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Activation of AMP-Activated Protein Kinase by A769662 Ameliorates Sepsis-Induced Acute Lung Injury in Adult Mice

Kitzmiller, Laura*; Ledford, John R.; Hake, Paul W.; O’Connor, Michael; Piraino, Giovanna; Zingarelli, Basilia†,‡

doi: 10.1097/SHK.0000000000001303
Basic Science Aspects
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ABSTRACT A serious consequence of sepsis is acute lung injury, whose severity is particularly impacted by the age of the patient. AMP-activated protein kinase (AMPK) is a crucial regulator of cellular metabolism, which controls mitochondrial biogenesis and autophagy. Here, we investigated the effect of pharmacological activation of AMPK with A769662 on lung injury by using a model that would preferably mimic the clinical condition of adult patients. Male C57BL/6 retired breeder mice (7–9 months old) were subjected to sepsis by cecal ligation and puncture (CLP). Mice received vehicle or A769662 (10 mg/kg) intraperitoneally at 1 h after CLP. At 6 h after CLP, vehicle-treated mice exhibited severe lung injury and elevation of plasma pro-inflammatory cytokines when compared with control mice. At molecular analysis, lung injury was associated with downregulation of AMPKα1/α2 catalytic subunits and reduced phosphorylation of AMPKβ1 regulatory subunit. Treatment with A769662 ameliorated lung architecture, reduced bacterial load in lung and blood, and attenuated plasma levels of interleukin-6. This protective effect was associated with nuclear phosphorylation of AMPKα1/α2 and AMPKβ1, increased nuclear expression of peroxisome proliferator-activated receptor γ co-activator-α and increased autophagy, as evaluated by the light-chain (LC)3B-I and LC3B-II content, without changes in sirtuin-1 cellular dynamics. Treatment with A769662 alone or in combination with the antimicrobial agent imipenem (25 mg/kg) increased survival rate (29% and 51%, respectively) when compared with vehicle treatment (10%) at 7 days after CLP. These data suggest that pharmacological activation of AMPK might be a beneficial approach for the treatment of sepsis in adult population.

*Division of Critical Care Medicine, Children's Hospital of Michigan, Wayne State University School of Medicine, Detroit, Michigan

Division of Critical Care Medicine, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio

Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, Ohio

Address reprint requests to Basilia Zingarelli, MD, PhD, Division of Critical Care Medicine, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail: basilia.zingarelli@cchmc.org

Received 28 September, 2018

Revised 16 October, 2018

Accepted 5 December, 2018

This work was supported by grant R01 GM-067202 from the National Institutes of Health to Basilia Zingarelli.

The authors report no conflicts of interest.

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INTRODUCTION

Sepsis is a life-threating organ dysfunction caused by dysregulated host responses to infection (1). Although comorbidities and chronic diseases contribute to the clinical variability, age is an independent risk factor of increased mortality to sepsis (2). The incidence of sepsis and the risk of sepsis-related mortality increases disproportionately in older adults when compared with patients younger than 50 years of age (3). These age-related differences among the adult population are observed both in early mortality during hospitalization and in 90-day mortality (3, 4).

Acute lung injury is a frequent complication of sepsis and is a leading cause of short-term mortality and long-term reduction in quality of life (5). Pathogenesis of acute lung injury is explained by injury to both the vascular endothelium and alveolar epithelium (6). However, the relative contribution of age-dependent mechanisms to susceptibility and outcomes of sepsis-induced lung injury remains unclear. Furthermore, very few effective strategies are available for treatment (5, 6).

