Dihydromyricetin improves mitochondrial outcomes in the liver of alcohol-fed mice via the AMPK/Sirt-1/PGC-1α signaling axis

Highlights

• Dihydromyricetin (DHM), a bioactive flavonoid, improved mitochondrial outcomes in the liver of male C57BL/6J mice after chronic alcohol feeding.

• Alcohol inhibition of key metabolic enzymes, AMPK and Sirtuins, and mitochondrial injury are reversed with DHM administration.

• These findings support the utility of DHM, a dietary supplement, as a novel therapeutic for the reduction/prevention of alcohol-related mitochondrial injury in the liver.

• This dietary flavonoid provides key metabolic responses that support its use for other mitochondria-related disorders.

Abstract

Alcoholic liver disease (ALD), due to the multifactorial damage associated with alcohol (ethanol) consumption and metabolism, is one of the most prevalent liver diseases in the United States. The liver is the primary site of ethanol metabolism and is subsequently injured due to the production of reactive oxygen species (ROS), acetaldehyde, and metabolic stress. Building evidence suggests that dihydromyricetin (DHM), a bioactive flavonoid isolated from Hovenia dulcis, provides hepatoprotection by enhancing ethanol metabolism in the liver by maintaining hepatocellular bioenergetics, reductions of oxidative stress, and activating lipid oxidation pathways. The present study investigates the utility of DHM on hepatic mitochondrial biogenesis via activation of the AMP-activated protein kinase (AMPK)/Sirtuin (Sirt)-1/PPARG coactivator 1 (PGC)-1α signaling pathway. We utilized a forced drinking ad libitum study that chronically fed 30% ethanol to male C57BL/6J mice over 8 weeks and induced ALD pathology. We found that chronic ethanol feeding resulted in the suppression of AMPK activation and cytoplasmic Sirt-1 and mitochondrial Sirt-3 expression, effects that were reversed with daily DHM administration (5 mg/kg; intraperitoneally [i.p.]). Chronic ethanol feeding also resulted in hepatic hyperacetylation of PGC-1α, which was improved with DHM administration and its mediated increase of Sirt-1 activity. Furthermore, ethanol-fed mice were found to have increased expression of mitochondrial transcription factor A (TFAM), reduced mitochondrial content as assessed by mitochondrial DNA to nuclear DNA ratios, and significantly lower levels of hepatic ATP. In contrast, DHM administration significantly increased TFAM expression, hepatic ATP concentrations, and induced mitochondrial expression of respiratory complex III and V. In total, this work demonstrates a novel mechanism of DHM that improves hepatic bioenergetics, metabolic signaling, and mitochondrial viability, thus adding to the evidence supporting the use of DHM for treatment of ALD and other metabolic disorders.

Introduction

Alcoholic liver disease (ALD) is one of the most prevalent liver diseases in the United States, and it includes a spectrum of diseases ranging from reversible fatty liver to alcoholic hepatitis, and cirrhosis (Mellinger et al., 2018). The majority of patients diagnosed with ALD are also suffering from alcohol use disorder (AUD), where this latter condition affects over 17 million people in the United States. A concerning trend is the increased incidences of younger adults being diagnosed with ALD due to increasing rates of alcohol (ethanol) abuse. The mechanisms involved in the development of ALD are multifaceted, and it is becoming evident that the disease spectrum and subsequent progress to late-stage ALD with prolonged ethanol abuse results from the multifactorial injurious responses that occur throughout the body (Rehm et al., 2010; Rehm, Samokhvalov, & Shield, 2013; Seitz et al., 2018). Ongoing cycles of high levels of ethanol abuse and ethanol metabolism represent a primary mechanism of organ damage and is one major contributing factor resulting in the pathology of ALD and subsequent development of late-stage ALD (Han et al., 2012).

