*3.1. PGC-1*α *Levels are Up-Regulated in Mice Livers after Inducing Acute Pancreatitis*

In order to determine the role of PGC-1α in liver tissue during AP, our first approach was to measure its transcriptional and protein levels. First, we confirmed the appropriate AP induction by a histological analysis of the pancreas and determined plasma amylase and lipase activity (see Figure S1 Supplementary Materials).

Interestingly, *Ppargc1a* mRNA expression was up-regulated in the livers of the mice with AP (Figure 1A). Then, we set out to verify whether this transcriptional up-regulation would result in a rise in protein levels. The Western blot analysis showed that PGC-1α protein expression was higher in the livers of the mice with cerulein-induced pancreatitis than in the control mice (Figure 1B).

#### *3.2. PGC-1*α *Restrains Nos2 Expression in the Liver after Acute Pancreatitis in Mice*

Having determined the induction of PGC-1α in the liver during AP, the inflammatory response of AP in liver tissue was studied using the PGC-1α deficient mice (Figure 2A).

For this purpose, the hepatic transcriptional expression of cytokines *Tnf*α and interleukin-6 (*Il6*), as well as *Nos2*, a marker of cellular stress, was analyzed. The *Tnf*α and *Il6* mRNA levels did not vary after administering cerulein to any experimental group (Figure 2B). *Nos2* gene expression remained unchanged in the wild-type (WT) mice after inducing AP (Figure 2B). However, and unlike the changes observed in the *Tnf*α and *Il6* gene expressions, a marked increase (around 6-fold) was detected in the transcription of *Nos2* in the livers of the PGC-1α deficient mice with AP (Figure 2B). Western blot also showed that NOS2 protein expression was higher in the livers of the KO mice with AP (Figure 2C).

**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 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. *Antioxidants* **2020**, *9*, x FOR PEER REVIEW 7 of 18

**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 2.** (**A**) Representative Western blot of PGC-1α in the livers of PGC-1α +/+ (WT—wild-type) and PGC-1α -/- (KO—knockout) mice. B-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). B-tubulin was used as the loading control. There were six mice per group. Statistical difference is indicated as *p* \* < 0.05 vs. Sham.

activation are involved in inducing *Nos2* in the liver upon PGC-1α deficiency.

PGC-1α-deficient mice compared to the WT under AP conditions (Figure 3A).

PGC-1α KO mice with pancreatitis [32], we considered studying whether NF-κB and STAT3

The Western blot analysis showed a dramatic increase in the phosphorylation of the p65 subunit of NF-κB in the livers of the PGC-1α KO mice after AP induction (Figure 3A). Regarding STAT3 activation, AP triggered an increase in its phosphorylated form in both the WT and KO mice in liver tissue (Figure 3A). Interestingly, the STAT3 phosphorylation levels were higher in the

It has been previously shown that the p65 subunit of NF-κB may complex with p-STAT3 favoring the expression of *Nos2* [33]. Additionally, our research group has shown that PGC-1α forms a protein complex with p65 in the pancreas during AP [32]. Based on this background, we decided to evaluate the role of PGC1alpha in p65-binding to p-STAT3 in the liver during AP. According to our immunoprecipitation studies, the augmented levels of PGC-1α found in the liver after the induction of AP bound to p65 (Figure 3B) as we previously reported in pancreatic tissue. However, the lack of

*Experimental Acute Pancreatitis* 

*3.3. PGC-1α Avoids the Assembly of the Complex between p65 and Phospho-STAT3 in the Liver during* 

We previously reported that *Il6* mRNA levels were selectively up-regulated in the pancreas of the PGC-1α KO mice with AP compared to their WT littermates, conversely to other pro-inflammatory cytokines [32]. Here, we observed that although *Nos2* mRNA expression was up-regulated in the pancreas upon pancreatitis induction, no significant differences appeared between the PGC-1α KO mice and the WT mice (Figure 2D). Similarly, the Western blot also showed NOS2 induction during AP, although there was no change between WT and KO mice with pancreatitis (Figure 2D).

### *3.3. PGC-1*α *Avoids the Assembly of the Complex between p65 and Phospho-STAT3 in the Liver during Experimental Acute Pancreatitis*

By taking into account our previous studies, in which the plasma levels of IL-6 increased in the PGC-1α KO mice with pancreatitis [32], we considered studying whether NF-κB and STAT3 activation are involved in inducing *Nos2* in the liver upon PGC-1α deficiency.