The AMP-activated protein kinase (AMPK) is a conserved kinase, which controls energy homeostasis. The protein consists of a catalytic α-subunit and two regulatory β- and γ-subunits. The enzyme is activated upon a decrease of ATP and an increase of AMP, which causes allosteric changes of the enzyme allowing the phosphorylation of the catalytic α-subunit by upstream kinases (7). Although the kinase is ubiquitously expressed, there are different isoforms of the subunits: two for the α- and β-subunits, and three for the γ-subunit, which display tissue-specific distribution. In the lung, the catalytic α1 and the regulatory β1 and γ1 isoforms are abundantly expressed (7, 8). One of the mechanisms by which the kinase ensures energy homeostasis is through regulation of mitochondrial biogenesis and quality control by direct activation of the nuclear transcription cofactor, the peroxisome proliferator-activated receptor γ co-activator α (PGC-1α) (9). AMPK also controls the disposal of damaged mitochondria by autophagy, which allows for regulated cellular degradation and recycling of cellular parts for energy recovery (10).

Experimental studies have demonstrated that the activation capacity of AMPK signaling declines with aging, which impairs the maintenance of efficient energy homeostasis (11). Similarly, age-related dysregulation of autophagy has been observed and can contribute to cellular dysmetabolism (12). In previous experimental studies, we have demonstrated that age-dependent impairment of AMPK activation plays a pathogenic role in multiple organ injury in mature adult male mice when compared with young animals in models of sepsis and hemorrhagic shock (13–17).

To extend our understanding of the mechanisms that contribute to sepsis-induced acute lung injury, we sought to determine whether AMPK-dependent signaling pathways are altered in polymicrobial sepsis in mature adult mice (7–9 months), e.g., retired breeder mice at a stage where reproductive performance has ceased but senescence has not yet fully reached (18). We also wanted to investigate the therapeutic efficacy of AMPK activation by using the novel selective activator, A769662, that mimics the function of AMP on the β subunit, causing AMPK allosteric activation and inhibition of dephosphorylation of the catalytic subunit (19).

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MATERIALS AND METHODS

Murine model of polymicrobial sepsis

The investigation conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th edition, 2011) and had the approval of the Institutional Animal Care and Use Committee. Male C57BL/6 mice at an age of 7 to 9 months were obtained from Charles River Laboratories (Wilmington, Mass) and were acclimatized for at least 48 h. All mice were allowed free access to water and a maintenance diet in a 10-h light/14-h dark cycle, with room temperature at 21 ± 2°C. Mice were anesthetized by inhaled 1% isoflurane and polymicrobial sepsis was induced by an established model of cecal ligation and puncture (CLP) (20). After opening the abdomen, the cecum was exteriorized and ligated at its base without obstructing intestinal continuity. The cecum was punctured twice with a 23-G needle and squeezed to excrete a small amount of fecal material into the peritoneal cavity. The cecum was then returned into the peritoneal cavity and the abdominal incision was closed with a silk ligature suture. After the procedure, mice were randomly assigned to two treatment groups: the vehicle-treated group received 0.5% dimethyl sulfoxide (200 μL/mouse) intraperitoneally (i.p.); the A769662-treated group received the selective AMPKβ activator A769662 (10 mg/kg i.p.) at 1 h after CLP. All groups of mice also received fluid resuscitation (35 mL/kg normal saline with 5% dextrose subcutaneously) immediately after, at 3 and 12 h after the CLP procedure. To minimize pain at the surgical incision site, lidocaine hydrochloride (1%, 4 mg/kg total dose) was applied locally. Control mice did not undergo any surgical procedure. Mice were then sacrificed at 6 and 18 h after CLP and blood, lung, liver, spleen, and peritoneal fluid were collected for biochemical assays and bacterial clearance.

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Survival study

In a separate study, another cohort of mice was used for assessing survival rate in the presence or absence of antibiotics. Mice were divided into four treatment groups: a vehicle group, an imipenem group (25 mg/kg i.p.), an A769662 group (10 mg/kg i.p.), and a combined treatment A769662/imipenem group; treatment was given at 1 h and every 24 h after the CLP procedure up to 72 h. All groups of mice also received fluid resuscitation (35 mL/kg normal saline with 5% dextrose subcutaneously) immediately after, at 1 h and every 24 h after the CLP procedure up to 72 h. To minimize pain at the surgical incision site, lidocaine hydrochloride (1%, 4 mg/kg total dose) was applied locally immediately after the procedure and every 12 h up to 48 h. Survival was monitored for 7 days.

