Figure 1. Ser-318 phosphorylation in mouse muscle and human peripheral mononuclear cells. A) Western blot analysis of Ser-318 and Irs1 in skeletal muscle tissue from C57Bl/6 mice weaned on a high fat (HFD) or low fat (LFD) diet for 60 days. Animals were stimulated intravenously (iv) with insulin or leptin. Each lane was loaded with total protein from one animal, and is representative of 3 independent experiments. B) Peripheral blood mononuclear cells were isolated from human blood at the beginning (basal) and at the end (clamp) of an euglycemic hyperinsulinemic clamp. Lysates were immunoblotted with anti-Ser-318 and anti-Irs1 antibodies. Data represent 2 out of 47 individuals tested. Relationship of basal Ser-318 phosphorylation (C) with age and percentage of body fat (D) in mononuclear cells isolated from human subjects. E, F) Relationship of Ser-318 phosphorylation (fold increase over basal) in human lymphocytes with increment in plasma insulin during an euglycemic hyperinsulinemic clamp (steady state-basal) adjusted for age and percentage of body fat in lean (E, lower quartile of percentage of body fat) and obese (F, upper quartile of percentage of body fat) human subjects.