Ethanol Exposure Impairs AMPK Signaling and Phagocytosis in Human Alveolar Macrophages: Role of Ethanol Metabolism

Abstract

Background: Chronic alcohol consumption impairs alveolar macrophage's (AM) function and increases risk for developing lung infection and pneumonia. However, the mechanism and metabolic basis of alcohol-induced AM dysfunction leading to lung infection are not well defined, but may include altered ethanol (EtOH) and reactive oxygen species metabolism and cellular energetics. Therefore, oxidative stress, endoplasmic reticulum (ER) stress, the formation of fatty acid ethyl esters [FAEEs, nonoxidative metabolites of EtOH], AMP-activated protein kinase (AMPK) signaling, and phagocytic function were examined in freshly isolated AM incubated with EtOH.Methods: AMs separated from bronchoalveolar lavage fluid samples obtained from normal volunteers were incubated with EtOH for 24 hours. AMPK signaling and ER stress were assessed using Western blotting, FAEEs by GC-MS, oxidative stress by immunofluorescence using antibodies to 4hydroxynonenal, and phagocytosis by latex beads. Oxidative stress was also measured in EtOH-treated AMs with/without AMPK activator [5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)] or inhibitor (Compound C), and in AMs incubated with FAEEs. mRNA expression for interleukins (IL-6 and IL-8), monocyte chemoattractant protein (MCP)-1, and transforming growth factor (TGF)-b was measured in AM treated with EtOH or FAEEs using RT-PCR.Results: EtOH exposure to AM increased oxidative stress, ER stress, and synthesis of FAEEs, decreased phosphorylated AMPK, and impaired phagocytosis. Attenuation or exacerbation of EtOHinduced oxidative stress by AICAR or Compound C, respectively, suggests a link between AMPK signaling, EtOH metabolism, and related oxidative stress. The formation of FAEEs may contribute to EtOH-induced oxidative stress as FAEEs also produced concentration-dependent oxidative stress. An increased mRNA expression of IL-6, IL-8, and MCP-1 by FAEEs is key finding to suggest a metabolic basis of EtOH-induced inflammatory response.Conclusions: EtOH-induced impaired phagocytosis, oxidative stress, ER stress, and dysregulated AMPK signaling are plausibly associated with the formation of FAEEs and may participate in the pathogenesis of nonspecific pulmonary inflammation.

Activation of AMP-activated protein kinase attenuates ethanol-induced ER/oxidative stress and lipid phenotype in human pancreatic acinar cells

“…Extraction and analysis of FAEEs formed by hPACs treated with 1,2-13 C-EtOH in the presence/absence of AMPKα activator (AICAR) were performed as described previously [24]. Triglyceride content in hPACs treated with EtOH with/ without AICAR was measured using a triglyceride colorimetric assay kit as per manufacturer's instructions (Cat # 10010303, Cayman Chemical, Ann Arbor, MI).…”

“…AMPKα is a crucial regulator of energy metabolic homeostasis, and its inactivation plays a significant role in key cellular events that are involved in the pathogenesis of various diseases [23]. More recently, EtOH-induced AMPKα dysregulation has been linked to ER stress in alveolar macrophages and the liver [24,25]. However, the role of AMPKα in ER stress and its link towards the initiation and progression of ACP is not well understood.…”

Differential cytotoxicity, ER/oxidative stress, dysregulated AMPKα signaling, and mitochondrial stress by ethanol and its metabolites in human pancreatic acinar cells

“…Experimental evidence suggests that dysregulation of unfolded protein response (UPR) induced by endoplasmic reticulum (ER) stress in acinar cells after chronic alcohol consumption promotes the development of pancreatitis (Lugea et al, 2017a; Pandol et al, 2010). On the other hand, dysregulation of AMP‐activated protein kinase (AMPKα), a major regulator of cellular metabolism and ER/oxidative stress (Kim et al, 2015; Steinberg & Kemp, 2009), plays a key role in the pathogenesis of various diseases, including EtOH‐related disorders (Chen et al, 2010; Kaphalia et al, 2019; Liangpunsakul et al, 2010; Shearn et al, 2014; Srinivasan et al, 2019). An underlying link between EtOH‐induced AMPKα inactivation and ER/oxidative stress in relation to pancreatic acinar cell injury, and its inflammatory responses and cellular bioenergetics could be key factors for the initiation of ACP (Srinivasan et al, 2020).…”

RiboTag-Seq Reveals the Activation of the Unfolded Protein Response in Striatal Microglia Induced by Ethanol Withdrawal

“…In order to further interpret this gene module, we performed over-representation analysis on this gene set, which is analogous to GSEA and we found that, among these were gene sets relating to the unfolded protein response (UPR). This result is intriguing because the UPR has been associated with ethanol toxicity and withdrawal in other tissues 42 and other cell types including macrophages 43 but has not been identified in microglia in the central nervous system. Interestingly, Erickson and colleagues detected altered expression of a module involving response to unfolded protein in both cortical microglia and astrocytes in acutely intoxicated mice after CIE whereas we observed this module to be reduced in EtOH-0h animals compared to Air controls but increased after withdrawal 38 .…”