Skeletal muscle represents an important site for insulin-stimulated glucose disposal. It is well known that insulin-stimulated muscle glucose uptake is impaired in the presence of inflammatory mediators. While it has been suggested from in vitro
studies that the underlying factors leading to this impairment is defective insulin signaling, the mechanism in vivo
remains to be clearly elucidated 
. The aim of the present study was to use a conscious mouse model to test the hypothesis that there was a significant vascular component to impaired insulin action in vivo
resulting from a LPS challenge. Our findings indicate that the inflammatory stress induced by LPS in the mouse impairs MGU in vivo
without affecting insulin signaling. The impairment in MGU was associated with decreases in muscle blood flow, suggesting that alterations in muscle glucose delivery play a critical role in insulin resistance when the cardiovascular system is altered.
Hyperinsulinemic-euglycemic clamps in chronically catheterized conscious mice were used to determine insulin-stimulated glucose uptake. In initial studies vehicle and high-dose LPS treated mice were both infused with 4 mU·kg−1
of insulin during the clamp. There was no difference in glucose requirements between the groups. However, arterial insulin was two-fold higher in mice that received LPS (5.0 vs. 2.5 ng/ml; LPS vs. vehicle) implying that LPS impaired insulin clearance. In subsequent studies the insulin infusion rate was reduced to 2.5 mU·kg−1
in an attempt to match circulating insulin levels in LPS treated mice to those having received vehicle. Although insulin levels remained somewhat higher, glucose requirements were reduced by 35% and there was a significant decrease in glucose uptake in multiple muscles. When the dose of LPS was decreased to 1 mg/kg impairments in insulin-stimulated MGU were not as robust. These results are consistent with findings in a variety of species 
. Our results suggest that decreased insulin clearance by LPS may serve to normalize insulin-stimulated glucose uptake. A recent study Park et al.
found that during a hyperinsulinemic-euglycemic clamp LPS did not affect insulin action in vivo 
. In the presence of LPS, there was either an increase or no difference in the glucose infusion rate versus animals that received saline. Similar results were seen when they examined whole-body glucose disposal. They found that while LPS lead to a decrease in hepatic glucose production, muscle glucose uptake was unaltered. Interestingly insulin concentrations were not reported 
. Pilon et al.
did observe a decrease in whole body glucose uptake during the hyperinsulinemic-euglycemic clamp 6 h after administering LPS. In contrast to our study, insulin-stimulated glucose uptake by the soleus (other tissues were not evaluated) was markedly decreased however they utilized a higher dose of LPS than our high dose group (20 mg/kg). Like Park et al.
, they also did not report insulin concentrations during the clamp 
. We found that LPS impaired insulin clearance therefore the insulin infusion rate during the clamp was decreased to maintain similar arterial insulin concentrations between groups. Thus, it is likely that in their study impairments in insulin clearance and the consequent increase in arterial insulin concentrations following LPS treatment offset any underlying impairment in insulin stimulated tissue glucose uptake.
Defects in insulin signaling through the PI3-kinase pathway in the muscle during a LPS challenge do not contribute to impaired glucose uptake. Consistent with our previous work LPS treatment increases the plasma levels of inflammatory cytokines 
. de Alvaro et al.
found that in cultured rat skeletal muscle cells TNF-α lead to a decrease in insulin signaling through the IR/IRS-1/AKT pathway 
. Impairments in insulin signaling are mediated in part by increased cytokines and subsequent NFκB pathway activation that occurs in response to LPS 
. We observed an increased expression of iNOS in muscle, which is consistent with recent reports 
. Despite the robust activation of the inflammatory response, Akt phosphorylation in gastrocnemius muscle at the end of the clamp was unaltered by LPS. Pilon et al.