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Histopathologic analysis

Lungs were fixed in 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin and evaluated by three independent observers. Lung injury was analyzed by a semiquantitative score based on the following histologic features: alveolar capillary congestion, infiltration of red blood cells, and inflammatory cells into the airspace, alveolar wall thickness, and hyaline membrane formation (17). A score of 0 represented normal findings and scores of 1, 2, 3, and 4 represented minimal (<25% lung involvement), mild (25%–50% lung involvement), significant (50%–75% lung involvement), and severe (>75% lung involvement) injury, respectively. The four variables were summed to represent the lung injury score (total score, 0–16).

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Myeloperoxidase activity

Myeloperoxidase (MPO) activity was measured as an indicator of neutrophil infiltration in lung tissue after septic shock. Tissues were homogenized in a solution containing 0.5% hexa-decyl-trimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7.0) and centrifuged for 30 min at 4,000 × g at 4°C. An aliquot of the supernatant was allowed to react with a solution of tetra-methyl-benzidine (1.6 mM) and hydrogen peroxide (0.1 mM). The rate of change in absorbance was measured by spectrophotometry at 650 nm. MPO activity was defined as the quantity of enzyme degrading 1 μmol of hydrogen peroxide/min at 37°C and expressed in units per 100 mg weight of tissue.

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Cytosol and nuclear protein extraction

Lungs were homogenized in a buffer containing 0.32 M sucrose, 10 mM Tris-HCl (pH 7.4), 1 mM EGTA, 2 mM EDTA, 5 mM NaN3, 10 mM β-mercaptoethanol, 20 μM leupeptin, 0.15 μM pepstatin A, 0.2 mM phenylmethanesulfonyl fluoride, 50 mM NaF, 1 mM sodium orthovanadate, and 0.4 nM microcystin. Samples were centrifuged at 1,000 × g for 10 min at 4°C and the supernatants collected as cytosol extracts. The pellets were then solubilized in Triton buffer (1% Triton X-100, 250 mM NaCl, 50 mM Tris HCl at pH 7.5, 3 mM EGTA, 3 mM EDTA, 0.1 mM phenylmethanesulfonyl fluoride, 0.1 mM sodium orthovanadate, 10% glycerol, 2 mM p-nitrophenyl phosphate, 0.5% NP-40, and 46 uM aprotinin). The lysates were centrifuged at 15,000 × g for 30 min at 4° C and the supernatant collected as nuclear extracts.

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Western blot analysis

Cytosol and nuclear content of AMPKα1/2, AMPKβ1, and their phosphorylated forms pAMPKα1/2 and pAMPKβ1, PGC1-α, and sirtuin-1 (SIRT1) were determined by immunoblot analyses. Extracts were heated at 70°C in equal volumes of ×4 Protein Sample Loading Buffer. Twenty-five μg of protein were loaded per lane on a 10% Bis-Tris gel. Proteins were separated electrophoretically and transferred to nitrocellulose membranes. For immunoblotting, membranes were blocked with Odyssey blocking buffer and incubated with specific primary antibodies; β-actin was concomitantly probed as a loading control for both cytosol and nuclear proteins. Membranes were washed in PBS with 0.1% Tween 20 and incubated with LI-COR secondary antibodies. The Odyssey LI-COR scanner (LI-COR Biotechnology, Lincoln, Nebr) was used for detection and fold changes of relative intensity of proteins were calculated versus mean value of control mice upon data normalization with β-actin.

Cytosol content of the light-chain (LC)3B-I and LC3B-II was also determined by immunoblot analyses. Extracts were boiled in equal volumes of NuPAGE LDS Sample Buffer (×4) and 40 μg of protein loaded per lane on a 16% Tris-glycine gradient gel. Proteins were separated electrophoretically and transferred to nitrocellulose membranes. For immunoblotting, membranes were blocked with 5% nonfat dried milk in Tris-buffered saline (TBS) for 1 h and incubated with primary antibodies for 24 h. Membranes were washed in TBS with 0.1% Tween 20 and incubated with secondary peroxidase-conjugated antibody; the immunoreaction was visualized by chemiluminescence and x-ray. Membranes were also reprobed with primary antibody against β-actin to ensure equal loading for both cytosol and nuclear proteins. Densitometric analysis of blots was performed using Quantity One (Bio-Rad Laboratories, Des Plaines, Ill).

