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, 25 (3), 492-502

Role of Reactive Oxygen Species in Injury-Induced Insulin Resistance

Affiliations

Role of Reactive Oxygen Species in Injury-Induced Insulin Resistance

Lidong Zhai et al. Mol Endocrinol.

Abstract

Acute insulin resistance is common after injury, infection, and critical illness. To investigate the role of reactive oxygen species (ROS) in critical illness diabetes, we measured hepatic ROS, which rapidly increased in mouse liver. Overexpression of superoxide dismutase 2, which decreased mitochondrial ROS levels, protected mice from the development of acute hepatic insulin resistance. Insulin-induced intracellular signaling was dramatically decreased, and cellular stress signaling was rapidly increased after injury, resulting in the hyperglycemia of critical illness diabetes. Insulin-induced intracellular signaling, activation of stress (c-Jun N-terminal kinase) signaling, and glucose metabolism were all normalized by superoxide dismutase 2 overexpression or by pretreatment with antioxidants. Thus, ROS play an important role in the development of acute hepatic insulin resistance and activation of stress signaling after injury.

Figures

Fig. 1.
Fig. 1.
Characterization of hepatic insulin resistance after trauma and hemorrhage in mice. Mice were subjected to trauma alone (T) or trauma and hemorrhage (TH) as described in the Materials and Methods. A and B, At 0, 30, and 90 min, fasting glucose (A) and insulin (B) levels were measured from femoral arterial blood. Data are presented as mean ± sem of samples from nine to 10 mice in each group. In this and all the other figures, a single symbol (# or $) represents P < 0.05; a double symbol (**, ##, or $$) represents P < 0.01; a triple symbol (### or $$$) represents P < 0.001, and no symbol or ns represents not significant. #, Statistics vs. T0′; $, statistics vs. time-matched trauma alone. C, At 0 and 30 min, either saline (−Ins) or 1 U insulin (+Ins) was injected into the inferior vena cava of C57BL/6 mice. After 4 min, the liver was removed and frozen in liquid nitrogen. Liver tissue lysates were later prepared and subjected to Western blotting. D–F, Multiple autoradiographs were quantified by scanning densitometry. Data are presented as mean ± sem of samples from three to seven mice in each group. #, Statistics vs. T0′+Ins; $, statistics vs. T30′+Ins. G, Time-dependent decrease in insulin sensitivity in the liver after trauma and hemorrhage as measured by insulin's ability to induce Akt phosphorylation. Data are presented as mean ± sem of samples from three to seven mice in each group. $, Statistics vs. time-matched trauma-only group T30′, T60′, or T90′. # or $, P < 0.05; **, ##, or $$, P < 0.01; ### or $$$, P < 0.001; ns, not significant.
Fig. 2.
Fig. 2.
Correlation between aconitase activity and insulin sensitivity after trauma and hemorrhage. A, As described in the legend to Fig. 1, mice were subjected to trauma alone (T) or trauma and hemorrhage (TH), and at the T0, TH15, TH30, TH60, and TH90 time points, the liver was removed and frozen in liquid nitrogen. Tissue lysates were prepared, and aconitase activity was assayed (n = 6). #, Statistics vs. T0′. ##, P < 0.01; ###, P < 0.001. B, The regression line comparing insulin-induced phosphorylation of Akt after trauma and hemorrhage and aconitase activity (and also on the x-axis is an indication of the increasing time of hemorrhage) .
Fig. 3.
Fig. 3.
The effect of overexpression of SOD2 on ROS, aconitase activity, and blood glucose and insulin levels after trauma and hemorrhage. A and B, Genotyping (A) and Western blot analysis (B, top panel) were performed as described to confirm the overexpression SOD2 in transgenic mice as compared with WT mice. Positive and negative controls are presented for the PCR in A. Also in B is a bar graph of the total level of SOD2, using a mouse antibody that cross-reacts with both endogenous mouse SOD2 and expressed human SOD2. As described in Fig. 1, either trauma alone (T90′) or trauma and hemorrhage (TH90′) was performed in C57BL/6 (WT) or mice overexpressing SOD2. C and D, ROS levels were assessed by direct DHE staining in situ (C) (n =3) and by indirect measurement of aconitase activity (D) (n =6). E and F, Fasting blood glucose levels (E) (n = 8–11) and insulin levels (F) (n =5–7) were also measured in these mice. #, Statistics vs. WT-T90′; *, statistics vs. WT-T90′; $, vs. WT-TH90′; ns, not significant vs. SOD2–T90′. # or $, P < 0.05; **, ## or $$, P < 0.01; ### or $$$, P < 0.001.
