All subjects had normal glucose tolerance and whole-body insulin sensitivity and were matched for physical fitness. Muscle biopsies were obtained prior to and following a 4-hour hyperinsulinemic euglycemic clamp and expression, and phosphorylation of key insulin signaling proteins was determined by Western blotting. We have previously shown that young and healthy, low birth weight men, exhibit multiple abnormalities in their skeletal muscle in the insulin signalling metabolic pathway downstream of the insulin receptor, under fasting conditions. In this study we aimed to investigate whether any further alterations in either mitogenic or metabolic pathways downstream of insulin receptor can be detected in skeletal muscle in response to in vivo insulin stimulation that could explain why are LBW men at higher risk of developing insulin resistance and type 2 diabetes in later life. The main finding of this study is marked insulin-mediated up-regulation in LBW subjects of the PI3K p85a and p110b subunits and reduced expression/phosphorylation of its two main effectors, PKCf and phospho-Akt at Ser 473, both of which are required for insulin-stimulated glucose uptake. Previously, we have reported reduced abundance of p85a, p110b, PKCf and GLUT4 proteins in skeletal muscle of LBW men under fasting conditions. Similar reductions of p85a, p110b and GLUT4 were observed in subcutaneous abdominal fat biopsies from an independent LBW cohort, together with a marked reduction of IRS-1. IRS molecules are key WZ8040 mediators of insulin signalling and play a central role in maintaining basic cellular functions such as growth, survival and metabolism. Using siRNA – mediated knockdown of IRS proteins, it was found that IRS-1, rather than IRS-2 is required for insulin-stimulated Akt1 phosphorylation, GLUT4 translocation and glucose uptake. Here we show that skeletal muscle IRS-1 expression was similar in control and LBW men. This is consistent with previous reports of normal IRS1 expression in the skeletal muscle from diabetic subjects. We also show that insulin-stimulated expression of IRS-1 was increased in LBW men but not in controls. Recent studies in L6 myotubes showed that, prior to its down-regulation by degradation, IRS-1 protein is acutely induced by insulin stimulation. Thus, the inappropriately increased IRS-1 in LBW at 4 h of insulin stimulation may be a consequence of delayed/protracted stimulation and/or delayed degradation. Despite lower expression in the basal state, insulin stimulation increased expression of PI3K subunits p110b and p85a in LBW subjects but not in controls. The mechanistic basis of this paradoxical increase following insulin infusion is unknown. Increased expression of p85a has been implicated in insulin resistance as knockout of p85a improves insulin sensitivity in vitro and in vivo. Conversely, over-expression of p85a is correlated with skeletal muscle insulin resistance in obesity and type 2 diabetes and has been reported in insulin-resistant states induced by e.g. growth hormone R428 excess and short-term overfeeding. It is thought that excess p85a may exert these effects by sequestration of IRS-1 and PI3K enzymatic activity into inert cellular foci incapable of PI-3, 4, 5-triphosphate generation. PI3K is thought to mediate many of its metabolic actions of insulin through phosphorylation of Akt. Phosphorylation of Akt at Ser473 was reduced in the LBW group compared to controls following insulin infusion, suggesting impaired PI3K activity. Limited sample availability meant that actual activity could not be determined. Decreased insulin stimulated Akt kinase activity has been previously reported in type 2 diabetic subject and non-obese spontaneously diabetic Goto-Kakizaki rats. As well as a reduction in PI3K activity, reduced Akt1 expression could also contribute to reduction in phospho-Akt detected in the insulin stimulated LBW muscle. However, Akt2 is the prominent isoform in skeletal muscle thus phosphorylated Akt detected primarily represents phospho-Akt2. Our findings of no difference in the basal and insulin stimulated expression of Akt2 between control and LBW confirm previous finding that insulin stimulation has no effect on total Akt expression. Knockout and siRNA studies have revealed that whereas Akt2 is indispensable for glucose homeostasis, Akt1 is essential for growth and may play a role in lipid metabolism.