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Bacterial colony counts

Bacterial clearance was indirectly assessed by counting bacterial colonies in whole blood, peritoneal fluid, and homogenized samples of spleen, liver, and lung at 18 h after CLP surgery. Serial dilutions of samples were cultured on 5% sheep blood trypticase soy agar plates (BBL Stacker Plate TSA II, BD Biosciences, Sparks, Md) and incubated at 37°C in aerobic conditions for 24 h. Colony forming units (CFU) were counted and log-transformed to obtain a normal distribution. Results were expressed as log CFU per milliliter of blood or peritoneal lavage fluid and as log CFU per gram of tissues.

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Plasma levels of cytokines

Plasma levels of interleukin (IL) 1β, IL-10, IL-6, and tumor necrosis factor (TNF)α were evaluated by a commercially available multiplex array system (Milliplex, Millipore Corporation, Billerica, Mass) using the protocols recommended by the manufacturer.

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Materials

The AMPK activator A769662 was obtained from LC Laboratories (Woburn, Mass). The primary antibodies directed at AMPKα1/2, pAMPKα1/2, AMPK β1/2, pAMPKβ1, LC3B-I, and LC3B-II were obtained from Cell Signaling (Beverly, Mass); the primary antibody directed at PGC-1α, was obtained from Abcam (Cambridge, Mass). The primary antibodies directed at SIRT1 and β-actin and the secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif). The Odyssey blocking and loading buffers and LI-COR secondary antibodies were obtained from LI-COR Biotechnology (Lincoln, Nebr). All other chemicals were obtained from Sigma-Aldrich (St. Louis, Mo).

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Statistical analysis

Statistical analysis was performed using SigmaPlot 13.0 (Systat Software, San Jose, Calif). Data in figures and text are expressed means ± SEM or median with 25th and 75th percentiles of n observations (n = 4–8 animals for each group). The results were examined by analysis of variance followed by the Student–Newman–Keuls's correction post hoc t test. Statistical analysis of damage scores was performed using the Mann–Whitney Rank Sum test; when normality and equal variance passed, data were further analyzed by t test. The Gehan–Breslow and log-rank tests were used to compare differences in survival rates (n = 20–35 animals for each group). A value of P < 0.05 was considered significant.

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RESULTS

Treatment with A769662 ameliorates lung injury and reduces neutrophil infiltration

At 6 h after CLP, lung injury in vehicle-treated mice was characterized by large hemorrhagic areas, infiltration of inflammatory cells, and reduction of alveolar air space when compared with lungs of healthy control mice (Fig. 1, A and B). Treatment with A769662 significantly reduced lung architecture derangement when compared with vehicle treatment (Fig. 1, C and D). To further confirm the degree of neutrophil infiltration, we measured activity of MPO, a lysosomal enzyme specific to neutrophils. Vehicle-treated mice exhibited a marked increase in MPO activity at 6 h after CLP when compared with lungs of control mice. Treatment with A769662 significantly reduced lung MPO activity (Fig. 1E).

Fig. 1

Fig. 1

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Treatment with A769662 reduces plasma levels of IL-6

To evaluate the sepsis-induced systemic inflammatory response, a panel of Th1/Th2 cytokines was measured. At 6 h after CLP, plasma levels of IL-1β, IL-6, IL-10, and TNFα were significantly increased in vehicle-treated mice when compared with control mice. Treatment with A769662 significantly reduced plasma levels of IL-6 when compared to vehicle treatment; whereas it did not affect plasma levels of TNFα, IL-1β or IL-10 (Fig. 2).

