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Hepatocyte-Specific Deletion of AMPKα1 Results in Worse Outcomes in Mice Subjected to Sepsis in a Sex-Specific Manner

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Hepatocyte-Specific Deletion of AMPKα1 Results in Worse Outcomes in Mice Subjected to Sepsis in a Sex-Specific Manner

Satoshi Kikuchi et al. Front Immunol.

Abstract

Alterations in the energy homeostasis contribute to sepsis-mediated multiple organ failure. The liver plays a central role in metabolism and participates to the innate immune and inflammatory responses of sepsis. Several clinical and experimental studies have suggested that females are less susceptible to the adverse outcome of sepsis. However, underlying mechanisms of organ damage in sepsis remain largely undefined. AMP-activated protein kinase (AMPK) is an important regulator of mitochondrial quality control. The AMPK catalytic α1 isoform is abundantly expressed in the liver. Here, we determined the role of hepatocyte AMPKα1 in sepsis by using hepatocyte-specific AMPKα1 knockout mice (H-AMPKα1 KO) generated with Cre-recombinase expression under the control of the albumin promoter. Using a clinically relevant model of polymicrobial sepsis by cecal ligation and puncture (CLP), we observed that male H-AMPKα1 KO mice had higher plasma levels of tumor necrosis factor-α and interleukin-6 and exhibited a more severe liver and lung injury than male H-AMPKα1 WT mice, as evaluated by histology and neutrophil infiltration at 18 h after CLP. Plasma levels of interleukin-10 and the keratinocyte-derived chemokine were similarly elevated in both KO and WT male mice. At transmission electron microscopy analysis, male H-AMPKα1 KO mice exhibited higher liver mitochondrial damage, which was associated with a significant decrease in liver ATP levels when compared to WT mice at 18 h after sepsis. Mortality rate was significantly higher in the male H-AMPKα1 KO group (91%) when compared to WT mice (60%) at 7 days after CLP. Female H-AMPKα1 WT mice exhibited a similar degree of histological liver and lung injury, but significantly milder liver mitochondrial damage and higher autophagy when compared to male WT mice after CLP. Interestingly, H-AMPKα1 KO female mice had lower organ neutrophil infiltration, lower liver mitochondrial damage and lower levels of cytokines than WT female mice. There was no significant difference in survival rate between WT and KO mice in the female group. In conclusion, our study demonstrates that AMPKα1 is a crucial hepatoprotective enzyme during sepsis. Furthermore, our results suggest that AMPK-dependent liver metabolic functions may influence the susceptibility to multiple organ injury in a sex-dependent manner.

Keywords: AMPKα1; Cre-lox; cecal ligation and puncture; female sex; liver injury; lung injury; mitochondria.

Figures

Figure 1
Figure 1
Representative histology photomicrographs of liver sections of hepatocyte-specific (H-AMPKα1) wild-type (WT) and knock-out (KO) mice at basal control condition and at 18 h after cecal ligation and puncture (CLP). Normal liver architecture in control male WT (A) and male KO (E), and in control female WT (C) and female KO (G) mice. Liver damage in male (B) and female (D) H-AMPKα1 WT mice with modest areas of sinusoid congestion (asterisks). Liver damage in male (F) and female (H) H-AMPKα1 KO mice with necrosis (arrows) and infiltration of inflammatory cells (arrowheads). Magnification ×100; scale bar = 100 μm. A similar pattern was seen in n = 4–8 different tissue sections in each experimental group. Liver myeloperoxidase (MPO) activity (I), plasma ALT levels (J) in male and female H-AMPKα1 WT and KO mice at 18 h after CLP. Data represents the mean ± SEM of 4–8 mice for each group. *P < 0.05 vs. sex-matched control mice; #P < 0.05 vs. sex-matched WT mice.
Figure 2
Figure 2
Transmission electron microscopy of hepatocytes in hepatocyte-specific (H-AMPKα1) wild-type (WT) and knock-out (KO) male (A–D) and female (E–H) mice at basal control condition or 18 h after cecal ligation and puncture (CLP). Arrows, damaged mitochondria presenting translucent matrix, disrupted membrane and cristae; arrow heads, elongated mitochondria; asterisk, autophagic vescicles packed with mitochondria; N, nucleus. Quantification of damaged mitochondria (I), elongated mitochondria (J), and autophagosomes (K) of hepatocytes of hepatocyte-specific (H- AMPKα1) wild-type (WT) and knock-out (KO) mice at 18 h after cecal ligation and puncture (CLP). Liver sections were examined at transmission electron microscopy. Damaged, elongated mitochondria and autophagosomes were determined by using the NIH Image J software and expressed as percentage of total number of mitochondria in nine consecutive cells. Data are means ± SEM of 3–4 mice for each group. Liver ATP content (L) of hepatocyte-specific (H-AMPKα1) wild-type (WT) and knock-out (KO) mice at 18 h after cecal ligation and puncture (CLP). Data represents the mean ± SEM of 4–6 mice for each group. *P < 0.05 vs. sex-matched control mice; #P < 0.05 vs. sex-matched WT mice; P < 0.05 vs. male group of the same genotype.
Figure 3
Figure 3
Representative Western blots of protein expression of LC3B-I and LC3B-II in liver cytosol extracts; GADPH was used as loading control protein (A). Image analyses of cytosol relative intensity of LC3B-I (B), LC3 B-II (C) and LC3B-II/LC3B-I (D) as determined by densitometry. Each data represents the mean ± SEM of 3–4 animals for each group. *P < 0.05 vs. sex-matched control mice; #P < 0.05 vs. sex-matched WT mice; P < 0.05 vs. male group of the same genotype.
Figure 4
Figure 4
Representative histology photomicrographs of lung sections of hepatocyte-specific (H-AMPKα1) wild-type (WT) and knock-out (KO) male (A–D) and female (E–H) mice at baseline conditions (Control) or 18 h after cecal ligation and puncture (CLP). Lung damage in H-AMPKα1 WT male (B) and female (F) mice with modest infiltration of inflammatory cells (arrow heads) and reduced alveolar space (arrows). Lung damage in H- AMPKα1 KO male mice (D) with reduced alveolar space, bronchial congestion (asterisk) and accumulation of red and inflammatory cells. Mild lung damage in H-AMPKα1 KO female mice (H) with mild infiltration of inflammatory cells only. Magnification ×100; scale bar = 200 μm. A similar pattern was seen in n = 4–8 different tissue sections in each experimental group. Lung myeloperoxidase (MPO) activity (I) in male and female H- AMPKα1 WT and KO mice at 18 h after CLP. Data represents the mean ± SEM of 4–8 mice for each group. *P < 0.05 vs. sex-matched control mice; #P < 0.05 vs. sex-matched WT mice; P < 0.05 vs. male group of the same genotype.
Figure 5
Figure 5
Plasma levels of TNFα (A), IL-6 (B), IL-10 (C), and KC (D). Data represents the mean ± SEM of 4–8 mice for each group. *P < 0.05 vs. sex-matched control mice; #P < 0.05 vs. sex-matched WT mice; P < 0.05 vs. male group of the same genotype.
Figure 6
Figure 6
Survival rate of hepatocyte-specific (H- AMPKα1) wild-type (WT) and knock-out (KO) mice at 7 days after cecal ligation and puncture (CLP). Mice were subjected to CLP and received fluid resuscitation (35 ml/kg normal saline with 5% dextrose subcutaneously) immediately after, at 3 h and every 24 h after the CLP procedure up to 72 h. *P < 0.05 of KO males vs. WT males as determined by Gehan-Breslow analysis.

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