observed a decrease in insulin stimulated IRS-1 associated PI 3-kinase activity in soleus (other muscles were not examined) obtained from control and LPS treated mice (6 h after LPS administration) stimulated ex vivo
with insulin 
. Ex vivo
the vascular-dependent effects of insulin are absent. While in vitro
models can unmask insulin signaling defects, it prevents detection of any vascular dependent alterations in insulin action. An additional consideration is that the impairment in insulin signaling may be dependent upon the time of exposure to the inflammatory environment, the dose of LPS administered, and the specific muscle group examined. Pilon examined insulin action utilizing a 2-fold higher dose of LPS than our high-dose group 
. To assess whether the peak phosphorylation events were missed during the 120 min clamp study, Akt phosphorylation was examined 10 minutes following a bolus of insulin. This experiment confirmed the findings from the insulin clamp studies; LPS administration in vivo
did not blunt insulin signaling in the muscle. Since Akt phosphorylation can be influenced by multiple events, we next looked upstream in the insulin signaling pathway at IRS-1 phosphorylation. We found that LPS administration did not blunt tyrosine phosphorylation nor did it increase serine phosphorylation of IRS-1 compared to vehicle treatment. These data suggest that there is dissociation between insulin-stimulated muscle glucose uptake and phosphorylation of key proteins in the PI3 kinase insulin signaling pathway. This strongly suggests that other factors can limit muscle glucose uptake in vivo
Consistent with the insulin signaling data we found that despite the absolute decrease in MGU the fold increase above basal MGU was unaltered in several tissues. Insulin-stimulated MGU increased ~10 fold above vehicle infused mice irrespective of whether they are given LPS. This suggests that the tissues were very responsive to insulin but that other factors such as a decrease in metabolic demand or vascular delivery of glucose limited the absolute magnitude of glucose uptake. Cardiac output following low-dose LPS was not significantly altered. However, we found that high-dose LPS significantly decreased cardiac output by ~50% and mean arterial blood pressure fell by ~50%. In conjunction with these results, we found that VO2
was significantly decreased with high-dose LPS (~50%) while low-dose LPS (~20%) did not significantly decrease metabolic demand (i.e. VO2
). A correlation between metabolic demand and insulin stimulated glucose uptake is well established as increasing metabolic demand can compensate for underlying insulin resistance 
. This may in fact explain why in humans it has been reported that during endotoxemia insulin can initially improve insulin action 
. The low dose of LPS used in human studies induced modest cardiovascular alterations (tachycardia and mild hypotension). In contrast to the mouse in both the human and pig the dose of LPS used caused hyperthermia, presumably increasing the metabolic rate and limiting cardiovascular disturbances. Therefore depending on the species, the duration of exposure to, and the dose of LPS used, among other factors such as the metabolic rate and associated cardiovascular events, may offset any negative effects of LPS on tissue glucose uptake. Thus, the combined decrease in tissue blood flow and VO2
following LPS may explain the accompanying impairment in MGU seen in our mouse model. Low dose LPS had limited alterations in cardiac output yet they were insulin resistant. This could be due to subtle changes in tissue perfusion that combine with other defects downstream of AKT signaling (e.g. glucose transport or phosphorylation). Our data suggest that the accompanying cardiovascular events should be considered when interpreting the impact of LPS in vivo
Previous studies have shown that increased blood flow leads to increased glucose uptake provided the muscle membrane is sufficiently permeable to glucose, such as it is during insulin stimulation 
. The impairment in MGU following LPS administration could be due to the combined effects of reduced tissue blood flow and/or extraction of glucose. Using PAH and microspheres to measure tissue blood flow we found that after high-dose LPS muscle blood flow was significantly impaired compared to vehicle-treated animals. This would decrease muscle glucose delivery and in the absence of a compensatory increase in fractional glucose extraction, MGU. Blood flow and glucose uptake decreased proportionally in both the SVL and gastrocnemius muscle (~69%), which suggests that fractional glucose extraction was unaltered. In contrast, blood flow to the heart and soleus decreased to a greater extent (~70% and 65%, respectively) than the fall in glucose uptake (~50% and 25%, respectively). This suggests that these tissues had a compensatory increase in fractional extraction of glucose and were able to minimize the decrease in tissue glucose uptake. As fat is the preferred substrate in these tissues they may switch to glucose utilization as the preferred substrate in low flow states. Indeed, studies by Petersen et al.
demonstrated that the extracellular barrier to glucose uptake was lower in Type I muscle fibers 
. Thus tissues with greater oxidative capacity and capillary densities (heart and soleus) have a greater ability to extract glucose in setting of insulin resistance and low flow states.
An inflammatory state can lead to decreased microcirculatory flow velocity and the density of perfused capillaries in rat models both of which are important factors in determining MGU 
. Our data suggests that high-dose LPS decreases blood flow to the muscle which subsequently impairs MGU. This is consistent with data that there is a redistribution of blood flow during inflammatory stress 
. Unlike glucose, decreased muscle insulin delivery would not necessarily result in decreased insulin signaling because insulin is not consumed by the muscle. Therefore the concentration of insulin presented to muscle cells would be unaltered in a setting of decreased tissue blood flow. The lack of difference in IRS and Akt phosphorylation between vehicle- and LPS-treated mice indicates a disassociation of insulin-stimulated glucose uptake and insulin signaling though the PI3 kinase pathway in muscle. Similar results in insulin signaling were observed by Orellana et al.
in the neonatal pig 
. The impairment in tissue blood flow following LPS administration is consistent with studies suggesting that skeletal muscle perfusion could act as an independent determinant of insulin stimulated glucose uptake 
. The brain is not an insulin responsive tissue therefore during a hyperinsulinemic-euglycemic clamp we would not expect a decrease in the glucose uptake. Therefore a fall in brain glucose uptake under these conditions is also consistent with a decrease in tissue blood flow. Pilon et al.
observed that mice lacking iNOS are protected from LPS-induced insulin resistance due to diminished nitrosylation of IRS-1 in the soleus muscle 
. However, an equally plausible explanation is that these mice are protected from the hypotension and the decrease in tissue perfusion that accompanies the excess nitric oxide generation from LPS 
In summary, this study provides evidence that an LPS challenge that impairs cardiovascular function significantly impairs MGU in mice but does not affect the stimulation of insulin signaling in vivo despite the release of inflammatory cytokines. Thus impairments in effective tissue perfusion can contribute to the decreased glucose uptake observed during endotoxemia.