Fig. 2

Fig. 2

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Treatment with A769662 decreases bacterial load in lungs and blood

To determine the effect of A769662 on the capability to eliminate bacterial pathogens, we counted bacterial CFU in blood, peritoneal fluid, lung, spleen, and liver at 18 h after CLP. Notably, A769662 treatment yielded a significant reduction of bacterial CFU in lungs and blood when compared with vehicle treatment. There was no difference in the number of bacterial colonies in peritoneal fluid, spleen, or liver after treatment with A769662 when compared with vehicle-treated mice (Table 1).

Table 1

Table 1

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Treatment with A769662 improves survival rate

In survival studies, mice were treated with A769662 or the antimicrobial agent imipenem alone, or a combination of imipenem with the AMPK activator at 1 h and every 24 h after the CLP procedure up to 72 h. Survival rate was monitored up to 7 days. We observed high mortality within 48 h and only 10% of mice survived up to 7 days when treated with vehicle and fluid resuscitation only. Treatment with A769662 or antibiotics alone improved survival rate when compared with vehicle treatment (P < 0.05). Animals treated with the combination of antibiotics with A769662 had a better survival rate when compared with vehicle (P < 0.0001) or A769662 treatment alone (P < 0.05) (Fig. 3).

Fig. 3

Fig. 3

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Treatment with A769662 increases nuclear phosphorylation of AMPKα in the lung

To confirm the mechanism of action of A769662 on AMPK activation, we evaluated the intracellular localization and phosphorylation of both catalytic α1 and α2 subunits (Fig. 4). The cytosol and nuclear content of total AMPKα1/α2 and pAMPKα1/α2, but not the ratio, decreased at 6 h after sepsis in vehicle-treated mice when compared with baseline content of control healthy mice. Treatment with A769662 significantly increased the ratio of the phosphorylated/total forms in the nuclear compartment, while decreasing the ratio in the cytosol (Fig. 4, A and B), thus suggesting the occurrence of activation in the nucleus.

Fig. 4

Fig. 4

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Treatment with A769662 increases nuclear phosphorylation of AMPKβ1 in the lung

Since the autophosphorylation at serine 108 (Ser108) of the AMPKβ1 subunit is necessary for A769662 mechanism of action (19), we also evaluated intracellular changes of the AMPKβ1 subunit. The cytosol and nuclear content of the total AMPKβ1 was unchanged, whereas the phosphorylated form significantly decreased at 6 h after sepsis in vehicle-treated mice when compared with baseline content of control healthy mice. Treatment with A769662 significantly increased the ratio pAMPKβ1/AMPKβ1 in the nuclear compartment (Fig. 4, A and C).

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Treatment with A769662 increases nuclear expression of PGC1-α in the lung

We then investigated the nuclear translocation PGC-1α, an AMPK downstream molecule involved in metabolic recovery and mitochondrial biogenesis (9). At 6 h after sepsis there were no significant changes in cytosolic or nuclear levels of PGC-1α in the vehicle-treated mice when compared with baseline content of control healthy mice. Treatment with A769662 significantly decreased cytosolic levels of PGC-1α, while increasing its nuclear expression (Fig. 4, A and D).

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Treatment with A769662 increases nuclear expression of SIRT1 in the lung

Since SIRT1 may participate in activating PGC-1α (9), we evaluated the cellular localization of this deacetylase (Fig. 4, A and E). At 6 h after sepsis there were no significant changes in cytosolic levels of SIRT1 in vehicle-treated mice when compared with baseline content of control healthy mice. In the nucleus two SIRT1 bands were detected, a finding that is consistent with post-translational events such as glycosylation, phosphorylation, or ubiquitination (21). These bands were markedly downregulated at 6 h after sepsis in vehicle-treated mice when compared with baseline content of control healthy mice. Treatment with A769662 did not affect the cytosol or nuclear levels of SIRT1 (Fig. 4, A and E).

