasted animals (F) and animals that received different in vivo treatments (control/rimonabant) and surgical procedures (vagotomy/sham operated). *vs control.</p

Food Intake measures in rats after icv treatment with ghrelin/vehicle (6 µg/rat) and ip treatment with rimonabant (3 mg/kg ip)/vehicle under different nutritional statuses: A) <i>Ad libitum</i> fed, B) nocturnal fasting, c) animals subjected to overnight fasting and previously subjected to surgical vagotomy.

<p>*vs control, ^#vs. rimonabant+ghrelin.</p

Measures of pmTOR/mTOR in the gastric mucosae and representative Western blots from animals in the fasting state treated with ip AM281 or vehicle (A).

<p>Phospho-S6K1/S6K1 in the gastric mucosae and representative Western blots from animals in the fasting state treated with ip AM281 or vehicle (control) (B). The results are expressed as percentage over control (phospho-S6K1/S6K1). *p<0.05.</p

Gastric ghrelin secretion from tissue explants (A) and plasma ghrelin levels (B) obtained from 36-hour fasted animals that received in vivo treatment with AM281 (3 mg/Kg ip).

<p>**p<0.01.</p