[Effects of insulin receptor substrate-1 and its serine phosphorylation and tyrosine phosphorylation on insulin resistance in skeletal muscle cells in the state of sepsis: experiment with rats].
OBJECTIVE
To investigate the effects of insulin receptor substrate (IRS)-1 and its serine (Ser)(307) phosphorylation and tyrosine (Tyr) phosphorylation on insulin resistance in skeletal muscle cells in the state of sepsis.


METHODS
120 SD rats were randomly divided into 3 groups: 10% group, with 10% of the total cecal length ligated and punctured without use of antibiotic so as to make sepsis model; 30% group, with 30% of the total cecal length ligated and punctured; and control group, undergoing sham operation. Fasting venous blood samples were collected before the operation to detect the fasting plasma glucose (FPG). 0, 8, 16, 24, and 48 hours after the operation 8 rats in each group underwent fasting of food and without fasting of water for 8 hours, i.e., until the 8 th, 16 th, 24 th, 48 th, and 72 nd hours after the operation. Then the rats underwent anesthesia, with blood sample from the vena cava and specimen of gastrocnemius of the hind leg collected, and then killed. The levels of FPG, fasting plasma insulin (FINS), tumor necrosis factor (TNF)-alpha, and interleukin (IL)-6 were measured. HOMA method was used to calculate the insulin resistance index (Ig-IRI). Immunohistochemistry was used to quantitatively examine the IRS-1 protein and its Ser(307) phosphorylation and Tyr phosphorylation in the gastrocnemius. Western blotting and immunoprecipitation were used to semi-quantitatively examine the changes in contents of IRS-1 and its Ser(307) phosphorylation and Tyr phosphorylation in the gastrocnemius respectively.


RESULTS
The survival rates at different time points of the control group were all 100%, all significantly higher than those of the other 2 groups (all P < 0.01), and those of the 10% group were all significantly higher than those of the 30% group (all P < 0.01). The levels of TNF-alpha and IL-6 of the 10% and 30% groups at different time points were all significantly higher than those of the control group (all P < 0.01), and those of the 30% group were all significantly higher than those of the 10% group (all P < 0.01). The FPG, FINS, and IgIRI were not significantly different among the 3 groups before the operation, and those of the 10% and 30% groups at different time points after operation were all significantly higher than those of the control group (all P < 0.01) and peaked 8 h after the operation, with those of the 30% group all significantly higher than those of the 10% group (all P < 0.01). The degree of increase of FINS was remarkably higher than that of FPG. IRS-1 was positive and located in the cytoplasm of the gastrocnemius cells in both the control and 30% groups; IRS-1 Tyr phosphorylation was positive in the control group and sporadic positive in the 30% group. IRS-1 Ser(307) was negative in the control group and strong positive in the 30% group. Semi-quantitative examination showed that the IRS-1 level at different time points after operation of the 30% group were not significantly different from those of the control group (all P > 0.05), and IRS-1 Tyr phosphorylation degrees at different time points of the 30% group were all significantly lower than those of the control group (all P < 0.01), and the IRS-1 Ser(307) phosphorylation at different time points of the 30% group were all significantly higher than those of the control group (all P < 0.01). Spearman correlation analysis showed that IgIRI was significantly negatively correlated with IRS-1 Tyr phosphorylation (r = -0.957, P < 0.01), and significantly positively correlated with IRS-1 Ser(307) phosphorylation (r = -0.955, P < 0.01).


CONCLUSIONS
Under the status of sepsis the IRS-1 content in the skeletal muscle cells is unchanged, the level of IRS-1 Tyr phosphorylation level is decreased, and the IRS-1 Ser phosphorylation is increased. The degrees of such changes are closely related with the degree of insulin resistance.
Yan
Xiao-Wen
XW
Surgery Intensive Care Unit, Nanjing General Hospital, Nanjing University, Nanjing 210002, China.
Li
Wei-Qin
WQ
Wang
Xiao-Dong
XD
Li
Jie-Shou
JS
Li
Ning
N
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D055504:Insulin Receptor Substrate Proteins; D007333:Insulin Resistance; D008297:Male; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D018805:Sepsis / Q000097:blood / Q000378:metabolism* / Q000473:pathology; D012694:Serine / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D001786:Blood Glucose; D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D014443:Tyrosine; D012694:Serine