Ethanol is primarily metabolized in the liver by cytosolic alcohol dehydrogenase (ADH) and the inducible cytochrome P450 2E1 (CYP2E1) in mitochondria and endoplasmic reticulum (Lieber, Rubin, & DeCarli, 1970; Seitz et al., 2018). The metabolism of ethanol by ADH1 and CYP2E1 results in the oxidation of ethanol to acetaldehyde (ACH), a highly reactive and toxic metabolite, which is then further metabolized to acetate by aldehyde dehydrogenase (ALDH2) located in the mitochondria in the liver. Throughout this metabolic process, reactive oxygen species (ROS) formation occurs, with CYP2E1 oxidation contributing to much of the ROS stress in the liver (Leung & Nieto, 2013; Lieber, 1997; Neve & Ingelman-Sundberg, 2000). The combination of high rates of ethanol metabolism, increased ROS, and production of ACH results in multiple responses in the liver that dysregulate energy signaling and lipid metabolic pathways, alongside the induction of inflammatory responses (Ceni, Mello, & Galli, 2014; Seitz et al., 2018). Additionally, ACH and ROS alter mitochondrial structure and activity, thereby leading to functional impairment, including decreased oxidative phosphorylation (ATP generation), ROS exacerbation, and a decrease in ALDH2 activity, resulting in elevated ACH (O'Shea et al., 2010). Furthermore, the nicotinamide adenine dinucleotide (NAD)-dependent metabolism of ethanol/ACH, which results in the depletion of hepatic NAD⁺, contributes to additional metabolic stressors (French, 2016; Wang et al., 2018). In an effort to adapt to the ongoing hepatic injury from high levels of ethanol exposure, mitochondria undergo various responses, such as biogenesis, to maintain mitochondrial integrity and prevent further damage resulting from dysfunctional metabolic responses (Degli Esposti et al., 2012; Pessayre et al., 2012; Serviddio, Bellanti, Sastre, Vendemiale, & Altomare, 2010). Collectively, this work suggests that pharmacological agents that target these aspects of ethanol-mediated stress on mitochondria could be critical in protecting hepatocytes from energy impairments that result from mitochondrial-related stress in the liver.

One key regulator of mitochondrial biogenesis and cellular energy metabolism is the peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α), which belongs to the family of PGC-1 transcription coactivators (Handschin, 2009; Scarpulla, 2011). PGC-1α is present at low but inducible levels in the liver, where it also regulates most metabolic pathways, such as fatty acid β-oxidation, gluconeogenesis, and ketogenesis (Puigserver et al., 2003; Rhee et al., 2003; Yoon et al., 2001). Under normal physiological stress conditions (e.g., energy deprivation, fasting, and/or low temperatures), PGC-1α is activated via cyclic AMP response element-binding protein (CREB) and post-translationally via adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and NAD⁺-dependent sirtuin (SIRT)-1 deacetylation (Scarpulla, 2011). Notably, ethanol metabolism inhibits the AMPK-dependent phosphorylation of PGC-1α necessary for activation, and depletes NAD⁺ concentrations critical for Sirt-1-driven deacetylation, thereby inhibiting PGC-1α activation via ROS stress and energy depletion (Chaung, Jacob, Ji, & Wang, 2008; French, 2016; You, Jogasuria, Taylor, & Wu, 2015). This inhibition of mitochondrial biogenesis, in addition to mitochondrial stress and damage, results in the accumulation of dysfunctional mitochondria and reduced hepatic ability to selectively remove mitochondria by mitophagy (Eid, Ito, Maemura, & Otsuki, 2013; Williams, Ni, Ding, & Ding, 2015).

Dihydromyricetin (DHM), an active bioflavonoid isolated from Hovenia dulcis, has been used in Chinese traditional medicines for centuries, and has been shown to protect the liver against chemically induced liver damage via increased Sirt-1 signaling (Ma et al., 2019). In support of this hypothesis, we and others have shown that DHM can have multiple medicinal benefits, including anti-inflammatory, antioxidant, hepatoprotective, and anti-alcohol properties (Chen et al., 2015; Martínez-Coria, Mendoza-Rojas, Arrieta-Cruz, & López-Valdés, 2019; Qi et al., 2012; Shen et al., 2012; Silva et al., 2020). As part of this later effort, we found that DHM enhances ethanol metabolism in the liver, in part due to increased NAD⁺ concentrations. Moreover, we found that DHM can reduce hepatic lipid accumulation via AMPK activation in ethanol-fed male C57BL/6J mice (Silva et al., 2020). However, much remains to be elucidated regarding the mechanism by which these metabolic changes in response to ethanol occur. The current study addresses this issue by investigating the hepatoprotective role of DHM in the AMPK/Sirt-1/PGC-1α signaling axis and subsequent mitochondrial viability that is otherwise altered by chronic ethanol feeding.