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Mesenteric ischemia and reperfusion (I/R) injury can ensue from a variety of vascular diseases and represents a major cause of morbidity and mortality in intensive care units. It causes an inflammatory response associated with local gut dysfunction and remote organ injury. Adenosine monophosphate-activated protein kinase (AMPK) is a crucial regulator of metabolic homeostasis. The catalytic α1 subunit is highly expressed in the intestine and vascular system. In loss-of-function studies, we investigated the biological role of AMPKα1 in affecting the gastrointestinal barrier function. Male knock-out (KO) mice with a systemic deficiency of AMPKα1 and wild-type (WT) mice were subjected to a 30 min occlusion of the superior mesenteric artery. Four hours after reperfusion, AMPKα1 KO mice exhibited exaggerated histological gut injury and impairment of intestinal permeability associated with marked tissue lipid peroxidation and a lower apical expression of the junction proteins occludin and E-cadherin when compared to WT mice. Lung injury with neutrophil sequestration was higher in AMPKα1 KO mice than WT mice and paralleled with higher plasma levels of syndecan-1, a biomarker of endothelial injury. Thus, the data demonstrate that AMPKα1 is an important requisite for epithelial and endothelial integrity and has a protective role in remote organ injury after acute ischemic events.

The defective eradication of invading pathogens is a major cause of death in sepsis. As professional phagocytic cells, macrophages actively engulf/kill microorganisms and play essential roles in innate immune response against pathogens. Growth differentiation factor 3 (GDF3) was previously implicated as an important modulator of inflammatory response upon acute sterile injury. In this study, administration of recombinant GDF3 protein (rGDF3) either before or after CLP surgery remarkably improved mouse survival, along with significant reductions in bacterial load, plasma pro-inflammatory cytokine levels, and organ damage. Notably, our in vitro experiments revealed that rGDF3 treatment substantially promoted macrophage phagocytosis and intracellular killing of bacteria in a dose-dependent manner. Mechanistically, RNA-seq analysis results showed that CD5L, known to be regulated by liver X receptor α (LXRα), was the most significantly upregulated gene in rGDF3-treated macrophages. Furthermore, we observed that rGDF3 could promote LXRα nuclear translocation and thereby, augmented phagocytosis activity in macrophages, which was similar as LXRα agonist GW3965 did. By contrast, pre-treating macrophages with LXRα antagonist GSK2033 abolished beneficial effects of rGDF3 in macrophages. In addition, rGDF3 treatment failed to enhance bacteria uptake and killing in LXRα-knockout (KO) macrophages. Taken together, these results uncover that GDF3 may represent a novel mediator for controlling bacterial infection.

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.

Age represents a major risk factor for multiple organ failure, including cardiac dysfunction, in patients with sepsis. AMP-activated protein kinase (AMPK) is a crucial regulator of energy homeostasis that controls mitochondrial biogenesis by activation of peroxisome proliferator-activated receptor-γ coactivator-1α and disposal of defective organelles by autophagy. We investigated whether AMPK dysregulation contributes to age-dependent cardiac injury in young (2–3 mo) and mature adult (11–13 mo) male mice subjected to sepsis by cecal ligation and puncture and whether AMPK activation by 5-amino-4-imidazole carboxamide riboside affords cardioprotective effects. Plasma proinflammatory cytokines and myokine follistatin were similarly elevated in vehicle-treated young and mature adult mice at 18 h after sepsis. However, despite equivalent troponin I and T levels compared with similarly treated young mice, vehicle-treated mature adult mice exhibited more severe cardiac damage by light and electron microscopy analyses with more marked intercellular edema, inflammatory cell infiltration, and mitochondrial derangement. Echocardiography revealed that vehicle-treated young mice exhibited left ventricular dysfunction after sepsis, whereas mature adult mice exhibited a reduction in stroke volume without apparent changes in load-dependent indexes of cardiac function. At molecular analysis, phosphorylation of the catalytic subunits AMPK-α1/α2 was associated with nuclear translocation of peroxisome proliferator-activated receptor-γ coactivator-1α in vehicle-treated young but not mature adult mice. Treatment with 5-amino-4-imidazole carboxamide riboside ameliorated cardiac architecture derangement in mice of both ages. These cardioprotective effects were associated with attenuation of the systemic inflammatory response and amelioration of cardiac dysfunction in young mice only, not in mature adult animals. NEW & NOTEWORTHY Our data suggest that sepsis-induced cardiac dysfunction manifests with age-dependent characteristics, which are associated with a distinct regulation of AMP-activated protein kinase-dependent metabolic pathways. Consistent with this age-related deterioration, pharmacological activation of AMP-activated protein kinase may afford cardioprotective effects allowing a partial recovery of cardiac function in young but not mature age.

Despite therapeutic advances in hemorrhagic shock, mortality from multiple organ failure remains high. AMP-activated protein kinase (AMPK) is involved in cellular energy homeostasis. Two catalytic subunits, α1 and α2, have been identified, with α1 subunit largely expressed in major organs. Here, we hypothesized that genetic deficiency of AMPKα1 worsens hemorrhage-induced multiple organ failure. We also investigated whether treatment with metformin, a clinically used drug for metabolic homeostasis, affords beneficial effects. AMPKα1 wild-type (WT) and knock-out mice (KO) were subjected to hemorrhagic shock by blood withdrawing followed by resuscitation with shed blood and Lactated Ringer’s solution and treatment with vehicle or metformin. Mice were sacrificed at 3 hours after resuscitation. Compared to vehicle-treated WT animals, KO animals exhibited a more severe hypotension, higher lung and liver injury and neutrophil infiltration, and higher levels of plasma inflammatory cytokines. Metformin treatment ameliorated organ injury and mean arterial blood pressure in both WT and KO mice, without affecting systemic cytokine levels. Furthermore, metformin treatment reduced liver lipid peroxidation and increased levels of complex II co-substrate FAD and levels of ATP in WT and KO mice. Beneficial effects of metformin were associated with organ-specific nuclear-cytoplasmic shuttling and activation of liver kinase B1 and AMPKα2. Thus, our data suggest that AMPKα1 is an important regulator of hemodynamic stability and organ metabolic recovery during hemorrhagic shock. Our data also suggest that metformin affords beneficial effects, at least in part, independently of AMPKα1 and secondary to AMPKα2 activation, increase of Complex II function and reduction of oxidative stress.

