The main findings of the current study implicate, for the first time, attenuation of insulin stimulation of the p42/p44 MAP kinase pathway as an early defect in obesity-induced IR in skeletal muscle. We observed an impaired insulin-induced stimulation of skeletal muscle p42/p44 MAP kinase activity, in those individuals with higher BMI or lower whole body insulin sensitivity (M value) i.e. obese, IR individuals. Consistent with this finding, in the same individuals, we observed blunting of insulin-induced phosphorylation of p42/p44 MAP kinase at the regulatory residues (Tyr185, Thr183). Importantly, this signalling defect only became apparent when assessing the magnitude of response to insulin, and was not detectable by measuring p42/p44 MAP kinase activity or phosphorylation, in the post-absorptive state, without insulin stimulation. Our findings also suggest that multiple molecular mechanisms may mediate skeletal muscle IR within different overweight or obese individuals. Although defective p42/p44 MAP kinase signalling was the most prevalent in the volunteers studied there was clear evidence of defects in other signalling proteins including IRS1 and PKB in several individuals.
These observations are entirely consistent with previous analyses from this group demonstrating an association of p42/p44 MAP kinase signalling with stimulation of muscle glucose transport 
as well as a defective regulation of the p42/p44 MAP kinase pathway in different pathophysiological conditions associated with skeletal muscle IR and reduced glucose transport. First, in young women with PCOS, there was a severe attenuation of insulin stimulation of the p42/p44 MAP kinase pathway in muscle compared to controls (with p42/p44 MAP kinase activity actually reducing in response to insulin) although the group was too small to detect any correlation between BMI and the defective regulation of p42/p44 MAP kinase in the PCOS group 
. Others have reported that the p42/p44 MAP kinase pathway is constitutively activated both in vitro and in vivo in the skeletal muscle of women with PCOS 
. Similarly, in older healthy subjects and in patients with T2DM, in whom skeletal muscle uptake of 2-deoxyglucose was blunted compared with healthy young men, reduced stimulation of p42/44 MAP kinase phosphorylation was observed 
. In contrast, Cusi et al
reported that insulin stimulation of the p42/p44 MAP kinase was normal in obese and diabetic subjects 
. Furthermore, Jager et al
demonstrated that specific inactivation of p44 MAP kinase in obese, leptin deficient mice protected them against insulin resistance despite massive obesity. These animals exhibited relatively good whole-body insulin sensitivity and increased insulin action in skeletal muscle compared to control animals 
. However all of the studies suggest that this pathway exerts control over insulin action, where chronic deletion may generate compensatory insulin sensitising mechanisms but the initial loss of insulin induction of p42/p44 MAP kinase may be a marker of defective insulin action in muscle in response to obesity.
Others have suggested that defective IRS1 or IRS2 signalling is present in muscle of patients with T2DM. Supporting this hypothesis, a genetic variant near IRS1, that is associated with reduced basal levels of IRS1 protein and decreased insulin induction of IRS1-associated PI-3K activity in human skeletal muscle biopsies, is associated with type 2 diabetes, insulin resistance and hyperinsulinemia 
. We have previously reported a significant increase in IRS1 protein expression following acute insulin treatment of human muscle 
; however the fold induction of IRS1 expression in response to insulin in this study was not correlated with either BMI or M value. We cannot rule out abnormalities in one or more of the many post translational modifications of this protein (or its homologue IRS2), however we have focussed on distal signalling mechanisms where deficits in IRS1 function would still be detectable. For example, a mutation in PKB beta has been found to associate with severe IR and lipodystrophy, demonstrating the importance of the IRS-PI3K-PKB pathway to insulin sensitivity 
, although mutations in this protein appear to contribute to only a very small fraction of IR in the population 
. Our data suggest that there are relatively few cases of defective IRS1-PKB signalling that correlate with obesity induced insulin resistance in an otherwise healthy population. We measured protein expression, the phosphorylation (at a residue known to regulate activity in response to insulin) and where possible the inherent activity of PKB, p42/p44 MAPK GSK3, FOXO1 and p70S6K. Although we could not detect abnormalities in PKB activation by insulin, there was an indication that the phosphorylation of PKB at Ser473 may be higher in the muscle of the more insulin sensitive group, at least after exposure to insulin, although the differences were not significant. Indeed, the dissociation of whole body IR from defects in proximal insulin signaling in obese volunteers that we observe are also consistent with those observations of several other groups 
. GSK3 and FOXO are more distal downstream targets of PKB while S6 protein is regulated by the mTOR pathway 
. No significant differences were observed in basal or insulin-induced phosphorylation of these molecules. This provides added confidence that the PI-3K pathway is responding to insulin in a similar fashion across the population studied.
Despite the correlation of defective p42/p44 MAP kinase activation and poor insulin sensitivity there were still some obese, insulin resistant individuals in whom p42/p44 MAP kinase could readily be activated in response to insulin (). However, in all of the most insulin resistant subjects at least one major signalling defect in their muscle was evident, when assessed as response to insulin (but not when examined as activity or expression of a signalling molecule in the post-absorptive state
). However, the individuals with the greatest induction of these molecules tended to have lower BMI and higher M-values, and conversely the subjects with poorest responses to insulin at the molecular level generally also had low M-values and high BMI scores (). It is worth noting that the 10 individuals with lowest whole body insulin sensitivity had complete loss of insulin induction of at least one of the major signalling molecules. However it was not always the same molecular defect: for example in subjects 15, 5 and 8, p42/p44 MAP kinase activity was suppressed (instead of increased) in response to insulin, while subject 22 exhibited no insulin induction of PKB phosphorylation or IRS1 protein (despite strong induction of p42/p44 MAPK phosphorylation and activity). It is not immediately obvious why different signalling defects should arise in a relatively healthy obese population, however it may be related to dietary variations with different compositions of fatty acids altering signalling in different ways 
, or other lifestyle factors not apparent in our study. This aspect, as well as establishing whether one signalling defect is more liable to promote diabetes, deserves further investigation.
In summary, aberrant p42/p44 MAPK signalling was the most common problem found in obesity-induced insulin resistant skeletal muscle. However, multiple defects in insulin signal transduction were apparent in this group and it will be of interest to establish whether the p42/p44 MAPK defect is associated with progression to T2DM.