The present work extends previous studies showing firstly that decreased glucose uptake in muscle in response to 5 h of hyperglycemia is not accompanied by defects in insulin signaling in skeletal muscle [6
] and secondly that suppression in AMPK activity occurs in this model [7
]. We confirm these findings but go on to make further novel findings that help to characterize the early onset of insulin resistance in skeletal muscle in response to excess glucose availability. We demonstrate that the AMPK activity suppression precedes the onset of insulin resistance and occurs in concert with activation of the mTOR pathway, also involved in nutrient signaling. By prolonging the hyperglycemia beyond 5 h, a further reduction in red quadriceps muscle glucose uptake occurs. This took place with a selective perturbation of one component of the insulin signaling pathway, namely a 50 % reduction in the phosphorylation of Akt at Ser473
(a key site for maximal kinase activity). Interestingly, this reduction is not associated with decreases in either GSK3 or AS160 phosphorylation in vivo
and the stimulation of glucose transport by insulin in isolated soleus muscle from these rats was not diminished. In addition, by extending the infusion to 8 h we show that while similar metabolic changes occur in white muscle (quadriceps) to that in red muscle, these changes are delayed (significant after 8 h but not 5 h infusion). It is noteworthy that this occurs without significantly altered Akt phosphorylation that may occur later. Our studies do not establish causality among the early muscle AMPK and mTOR changes and later insulin resistance. However, based on the time course data obtained here, taken with a recent in vitro study where insulin resistance is prevented by maintenance of AMPK activity in the presence of glucose elevation [15
], we hypothesize that AMPK plays an important regulatory role influencing insulin potency in response to increased nutrient availability.
Under conditions of glucose oversupply, in the presence of insulin (e.g. during a glucose infusion in vivo), one mediator of decreased muscle glucose metabolism may be the degree of accumulation of muscle glycogen. Here we confirm previous reports demonstrating that muscle glycogen levels correlate negatively with glucose uptake () [16
]. While the precise mechanism for this remains uncertain, a number of reports attribute this to either inhibition of glucose transport, hexokinase and/or glycogen synthase activity [18
]. Hyperglycemia has been shown to lead to a shift of the rate limiting step from glucose transport to post-transport steps [21
AMPK 2 but not AMPK 1 activity was decreased in both red and white muscle prior to any decrease in glucose uptake and any changes to the insulin signaling pathway (). It recently has been hypothesized that when the pool of muscle glycogen is enlarged, more AMPK is bound via its glycogen binding domain to glycogen where it phosphorylates glycogen synthase on site 2, decreasing its activity [14
]. Since glucose incorporation into muscle glycogen is perhaps its major fate in a rat infused with glucose, a decrease in glycogen synthase activity could have been a major contributor to the decreased glucose uptake seen here.
A test of the role of suppression of AMPK activity on subsequent insulin resistance could conceivably be made by interventional studies such as concomitant AICAR infusion. This was investigated in pilot studies (not shown) but these confirmed that due to the inhibitory effects of AMPK activation on the pancreatic -cell to release insulin [22
], systemic infusion of AICAR cannot easily be interpreted in the current context. However, studies in isolated muscle strips have shown co-incubation in high glucose (+/− insulin) and AICAR can reverse the decreased AMPK activity, Akt phosphorylation [15
] and the decreased glucose uptake [24
] associated with hyperglycaemia. These studies, combined with our own data, suggest a role for AMPK in the development of insulin resistance due to hyperglycemia.
The present study indicates that decreased Akt phosphorylation is a relatively late marker of the decrease in glucose uptake by the red quadriceps muscle since it developed between 5 and 8 h of glucose infusion. Akt needs to be phosphorylated on two sites for full activation [25
]. Thus, the decrease in its phosphorylation at Ser473
should theoretically have been sufficient to decrease the kinase activity of the enzyme. However, there was no change in the phosphorylation state of two well described targets of Akt, GSK-3 or AS160 (). Furthermore, insulin stimulated glucose transport in isolated soleus muscle strips was unaltered at 8 h glucose infusion (), indicating that the glucose transport step is unaltered. Thus decreased Akt phosphorylation of the magnitude observed may not be responsible for the further decline in glucose uptake during the 5–8 h time period. The non-linearity of Akt phosphorylation and downstream responses has support in the literature both from cell and animal studies. Whitehead et al
] showed that in cultured adipocytes, only 20 % of the total Akt pool needs to be phosphorylated for maximal glucose uptake (as measured by 2DG uptake) to occur. More recently, Hoehn and colleagues [27
] showed that in L6 myotubes, GLUT4 translocation is maximal despite as little as 5 % of the Akt pool being phosphorylated. In rats, 3 days of high fat feeding caused adiponectin resistance in skeletal muscle and this occurred with a decrease in Akt Ser473
phosphorylation but without changes in glucose uptake [28
]. An important point from the current study is that although we observed a defect in Akt phosphorylation, the level was still a 2-fold increase over basal and appeared sufficient to maintain the ability of insulin to stimulate glucose transport.
In the red quadriceps we have shown an increased Tyr612
phosphorylation of IRS-1 from 3 h of glucose infusion (). We also show an increase in phosphorylation of mTOR and p70S6K at 5 h glucose infusion when the insulin resistance is occurring (). mTOR, and its downstream target p70S6K, are nutrient sensors that are known to negatively feedback on the insulin signaling cascade by phosphorylating IRS-1 on Ser307
]. This is interesting as multiple studies have shown that serine phosphorylation can interfere with subsequent tyrosine phosphorylation, disrupting signal transduction [32
]. However, a recent study by Paul et al
] showed a dissociation between tyrosine and serine phosphorylation. That is, animals fed a high fat diet had restored IRS-1 total tyrosine phosphorylation after a single bout of exercise and this occurred without a reduction in the increased Ser307
]. Hence it may be that under certain circumstances, as in the current study, there isn’t a linear relationship between serine and tyrosine phosphorylation of IRS-1. Lastly, the reciprocal variation between AMPK 2 activity and elevated mTOR and p70S6K, approximately coincident in time, supports a possible inverse cyclic interaction between these two pathways, as supported by recent genetic downregulation studies of S6 kinase [35
Due to the lack of alterations in the phosphorylation state of proximal signaling intermediates (IR, IRS-1) it was unclear what caused the decreased Akt phosphorylation observed in the RQ after 8 h of glucose infusion. The selectivity of this defect at Akt is similar to that seen in cells treated with palmitate, or ceramide [36
]. We observed a ~50 % increase in mean ceramide content in red gastrocnemius in the current study; however, neither the increase in ceramide nor its correlation with Akt phosphorylation was statistically significant.
Our data indicates that white glycolytic muscle may be less susceptible to the onset of insulin resistance than red oxidative muscle. White quadriceps showed a significant delay in development of insulin resistance compared to red quadriceps (after 8 vs 5 h infusion). This is consistent with less glucose uptake into white muscle under equivalent conditions of insulin elevation [9
]. Importantly and consistent with this finding was a delay in suppression of AMPK 2 activity in white versus red quadriceps muscle.
In conclusion, this study adds to the growing body of literature [6
] that suggests that defects in the insulin signaling cascade and glucose transport are not always associated with impaired glucose uptake and metabolism in skeletal muscle. Thus other mechanisms appear to be responsible for the decrease in muscle glucose uptake and glycogen storage during the early stages of a glucose infusion (i.e. prolonged hyperglycemia and hyperinsulinemia). The results suggest that reduced AMPK activity and increased glycogen are possible contributors to these mechanisms.