The Western blot analysis showed a dramatic increase in the phosphorylation of the p65 subunit of NF-κB in the livers of the PGC-1α KO mice after AP induction (Figure 3A). Regarding STAT3 activation, AP triggered an increase in its phosphorylated form in both the WT and KO mice in liver tissue (Figure 3A). Interestingly, the STAT3 phosphorylation levels were higher in the PGC-1α-deficient mice compared to the WT under AP conditions (Figure 3A). *Antioxidants* **2020**, *9*, x FOR PEER REVIEW 8 of 18 PGC-1α markedly increased the binding of p65 to p-STAT3 in KO mice with pancreatitis (Figure 3C), suggesting that the induction of PGC-1α in the liver during AP may inhibit the formation of the p65/p-STAT3 complex.

**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 **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.

pancreatitis (Cerulein). IgG was used as the loading control. There were six mice per group.

*3.4. PGC-1α Deficiency Downregulates Antioxidant Gene Expression and Increases Oxidative Stress in the Liver with Acute Pancreatitis*  Taking into account the role of PGC-1α in the regulation of antioxidant genes, the mRNA expression of *Sod2* and *Prdx3* was measured in the liver of the PGC-1α KO and WT mice. As expected, PGC-1α deficiency triggered the down-regulation of *Sod2* and *Prdx3* under basal conditions and during pancreatitis (Figure 4A). Additionally, the liver redox status was assessed by measuring disulfide pairs, Cyss/Cys, γ-glutamyl cystine/γ-glutamyl cysteine and GSSG/GSH. According to the antioxidant genes It has been previously shown that the p65 subunit of NF-κB may complex with p-STAT3 favoring the expression of *Nos2* [33]. Additionally, our research group has shown that PGC-1α forms a protein complex with p65 in the pancreas during AP [32]. Based on this background, we decided to evaluate the role of PGC1alpha in p65-binding to p-STAT3 in the liver during AP. According to our immunoprecipitation studies, the augmented levels of PGC-1α found in the liver after the induction of AP bound to p65 (Figure 3B) as we previously reported in pancreatic tissue. However, the lack of PGC-1α markedly increased the binding of p65 to p-STAT3 in KO mice with pancreatitis (Figure 3C), suggesting that the induction of PGC-1α in the liver during AP may inhibit the formation of the p65/p-STAT3 complex.

expression, the GSSG/GSH, γ-glutamyl cystine/γ-glutamyl cysteine and Cyss/Cys ratios were

### *3.4. PGC-1*α *Deficiency Downregulates Antioxidant Gene Expression and Increases Oxidative Stress in the Liver with Acute Pancreatitis*

Taking into account the role of PGC-1α in the regulation of antioxidant genes, the mRNA expression of *Sod2* and *Prdx3* was measured in the liver of the PGC-1α KO and WT mice. As expected, PGC-1α deficiency triggered the down-regulation of *Sod2* and *Prdx3* under basal conditions and during pancreatitis (Figure 4A). *Antioxidants* **2020**, *9*, x FOR PEER REVIEW 9 of 18

**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 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.

*3.5. PGC-1α Prevents Protein Nitration in the Liver after Inducing Experimental Acute Pancreatitis*  In accordance with the *Nos2* mRNA levels found in the pancreas and liver of the mice with AP, we observed that AP produced increased protein nitration in the pancreas, but not in the liver of the WT mice (Figure 5A). Remarkably, and consistently with the increased *Nos2* mRNA expression Additionally, the liver redox status was assessed by measuring disulfide pairs, Cyss/Cys, γ-glutamyl cystine/γ-glutamyl cysteine and GSSG/GSH. According to the antioxidant genes expression, the GSSG/GSH, γ-glutamyl cystine/γ-glutamyl cysteine and Cyss/Cys ratios were significantly higher in the livers of the PGC-1α KO mice than in the WT mice (Figure 4B), which supports the relevance of PGC-1α for maintaining redox homeostasis in liver during AP.