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Treatment with A769662 induces autophagy in the lung

We next determined the capacity to mount an autophagic process by measuring the conversion of LC3B-I to LC3B-II, a marker for autophagosome formation (10). At 6 h after CLP, LC3B-I significantly decreased, while LC3B-II increased in vehicle-treated mice when compared with baseline content of control healthy mice. Treatment with A769662 induced increase of both LC3B-I and LC3B-II expression in the lung, thus suggesting the capability to enhance autophagy (Fig. 5).

Fig. 5

Fig. 5

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DISCUSSION

Incidence of multiple organ failure and mortality rates associated with sepsis increases with adult and elderly age (2, 4). Even with the application of standard procedures of resuscitation and mechanical ventilation, lung injury still remains a treatment challenge in intensive care units (ICU) (5, 6). The limited availability of mouse models that truthfully mimic the course and progression of human sepsis in the adult population and their response to novel therapies has emerged as one of the most limiting factors of translational sepsis research (22). Recent studies from our laboratory demonstrated that AMPK is an important pathway to maintain energy metabolism under pathological processes of multiple organ failure (13–17). We have demonstrated that the metabolic AMPK pathway becomes dysfunctional with aging and this impairment correlates with liver and cardiac injury in mature adult male mice when compared with young animals after sepsis (13, 14). Similarly, we previously reported that age-dependent AMPK dysregulation plays a pathogenetic role in myocardial and lung injury in mature adult male mice after hemorrhagic shock (15–17). These findings have also implied novel therapeutic strategies of activating AMPK with chemical agonists. AMPK activators can be divided into three classes: AMP mimetics, such as 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), drugs inhibiting mitochondrial ATP production, such as the antidiabetic drug metformin, and allosteric activators such as A769662 (23). In our previous studies we have demonstrated the therapeutic advantages of AICAR and metformin in sepsis- or hemorrhage-induced multiple organ failure (13–17). To confirm the potential clinical translation of pharmacological AMPK targeting in an aging population, we have extended these initial findings in the current study by demonstrating that impairment of AMPK activation is a likely underlying mechanism of sepsis-induced lung injury in retired breeder mice. Furthermore, we have demonstrated for the first time that the scaffold regulatory subunit AMPKβ1 is a potential therapeutic target as the selective pharmacological AMPKβ1 activator A769662 exerted pulmonary protective effects. We found, in fact, that A769662 treatment attenuated lung injury and infiltration of inflammatory cells, reduced the systemic release of the pro-inflammatory cytokine IL-6, and, when given as an adjunctive treatment to the standard fluid resuscitation and antibiotics, improved survival rate of septic mice.

We investigated the potential mechanisms of action of A769662 by analyzing the contribution of the AMPK signaling pathway. AMPK is a trimeric molecule consisting of one catalytic α-subunit and two regulatory β- and γ-subunits. Each subunit is expressed as different isoforms (two isoforms α1 and α2, two isoforms β1 and β2, and three isoforms γ1, γ2, and γ3), which display tissue-specific distribution (7, 8). AMPK is regulated both allosterically upon AMP binding to the γ-subunit and by post-translational modifications, such as phosphorylation of the catalytic α-subunit by upstream kinases. AMPKβ subunits are also phosphorylated at multiple sites. In the case of AMPKβ1, phosphorylation of Ser108 increases the activity of the AMPK heterotrimeric complex (24). The thienopyridone drug A769662 has previously been reported to activate AMPK both allosterically and by inhibiting AMPK dephosphorylation (19). Its mode of action does not involve binding to the γ subunit, in contrast to other AMPK activators, such as AMP or AICAR. On the contrary, its effect depends on the presence of the β1-subunit and the phosphorylation of the β1-subunit on Ser108 because a S108A mutation in the β1-subunit almost completely abolishes activation (19). Furthermore, several studies, including ours, have demonstrated that the heterotrimeric complex of AMPK is present both in the nucleus and cytoplasm and its distribution changes according to the need of metabolic recovery (13–17, 25, 26). As exemplified in Figure 6, in our study, we observed that sepsis-induced lung injury was associated with failure to upregulate activity of AMPK most probably consequent to the marked reduction of total content of AMPKα1/α2 in both cytosol and nuclear compartments in vehicle-treated mice. Sepsis-induced changes in AMPKβ1 phosphorylation could also play a role in the inability of the kinase to respond to the cellular stress, since we also observed a marked downregulation of the Ser108-phosphorylated form of AMPKβ1 in both cytosol and nuclear compartments. This AMPK failure in sepsis-induced lung injury may be related to the aging process of retired breeder mice. Our data are, in fact, consistent with our previous findings demonstrating that multi-organ inability of AMPK to respond to acute stressor of sepsis or hemorrhage occurs only in aging adult mice but not in young animals (13–17). Interestingly, treatment with A769662 significantly increased phosphorylation of the catalytic and the regulatory AMPK subunits in the nuclear compartment. The drug-induced AMPK nuclear localization and activation was paralleled by the increased expression of PGC-1α, the nuclear metabolic effector of AMPK (9), thus raising the possibility that A769662 might improve lung recovery through activation of mitochondrial biogenesis.

