Impairment of PGC-1 Alpha Up-Regulation Enhances Nitrosative Stress in the Liver during Acute Pancreatitis in Obese Mice

Abstract

Acute pancreatitis is an inflammatory process of the pancreatic tissue that often leads to distant organ dysfunction. Although liver injury is uncommon in acute pancreatitis, obesity is a risk factor for the development of hepatic complications. The aim of this work was to evaluate the role of PGC-1α in inflammatory response regulation in the liver and its contribution to the detrimental effect of obesity on the liver during acute pancreatitis. For this purpose, we induced acute pancreatitis by cerulein in not only wild-type (WT) and PGC-1α knockout (KO) mice, but also in lean and obese mice. PGC-1α levels were up-regulated in the mice livers with pancreatitis. The increased PGC-1α levels were bound to p65 to restrain its transcriptional activity toward Nos2. Lack of PGC-1α favored the assembly of the p65/phospho-STAT3 complex, which promoted Nos2 expression during acute pancreatitis. The increased transcript Nos2 levels and the pro-oxidant liver status caused by the down-regulated expression of the PGC-1α-dependent antioxidant genes enhanced nitrosative stress and decreased energy charge in the livers of the PGC-1α KO mice with pancreatitis. It is noteworthy that the PGC-1α levels lowered in the obese mice livers, which increased the Nos2 mRNA expression and protein nitration levels and decreased energy charge during pancreatitis. In conclusion, obesity impairs PGC-1α up-regulation in the liver to cause nitrosative stress during acute pancreatitis.

Keywords: PGC-1α; acute pancreatitis; liver; nitrosative stress; obesity.

Figure 1

(A) mRNA relative expression of Ppargc1a versus Tbp (TATA-binding protein; housekeeping) in the livers of the control (Sham) and 1 h after the cerulein-induced acute pancreatitis (Cerulein) mice. (B) Representative Western blot of PGC-1α in the livers of the control (Sham) and 1 h after the cerulein-induced acute pancreatitis (Cerulein) mice. β-tubulin was used as the loading control. There were six mice per group. Statistical difference is indicated as * p < 0.05 vs. Sham.

Figure 2

(A) Representative Western blot of PGC-1α in the livers of PGC-1α^+/+ (WT—wild-type) and PGC-1α^-/- (KO—knockout) mice. Β-tubulin was used as the loading control. (B) mRNA relative expression of Tnfa, Il6 and Nos2 versus Tbp (TATA binding protein; housekeeping) in the livers of the sham PGC-1α^+/+ (WT) and PGC-1α^-/- (KO) mice and at 1 h after cerulein-induced acute pancreatitis (AP) (Cerulein). (C) Representative Western blot of NOS2 in the livers of the sham PGC-1α^+/+ (WT) and PGC-1α^-/- (KO) mice and at 1 h after cerulein-induced AP (Cerulein). GAPDH was used as the loading control. (D) mRNA relative levels of Nos2 versus Tbp in the pancreas of the sham PGC-1α^+/+ (WT) and PGC-1α^-/- (KO) mice and at 1 h after cerulein-induced AP (Cerulein). (E) Representative Western blot of NOS2 in the pancreas of the sham PGC-1α^+/+ (WT) and PGC-1α^-/- (KO) mice and at 1 h after cerulein-induced AP (Cerulein). Β-tubulin was used as the loading control. There were six mice per group. Statistical difference is indicated as p * < 0.05 vs. Sham.

Figure 3

(A) Representative Western blot of p-p65 (Ser536), p65, p-STAT3 (Tyr705) and STAT3 in the livers of the sham PGC-1α^+/+ (WT) and PGC-1α^-/- (KO) mice and at 1 h after cerulein-induced AP (Cerulein). GAPDH was used as the loading control. (B) Representative Western blot of p65 and PGC-1α in the PGC-1α immunoprecipitate of the livers of the sham PGC-1α^+/+ (WT) mice and at 1 h after cerulein-induced AP mice. (C) Representative Western blot of p-STAT3 (Tyr705) and p65 in the p65 immunoprecipitate of the livers of the PGC-1α^+/+ (WT) mice and PGC-1α^-/- (KO) mice with pancreatitis (Cerulein). IgG was used as the loading control. There were six mice per group.

Figure 4

(A) mRNA relative expression of Sod2 and Prx3 versus the Tbp (TATA-binding protein; housekeeping) in the livers of the sham PGC-1α^+/+ (WT) and PGC-1α^-/- (KO) mice and at 1 h after cerulein-induced AP (Cerulein). (B) The GSSG/GSH, g-glutamilcystine/g-glutamilcysteine and cystine/cysteine ratios in the livers of the sham PGC-1α^+/+ (WT) and PGC-1α^-/- (KO) mice and at 1 h after cerulein-induced AP (Cerulein). There were six mice per group. The statistical difference is indicated as * p < 0.05 and ** p < 0.001.

Figure 5

(A) Representative Western blot of 3-Nitrotyrosine (NT) in the pancreas and liver of the control (Sham) and 1 h after cerulein-induced acute pancreatitis (Cerulein) mice. Ponceau was used as the loading control. (B) Determination of the 3NO2-Tyr/p-Tyr ratio in the livers of the sham PGC-1α^+/+ (WT) and PGC-1α^-/- (KO) mice and at 1 h after cerulein-induced AP (Cerulein). (C) Representative Western blot of 3-NT in the liver after inducing acute pancreatitis (Cerulein) in the PGC-1α^+/+ (WT) and PGC-1α^-/- (KO) mice. Ponceau was used as the loading control. (D) Energy charge in the livers of the sham PGC-1α^+/+ (WT) and PGC-1α^-/- (KO) mice and at 1 h after cerulein-induced AP (Cerulein). There were six mice per group. The statistical difference is indicated as ** p < 0.001.

Figure 6

(A) mRNA relative expression of Ppargc1a versus Tbp (TATA-binding protein; housekeeping) in the livers of the sham lean and obese mice and 1 h after cerulein-induced acute pancreatitis (Cerulein). (B) mRNA relative expression of Nos2 versus Tbp in the livers of the sham lean and obese mice and 1 h after cerulein-induced acute pancreatitis (Cerulein). (C) Representative Western blot of p-STAT3 (Tyr705) and p65 in p65 immunoprecipitate of the sham lean and obese mice and 1 h after cerulein-induced acute pancreatitis (Cerulein). IgG was used as the loading control. (D) Representative Western blot of 3-NT in the livers of the sham lean and obese mice and 1 h after cerulein-induced acute pancreatitis (Cerulein). Ponceau was used as the loading control. (E) Energy charge in the livers of the sham lean and obese mice and 1 h after cerulein-induced acute pancreatitis (Cerulein). There were five mice per group. Statistical difference is indicated as * p < 0.05 and ** p < 0.001.