#### observed in the livers of the PGC-1α KO mice with pancreatitis, we detected higher levels of 3-nitrotyrosine, a marker of protein nitration measured by mass spectrometry, in the livers of these *3.5. PGC-1*α *Prevents Protein Nitration in the Liver after Inducing Experimental Acute Pancreatitis*

(see Figure S2A in the supplementary materials) [34].

(see Figure S2B in the supplementary materials).

mice compared to the other groups (Figure 5B). This increase in 3-nitrotyrosine was confirmed by Western blot in the livers of the PGC-1α KO mice with cerulein-induced pancreatitis (Figure 5C). In accordance with the *Nos2* mRNA levels found in the pancreas and liver of the mice with AP, we observed that AP produced increased protein nitration in the pancreas, but not in the liver of the WT

However, we did not observe changes in 3-chlorotyrosine, parameter associated with inflammation

susceptible to the effects of nitrosative stress [35], we set out to calculate the energy charge in liver tissue during AP. The results revealed a significant drop in this parameter in the livers of the KO mice with pancreatitis (Figure 5D), although these changes did not alter the serum AST levels in AP

3).

mice (Figure 5A). Remarkably, and consistently with the increased *Nos2* mRNA expression observed in the livers of the PGC-1α KO mice with pancreatitis, we detected higher levels of 3-nitrotyrosine, a marker of protein nitration measured by mass spectrometry, in the livers of these mice compared to the other groups (Figure 5B). This increase in 3-nitrotyrosine was confirmed by Western blot in the livers of the PGC-1α KO mice with cerulein-induced pancreatitis (Figure 5C). However, we did not observe changes in 3-chlorotyrosine, parameter associated with inflammation (see Figure S2A in the Supplementary Materials) [34]. *Antioxidants* **2020**, *9*, x FOR PEER REVIEW 10 of 18

**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 **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.

as \*\* *p* < 0.001. *3.6. PGC-1α Levels Lower in the Livers of Obese Mice under Basal Conditions and during Pancreatitis*  Considering that mitochondria are the main source of peroxynitrite and are also highly susceptible to the effects of nitrosative stress [35], we set out to calculate the energy charge in liver tissue during AP. The results revealed a significant drop in this parameter in the livers of the KO mice with pancreatitis

PGC-1α has been previously reported to be dysregulated in obese animals and patients [18]. As we know that obesity increases the risk of systemic complications in AP [36], we decided to measure

First, we corroborated that high-fat diet induced weight gain, hyperglycemia and fatty liver (Table

(Figure 5D), although these changes did not alter the serum AST levels in AP (see Figure S2B in the Supplementary Materials).

#### *3.6. PGC-1*α *Levels Lower in the Livers of Obese Mice under Basal Conditions and during Pancreatitis*

PGC-1α has been previously reported to be dysregulated in obese animals and patients [18]. As we know that obesity increases the risk of systemic complications in AP [36], we decided to measure the PGC-1α levels in the livers of the lean and obese mice under basal conditions and during AP. First, we corroborated that high-fat diet induced weight gain, hyperglycemia and fatty liver (Table 3).


**Table 3.** Parameters of high-fat diet-induced obese mice.

The statistical difference is indicated as \* *p* < 0.05 and \*\* *p* < 0.001.

The *Ppargc1a* mRNA levels were markedly down-regulated in the livers of the obese mice under basal conditions (Figure 6A). Interestingly, the obese mice did not exhibit any increased *Ppargc1a* expression, which was found in the lean mice after inducing AP (Figure 6A).

After bearing in mind our findings in the PGC-1α KO mice, we decided to study whether the PGC-1α deficiency detected in the obese mice would impact nitrosative stress in the livers of these mice during AP. We observed that *Nos2* gene expression was up-regulated in the obese mice under basal conditions (Figure 6B). We stress that this up-regulation was higher in the obese mice with pancreatitis (Figure 6B). We also observed a marked increase in the binding of p65 to p-STAT3 in the obese mice with pancreatitis (Figure 6C), which supports the notion that lack of PGC-1α in obese mice livers may be associated with inducing *Nos2* during AP.

The protein nitration levels were higher in the livers of the obese mice than those of the lean mice with pancreatitis (Figure 6D). Interestingly, the obese mice showed a lower hepatic energy charge in relation to the lean mice with pancreatitis (Figure 6E). As we observed in the PGC-1α-deficient mice, this drop in energy charge did not change the AST levels in serum (see Figure S2C in the Supplementary Materials).