Fig. 6

Fig. 6

There is extensive evidence that SIRT1 shares several targets with AMPK and may increase the nuclear PGC-1α activity by deacetylation (9). SIRT1 resides mainly in the nucleus but can shuttle from the nucleus to the cytosol (27). In our study, we detected two SIRT1 bands in the nucleus, a finding that is consistent with post-translational events such as glycosylation, phosphorylation, or ubiquitination (21). Both bands were downregulated after sepsis in vehicle-treated mice, thus suggesting that different active forms of SIRT1 are impaired during lung injury. Treatment with A769662 did not affect the sepsis-induced downregulation of the nuclear SIRT1. Interestingly, in a recent study we have observed that pharmacological activation of AMPK may facilitate the nuclear translocation of SIRT1 in hemorrhage-induced lung injury in young mice only but not in adult middle-age mice (17). Taken together, these data suggest that SIRT1 activity is modulated by AMPK pathway in an age-dependent manner, since in aged organs pharmacological activation of AMPK fails to restore the nuclear localization of the sirtuin.

Extensive research has previously established that neutrophil accumulation along the endothelium of lung vasculature and infiltration into the interstitial and alveolar space is a critical pathophysiological event of sepsis-induced lung injury both in humans and animals (28). In our study, A769662 treatment of retired breeder mice resulted in amelioration of lung alveolar architecture after sepsis and significant decrease of parenchymal neutrophil infiltration. Thus, our results suggest that the metabolic effects of AMPK activation may extend to improvement of endothelial barrier function. This result agrees with another report demonstrating that activation of AMPK inhibits endotoxin-induced pro-inflammatory responses in murine neutrophils and diminishes the severity of endotoxin-induced acute lung injury in young mice (29).

In previous studies we have demonstrated that pharmacological activation of AMPK by the AMP mimetic, AICAR, had an age-dependent effect on cytokine release since it did not affect plasma levels of cytokines in mature adult mice, whereas it reduced Th2 and Th17 immune response in young mice at 18 h after sepsis (14). In the current study, to further elucidate the possible protective mechanisms triggered by the AMPK allosteric activator A769662 in retired breeder mice, we analyzed the production of pro- and anti-inflammatory cytokines at an early time after sepsis (i.e., 6 h after CLP). Interestingly, treatment with A769662 significantly reduced the plasma elevation of the pro-inflammatory IL-6, without affecting other cytokines. It should be noted, however, that we did not measure cytokines at later time points after sepsis. As we have previously shown, in our model of sepsis, plasma levels of IL-1β and IL-6 are sustained at high levels, whereas plasma levels of TNFα and IL-10 further increase as the sepsis severity progresses at 18 h after CLP (14). Thus, it is possible that our observation of a lack of effect of A769662 on other cytokines could of be due to the one time point sampling after the onset of disease. Nevertheless, elevated plasma levels of IL-6 are associated with morbidity and mortality in patients with acute lung injury (30). Thus, our results suggest that reduction of IL-6 production may contribute to the beneficial effects of the allosteric AMPK activator. Although the cellular sources and mechanisms of IL-6 inhibition need further investigation, previous reports have provided evidence that A769662 may inhibit IL-6 secretion by human endothelial cells in both AMPK-dependent and -independent mechanisms (31).