Age is an independent risk factor of multiple organ failure in patients with sepsis. However, the age-related mechanisms of injury are not known. AMPK is a crucial regulator of energy homeostasis, which controls mitochondrial biogenesis by activation of peroxisome proliferator-activated receptor-γ coactivator-α (PGC-1α) and disposal of defective organelles by autophagy. We investigated whether AMPK dysregulation might contribute to age-dependent liver injury in young (2–3 mo) and mature male mice (11–13 mo) subjected to sepsis. Liver damage was higher in mature mice than in young mice and was associated with impairment of hepatocyte mitochondrial function, structure, and biogenesis and reduced autophagy. At molecular analysis, there was a time-dependent nuclear translocation of the active phosphorylated catalytic subunits AMPKα1/α2 and PGC-1α in young, but not in mature, mice after sepsis. Treatment with the AMPK activator 5-amino-4-imidazolecarboxamide riboside-1-β-d-ribofuranoside (AICAR) improved liver mitochondrial structure in both age groups compared with vehicle. In loss-of-function studies, young knockout mice with systemic deficiency of AMPKα1 exhibited greater liver injury than did wild-type mice after sepsis. Our study suggests that AMPK is important for liver metabolic recovery during sepsis. Although its function may diminish with age, pharmacological activation of AMPK may be of therapeutic benefit.—Inata, Y., Kikuchi, S., Samraj, R. S., Hake, P. W., O’Connor, M., Ledford, J. R., O’Connor, J., Lahni, P., Wolfe, V., Piraino, G., Zingarelli, B. Autophagy and mitochondrial biogenesis impairment contribute to age-dependent liver injury in experimental sepsis: dysregulation of AMP-activated protein kinase pathway.

The development of multiple organ failure in patients with hemorrhagic shock is significantly influenced by patient age. Adenosine monophosphate–activated protein kinase (AMPK) is a crucial regulator of energy homeostasis, which coordinates metabolic repair during cellular stress. We investigated whether AMPK-regulated signaling pathways are age-dependent in hemorrhage-induced lung injury and whether AMPK activation by 5-amino-4-imidazole carboxamide riboside (AICAR) affords lung protective effects. Male C57/BL6 young mice (3–5 mo), mature adult mice (9–12 mo), and young AMPKα1 knockout mice (3–5 mo) were subjected to hemorrhagic shock by blood withdrawing, followed by resuscitation with shed blood and lactated Ringer’s solution. Plasma proinflammatory cytokines were similarly elevated in C57/BL6 young and mature adult mice after hemorrhagic shock. However, mature adult mice exhibited more severe lung edema and neutrophil infiltration, and higher mitochondrial damage in alveolar epithelial type II cells, than did young mice. No change in autophagy was observed. At molecular analysis, the phosphorylation of the catalytic subunit AMPKα1 was associated with nuclear translocation of peroxisome proliferator-activated receptor γ co-activator-α in young, but not mature, adult mice. Treatment with AICAR ameliorated the disruption of lung architecture in mice of both ages; however, effects in mature adult mice were different than young mice and also involved inhibition of nuclear factor-κB. In young AMPKα1 knockout mice, AICAR failed to improve hypotension and lung neutrophil infiltration. Our data demonstrate that during hemorrhagic shock, AMPK-dependent metabolic repair mechanisms are important for mitigating lung injury. However, these mechanisms are less competent with age.

Severity of multiple organ failure is significantly impacted by age and gender in patients with hemorrhagic shock. However, the molecular mechanisms underlying the enhanced organ injury are not fully understood. AMP-activated protein kinase (AMPK) is a pivotal orchestrator of metabolic responses during stress. We investigated whether hemorrhage-induced myocardial injury is age and gender dependent and whether treatment with metformin, an AMPK activator, affords cardioprotective effects. C57/BL6 young (3–5 months) and mature (9–12 months) male and female mice were subjected to hemorrhagic shock by blood withdrawing followed by resuscitation with blood and Lactated Ringer’s solution. Vehicle-treated young and mature mice of both genders had a similar elevation of plasma inflammatory cytokines at 3 hours after resuscitation. However, vehicle-treated male mature mice experienced hemodynamic instability and higher myocardial damage than young male mice, as evaluated by echocardiography, histology and cardiovascular injury biomarkers. There was also a gender-dependent difference in cardiovascular injury in the mature group as vehicle-treated male mice exhibited more severe organ injury than female mice. At molecular analysis, vehicle-treated mature mice of both genders exhibited a marked downregulation of AMPKα activation and nuclear translocation of peroxisome proliferator-activated receptor γ co-activator α when compared with young mice. Treatment with metformin improved cardiovascular function and survival in mature animals of both genders. However, specific cardioprotective effects of metformin were gender-dependent. Metformin did not affect hemodynamic or inflammatory responses in young animals. Thus, our data suggest that targeting metabolic recovery with metformin may be a potential treatment approach in severe hemorrhage in adult population.