**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 **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.

pancreatitis (Cerulein). There were five mice per group. Statistical difference is indicated as \* *p* < 0.05

#### and \*\* *p* < 0.001. **4. Discussion**

**4. Discussion**  Of the different systemic complications to occur during AP, the commonest are those that affect the cardiovascular, renal, and pulmonary systems [14]. Although most of the pancreatic enzymes and mediators released by an inflamed pancreas pass through the liver before entering systemic circulation, liver failure is a rare pathophysiological condition in the course of AP with a significant prognostic value for its severity [15,16]. It is noteworthy that obesity, which is a risk factor for AP development, decisively contributes to the appearance of systemic complications, including those Of the different systemic complications to occur during AP, the commonest are those that affect the cardiovascular, renal, and pulmonary systems [14]. Although most of the pancreatic enzymes and mediators released by an inflamed pancreas pass through the liver before entering systemic circulation, liver failure is a rare pathophysiological condition in the course of AP with a significant prognostic value for its severity [15,16]. It is noteworthy that obesity, which is a risk factor for AP development, decisively contributes to the appearance of systemic complications, including those that affect liver function [13,16]. The present work highlights the role of PGC-1α in regulating the relation between obesity and liver injury in the course of AP. We particularly demonstrate that obesity impairs hepatic

that affect liver function [13,16]. The present work highlights the role of PGC-1α in regulating the relation between obesity and liver injury in the course of AP. We particularly demonstrate that PGC-1α up-regulation and, thus, enhances *Nos2* transcriptional expression and causes nitrosative stress in the liver during AP.

Our results show that AP induction in mice increases transcriptional and protein PGC-1α expressions in the liver, which is crucial for preventing *Nos2* up-regulation. In pancreatic tissue, our group has recently shown that PGC-1α binds to phospho-p65 to repress *Il6* gene expression during pancreatitis. Consequently, PGC-1α deficiency enhances NF-κB-dependent *Il6* transcription in the pancreas by augmenting circulating IL-6 levels and increasing local and systemic inflammatory responses [32]. In accordance with these findings, here we found higher levels for both NF-κB and STAT3 activation in the livers of the PGC-1α KO mice with pancreatitis. PGC-1α deficiency may be responsible for activating p65, as we have previously reported in pancreatic tissue [32]. It is also accompanied by STAT3 hyperactivation, probably due to the high IL-6 plasma levels achieved in PGC-1α KO mice during pancreatitis. Yet despite the activation of both the NF-κB and STAT3 signaling pathways in the livers of the PGC-1α KO mice, we did not observe any increase in the *Tnf*α or *Il6* expression levels in these mice. Strikingly, and unlike pancreatic tissue, lack of PGC-1α in the liver specifically induced *Nos2* transcription during AP development.

Although *Nos2* expression can be induced by different transcription factors (AP-1, C/EBP, CREB, IRF-1, NF-κB, NF-IL6, Oct-1, SRF, STAT1α) [37], its transcription has also been described to be specifically regulated by the formation of complexes between p65 and STAT3 in the promoter region of *Nos2* [33]. During pancreatitis, our results revealed that p65 bound to PGC-1α in the liver. The formation of this complex has been previously reported in both human cardiac cells and the heart, liver and pancreas of mice [32,38,39]. The present work shows that, as a consequence of PGC-1α deficiency, p65 bound to p-STAT3 in the liver, which could justify the particular increase in the *Nos2* transcription found in the livers of the PGC-1α KO mice.

The present results confirm the presence of nitrosative stress in the pancreas during AP in accordance with augmented pancreatic *Nos2* expression levels [8,9]. Nevertheless, we did not note any increase in the protein nitration levels in the liver after inducing pancreatitis, which is consistent with the PGC-1α-dependent repression of *Nos2* in this tissue. It is well-known that nitrosative stress is implied in the liver pathophysiology [40]. In particular, peroxynitrite accumulation in LPS-treated mice is one of the commonest causes for acute liver injury [41,42]. In fact, hepatocytes respond to LPS treatment with IL-6- and TNF-α-mediated NO production, which induces a hepatic acute phase response [43]. Furthermore, reactive nitrogen species (RNS) production contributes to nitrate critical amino acid residues, which render hepatocytes more susceptible to oxidative damage [40]. In our work, lack of PGC-1α in the liver lowered the antioxidant defense gene expression, which triggered increased oxidative stress. This pro-oxidant environment in the livers of the PGC-1α KO mice, together with specific *Nos2* induction, would explain the more marked nitration pattern observed in the livers of these mice with pancreatitis. Hence, these results demonstrate that PGC-1α is crucial for preventing nitrosative stress in the liver during AP development.