Another downstream pathway of AMPK activation is autophagy via inhibition of mTOR (the mammalian target of rapamycin) signaling. Autophagy is a cellular process that degrades damaged mitochondria and other organelles (10). At examination of molecular events, the conversion of LC3B from its free form (LC3B-I) to its phosphatidyl-ethanol-amine-conjugated form (LC3B-II) is a required step in autophagosome formation (10). Although autophagy has been shown to decline with age (12), in our study we found that LC3B-I and LC3B-II forms increased in the lung after sepsis in vehicle-treated retired breeder mice, thus suggesting the capability to mount an autophagic event in response to cellular stress. Remarkably, treatment with A769662 further increased the expression of the LC3B-I and LC3B-II proteins, suggesting that increase of autophagy may be an additional protective mechanism of the drug in mitigating lung injury.

Efficient microbial clearance is essential for survival from polymicrobial sepsis. Remarkably, even in the absence of antimicrobial therapy, a significant reduction in bacterial load was noted in the lungs and blood of A769662-treated mice at 18 h after the CLP procedure, suggesting the potential role of AMPK activation in pathogen clearance. Other studies have provided evidence that activators of AMPK, such as metformin, may improve the capacity for bacterial eradication in murine models of bacterial lung infection and sepsis (32). However, these studies have used juvenile mice of 10 to 12 weeks of age. Thus, our study is the first report that AMPK may improve that immune capability even in aged mice. The precise mechanisms by which AMPK activation may improve bacteria clearance remain to be elucidated. In vitro studies have demonstrated that pharmacological activation of AMPK by AICAR enhanced the bacterial phagocytic and intracellular killing activities of cultured microglia, macrophages, and neutrophils (33). However, in our study, reduction of bacterial load was not observed in liver, spleen, or peritoneal cavity after treatment with A769662. This inconsistent effect on bacterial clearance in various organs may be related to the time of bacterial detection after the CLP procedure. Our results are, in fact, in accordance with other studies in experimental models of sepsis demonstrating that differences in bacterial loads depend on the stage of the infectious disease (34). Another possible explanation is that AMPK activation may promote distinct organ-specific phagocytosis signaling, which needs to be investigated.

One of the most notable observations in our study was that treatment alone with A769662 improved survival in retired breeder mice subjected to sepsis. To mimic human sepsis management, mice were resuscitated with fluids and treated with antibiotics. Interestingly, the combination of antibiotics with A769662 further increased survival benefit resulting in 51% survived mice when compared with only 10% survival rate in the vehicle-treated group. Although we cannot exclude that maximal efficacy of this combined strategy may require prolonged treatment or dosage increase, our data showed that even with the early cessation of treatment at 72 h after CLP, the survival benefit was long lasting and was observed up to 7 days. It is important to note that several pharmacological agents have been used extensively to activate AMPK; among them, metformin activates AMPK through intracellular changes of AMP and is widely used to treat patients with type 2 diabetes (23). A recent retrospective clinical study reported that preadmission use of metformin, due to pre-existing dysmetabolic conditions, was associated with reduced 30-day mortality among adult ICU patients (35). Taking these observations into account, our findings suggest that pharmacological activation of AMPK may be a novel adjunctive approach for treating sepsis.

In conclusion, our data indicates that sepsis-induced acute lung injury during adult mature age in mice is associated with dysregulation of the AMPK metabolic pathway and that selective pharmacological activation of AMPKβ1 subunit exerts a pulmonary protective effect and affords a survival advantage. Thus, our data suggests that targeting AMPK may provide an effective adjunct therapy for the treatment of sepsis.

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Keywords:

AMPKβ; AMPKα; autophagy; LC3B; PGC-1α; sepsis; SIRT1

© 2019 by the Shock Society