| Background and ObjectiveInsulin resistance is a common pathological state in which target tissues fail to respond properly to normal levels of circulating insulin. Metabolic stress after surgery is always associated with alterations in carbohydrate metabolism characterized by the impairment in insulin-stimulated glucose disposal, which is named posttraumatic insulin resistance. Recent studies show that the development of posttraumatic insulin resistance is proportional to the magnitude of operation but negatively correlated to the prognosis. However, the molecular bases underlying posttraumatic insulin resistance still remain unclarified. Various researches have indicated that the main site for posttraumatic insulin resistance is peripheral tissue most likely skeletal muscle and a defect responsible for insulin resistance lies at the postreceptor level of insulin signaling. The purpose of this study is to unravel the mechanism of posttraumatic insulin resistance. To accomplish this goal, small intestine bowel resection was performed to establish the surgical trauma model in rats. We previously used this model to investigate whether skeletal muscle insulin sensitivity is affected by operation. Then we studied posttraumatic alterations in insulin signaling in skeletal muscle. Insulin-stimulated glucose transport in vitro in soleus muscles were also performed after surgical trauma. Finally, serum TNF-αwas measured to elucidate its contribution to inducing insulin resistance.Methods40 clean male Sprague-Dawley rats (200～250g) were randomized into two groups, the operation group (n=20) and the control group (n=20). Small intestinal resection was performed in the operation group. The control animals underwent anesthesia only. The following five different sets of experiments were performed. (1)Blood samples were obtained for determination of the level of blood glucose, serum insulin and serum TNF-α. Then the HOMA-IR index and the HOMA-βindex were calculated.(2)The Hyperinsulinemic-euglycemic clamp technique was also performed after surgical trauma to investigate the glucose disposal rate by periphery tissue. (3)Several key proteins in the insulin signaling such as insulin receptor substrate 1(IRS-1), phosphatidylinositol 3 kinase (PI3-K) and protein kinase B (PKB/AKT) in skeletal muscle were measured respectively. (4)The [3H] labeled glucose uptake experiment was carried out to evaluate the glucose transport function in both groups. (5)Finally, the expression and distribution of Glucose Transporter 4 (GLUT-4) in skeletal muscle were detected respectively.Results1. The level of blood glucose elevated significantly after small bowel resection in rats. The level of serum insulin decreased 30min after operation but elevated in the following time.2. The index of insulin resistance (HOMA-IR) in the operation group was significantly greater than in the control group, while the index of insulin secretion (HOMA-β) in the operation group was less than in the control group.3. The level of serum TNF-αin the operation group was significantly greater than in the control group. TNF-αpositively correlated with blood glucose, serum insulin, and the HOMA-IR index.4. The glucose disposal rate or clearance rate by the periphery tissue decreased 49% (7.41±0.20 mg·kg-1·min-1 vs. 14.50±0.12 mg·kg-1·min-1, F=5342.62, P=0.000) after small bowel resection.5. The total content of IRS-1 in skeletal muscle in both groups had no difference (246.75±5.41% vs. 248.76±6.29%, F=1.172, P=0.286). The phosphorylation of tyrosine (Tyr) residue of IRS-1 seen in the operation group was attenuated by 31% (88.62±45.06 vs. 127.61±53.88, F=6.165, P=0.018), whereas the phosphorylation of serine (Ser) residue of IRS-1 was significantly enhanced by 63% (153.56±44.59% vs. 94.38±36.07%, F=21.290, P=0.000) compared with the control group.6. No difference was observed for the level of p85 regulatory subunit of PI3-kinase in both groups (50.72±3.68% vs. 52.17±2.55%, F=2.103, P=0.155).7. The total content of PKB/Akt in skeletal muscle in two groups had no significant difference (115.78±8.87% vs. 115.99±11.23%, F=0.004, P=0.947). Yet the phosphorylation state of PKB/Akt (activated) was attenuated by 48% in the operation group (46.81±8.10 vs. 89.94±8.32, F=275.772, P=0.000).8. The level of serum TNF-αwas uncorrelated with the content of total IRS-1, p85 subunit, and PKB/Akt. TNF-αpositively correlated with the phosphorylation of serine (Ser) residue of IRS-1, while negatively correlated with the the phosphorylation of tyrosine (Tyr) residue of IRS-1 and the phosphorylation state of PKB/Akt.9. In contrast to the control group, the rate of 2-Deoxy-d-[1-3H] glucose uptaken by skeletal muscle in the operation group was significantly decreased (P＜0.05) under the same conditions.10. The expression of GLUT-4 mRNA in both groups had no difference (54.16±13.07%vs. 55.67±13.99%, F=0.063, P=0.805).11. In the early stage after operation (3h), there was no difference in the content of GLUT-4 in both groups (53.01±6.89% vs. 52.26±5.39%, F=0.149, P=0.702). However, with more observation in the 3rd day after surgery, the level of total GLUT-4 proteins were decreased in the operation group (48.07±12.08% vs. 57.33±10.49%, F=6.692, P=0.014).12. GLUT-4 proteins mostly resided in the cytoplasm and only a few of them located in the plasma membrane. After surgery, the translocation of GLUT-4 was blocked and the expression of GLUT-4 in the membrane was reduced than in the control group (Z=—2.191, P=0.028). ConclusionIn conclusion, we demonstrated that small intestinal resection can be ensured to establish the animal model of posttraumatic insulin resistance. The surgical trauma induced marked alterations in glucose metabolism during the immediate postoperative period. The later appearance of elevated serum insulin argued against the opinion that secretion of insulin was reduced after trauma. The research on insulin signaling suggested that insulin resistance was associated with enhanced Ser phosphorylation of IRS-1, which impaired its interaction with PI3-K and its downstream target PKB/Akt. Such impaired interactions abolished the ability of IRS-1 to undergo insulin-induced Tyr phosphorylation and further propagate the insulin receptor signal. Uncoupling of signal transduction led to decrease in glucose uptake which associated with a defect in insulin-stimulated glucose transport and GLUT-4 translocation. Elevation of serum TNF-αmay be responsible for such alteration after surgical trauma. |
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