Fig. 4.
Fig. 4.
Overexpression of SOD2 increased glucose tolerance and insulin sensitivity after trauma and hemorrhage. WT mice or mice overexpressing SOD2 were subjected to trauma alone (T90′) or trauma and hemorrhage (TH90′) as previously described. However, in these mice, glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed. A and B, In the glucose tolerance tests, after a 6-h fast, blood glucose was measured (the 0-min time point) after T90′ or TH90′, and then glucose (0.6 g/kg) was injected into the femoral vein, blood glucose levels were measured every 15 min (A), and the AUC were calculated as described (B). C and D, In the insulin tolerance tests, there was no fasting period, blood glucose was measured (the 0 min time point) after T90′ or TH90′, and then insulin (0.5 U/kg) was injected into the femoral vein, and blood glucose levels were measured (C) and analyzed (D). Data are presented as mean ± sem of three samples in each group. $, Statistics between WT-TH90′ and SOD2-TH90′; #, vs. WT-T90′. # or $, P < 0.05; ## or $$, P < 0.01; ### or $$$, P < 0.001.
Fig. 5.
Fig. 5.
Overexpression of SOD2 inhibited trauma- and hemorrhage-induced hepatic insulin resistance. After T90′ or TH90′, either saline (−Ins) or 1 U insulin (+Ins) was injected into the inferior vena cava, and after 4 min, the liver was removed and frozen in liquid nitrogen. A and B, Liver tissue lysates from WT or SOD2-overexpressing mice were subjected to Western blotting with P-Akt(S473), total (T)-Akt, T-ERK, P-IR (Y972), P-IRS1 (Y612), and T-IR antibodies. C–E, Autoradiographs were quantified by scanning densitometry. Data are presented as mean ± sem of samples from three to seven mice in each group. #, Statistics vs. WT-T90′+Ins; $, vs. WT-TH90′+Ins; ns, not significant vs. SOD2–T90′+Ins. # or $, P < 0.05; ### or $$$, P < 0.001.
Fig. 6.
Fig. 6.
NAC and MnTBAP prevented trauma- and hemorrhage-induced hepatic insulin resistance. WT mice were pretreated with NAC (300 mg/kg, ip) or MnTBAP (10 mg/kg, ip) before surgery. After 30 min of hemorrhage, either saline (−Ins) or 1 U insulin (+Ins) was injected into the inferior vena cava. A and B, Liver tissue lysates were subjected to Western blotting with P-Akt(S473), total (T)-Akt, and T-ERK antibodies. C and D, Autoradiographs were quantified by scanning densitometry. #, Statistics vs. TH30′+Ins; ns, not significant vs. T30′+Ins. E, Liver lysates were prepared, and aconitase activity was assayed; pretreatment with MnTBAP and NAC protected from the decrease in aconitase activity after trauma and hemorrhage. #, Statistics vs. T0′; *, statistics vs. TH30′. Data are presented as mean ± sem of samples from three to seven mice in each group. #, P < 0.05; ##, P < 0.01; ### or ***, P < 0.001.
Fig. 7.
Fig. 7.
Trauma- and hemorrhage-induced activation of the JNK signaling pathway in liver was inhibited by a decrease of ROS. A and B, As described in the legend to Fig. 1, WT mice were subjected to trauma alone (T) or trauma and hemorrhage (TH), and at the 0-, 30-, 60-, and 90-min time points, the liver was removed and frozen in liquid nitrogen. Liver lysates were subjected to Western blotting with P-JNK1/2 antibodies, and autoradiographs were quantified by scanning densitometry. Data are presented as mean ± sem of samples from four to eight mice in each group. C, As described in the legend to Fig. 3, WT and SOD2-overexpressing mice underwent trauma alone (T90′) or trauma and hemorrhage (TH90′), and liver lysates were subjected to Western blotting with anti-P-JNK1/2 and T-JNK antibodies. D and E, Autoradiographs were quantified by scanning densitometry. Data are presented as mean ± sem of samples from four to six mice in each group. #, Statistics vs. time-matched trauma-only group T30′, T60′, or T90′; $, vs. WT-TH90′; ns, not significant vs. SOD2–T90′. # or $, P < 0.05; **, ##, or $$, P < 0.01; ### or $$$, P < 0.001.

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