The mitochondrion is the organelle most susceptible to the consequences of nitrosative stress [35]. Numerous studies have shown that mitochondrial dysfunction participates both in the progression of organ failure and in the development of the systemic inflammatory response syndrome (SIRS) [44]. Interestingly, Trumbecakaite et al. showed that AP triggered mitochondrial failure in the pancreas, kidney, and lung, while the liver preserved mitochondrial function during the development of PA [45]. According to this work, our results reveal that the energy charge was unchanged in the liver of WT mice with pancreatitis. However, the induction of PA in mice deficient in PGC-1α caused a decrease in the energy charge in the liver of these mice, confirming the essential role of PGC-1α in the maintenance of mitochondrial homeostasis during inflammatory processes.

According to our results, *Nos2* transcription in the liver during pancreatitis seems to depend on PGC-1α levels. PGC-1α expression lowers in skeletal muscle in both mice with genetic obesity (ob/ob) and fat diet-induced obesity [46]. Furthermore, it has been found that a high-fat diet inhibits PGC-1α expression in mice liver and induces non-alcoholic fatty liver development [39]. Accordingly, our research group recently showed a drop in the protein and transcriptional levels of PGC-1α in the pancreas of obese Zucker rats and in mice with fat diet-induced obesity [32]. Here, we confirm that a high-fat diet in mice down-regulated *Ppargc1a* expression in the liver under basal conditions, and that this decrease was also maintained after inducing AP. Consequently, we observed higher *Nos2* gene expression and protein nitration levels and decreased energy charge in the livers of obese mice compared to the lean mice with pancreatitis.

Obesity is associated with increased *Nos2* expression in insulin-sensitive tissue in rodents and humans [47]. Abdominal obesity increases free fatty acids in the liver, which induces superoxide anion formation and up-regulates *Nos2* gene expression to result in peroxynitrite synthesis and to lead to both mitochondrial dysfunction and liver injury [48]. After taking into account that high-fat diet administration aggravates AP-induced hepatic injury via oxidative stress [49], our herein reported findings provide new insights into the detrimental effect of obesity on liver complications during AP, which highlights the key role of PGC-1α deficiency in this regard.

#### **5. Conclusions**

The present work demonstrates the essential role of PGC-1α in inflammatory response and nitrosative stress during AP, particularly in liver tissue. First, pancreatitis leads to marked PGC-1α induction in the liver. Our results also suggest that PGC-1α binds to p65 by acting as a repressor of NF-κB transcriptional activity and, in turn, prevents the formation of a transcriptional complex between p65 and p-STAT3. Therefore, PGC-1α deficiency triggers the increase in the *Nos2* expression mediated by the p65/p-STAT3 complex in the liver, and consequently results in higher protein nitration levels and a decrease in the energy charge. Finally, obesity triggers PGC-1α deficiency in the liver and enhances nitrosative stress during pancreatitis. Therefore, our results highlight the protective role of PGC-1α in the liver for preventing nitrosative stress during AP.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3921/9/9/887/s1, Figure S1 and Figure S2 are in the attached supplementary document.

**Author Contributions:** S.R.-P. and I.T.-C. induced AP in mice. S.R.-P. and I.T.-C. performed most of the assays. F.J.M. did the statistical data analysis. M.M. provided the PGC-1α KO mice and performed the genotyping of the KO and WT mice. S.R.-P., I.T.-C. and F.J.M. prepared all the figures in the manuscript. S.P. designed the study, supervised the experiments and wrote the manuscript. All the authors contributed to update and critically review the literature, wrote the manuscript, and supervised and approved the final version. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by Grant GV/2019/153 from the Generalitat Valenciana, Conselleria d'Educació, Investigació, Cultura I Esport.

**Conflicts of Interest:** The authors declare that there is no conflict of interest.

#### **References**


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