Lipid composition influences the biophysical properties of membrane bilayers. In turn, membrane fluidity controls diverse membrane-associated processes, including membrane—protein contacts and function, signal transduction, and membrane fusion and fission events that ultimately regulate organelle biogenesis and growth, dynamics, and vesicular trafficking [43, 44]. In addition, lipids can act as signaling intermediates that trigger specific signaling pathways, as best illustrated by the hydrolysis of sphingomyelin by sphingomyelinases.

Focusing on the molecular organization of membrane lipids, they can generally be classified as phospholipids, sphingolipids, and sterols [45]. Phospholipids, the most abundant lipids in bilayers, are formed by a glycerol backbone with one (or two, in CL) phosphate group(s) together with fatty acid (FA) side chains [46]. The main phospholipids of mitochondrial membranes are PC and PE, accounting for 40–45% and 25–30% respectively of the total lipid composition, with phosphatidylinositol (PI) and CL representing 10–15%, while PS is the less abundant phospholipid (3–5%) [18–21]. The amount of these lipids differs between the IMM and OMM. For example, the IMM is enriched in PE and CL [21, 24].

In contrast, sphingolipids are characterized by a sphingoid backbone formed by the condensation between palmitoyl-CoA with serine [47]. Sphingosine is the singular sphingoid precursor, consisting of the palmitoyl-CoA-serine backbone. Fatty acyl chains of varying length acylate the sphingosine backbone to form sphingolipids of which ceramides are the most intensively studied. Ceramides are a heterogeneous family of sphingolipids that exhibit a large degree of FA of different lengths determined by 6 ceramide synthases (CerS1–CerS6), which exhibit different affinity towards short or long-chain FA. Ceramides, like most sphingolipids, are synthesized in the ER and then undergo exchange and transport to different membrane bilayers in the cell, including mitochondria through the ER-mitochondria contact sites [48, 49].

Not only does the presence of diverse fatty acyl chains with different lengths and saturation regulates the flexibility and stiffness of the bilayer, but also the existence of cholesterol is key in membrane fluidity regulation [50]. For instance, although the amount of cholesterol in mitochondrial membranes is low compared to its abundance in plasma membranes, the OMM is relatively enriched in this sterol compared to IMM.

Fatty acyl chains in the backbone of lipids can be oxidized by lipid peroxidation, which results in membrane bilayer damage thereby affecting cell integrity and organelle performance [51–54]. Oxidative damage to the mitochondrial lipidome induces mitochondrial dysfunction [55]. As mentioned above, CL is crucial for OXPHOS [23, 27, 56, 57], mitochondrial dynamics [58], ATP production, and apoptosis [59, 60]. Due to this key role in mitochondrial physiology, decreased CL levels caused either by low biosynthesis of CL, as in the rare genetic disorder of Barth syndrome, or CL loss by lipid peroxidation due to its susceptibility to ROS attach, compromise mitochondrial function with low OXPHOS activity and ATP generation [61].

Mitochondrial dynamics are controlled by lipid composition via diacylglyceride (DAG), PE, PS, and cholesterol, promoting negative membrane bends essential for mitochondrial fusion [62]. In addition, the import of proteins is pivotal for mitochondrial synthesis and activity. Many mitochondrial proteins are produced as precursors on cytosolic ribosomes and then are transferred to mitochondria. Phospholipids possess important functions in protein trafficking through and into mitochondrial membranes. Concretely, CL, PE, and PC distinctly influence protein translocases of both OMM and IMM, such as the translocase of the OMM (TOM) complex activity, which depends on OMM phospholipids [17], and the dynamic translocase of the inner membrane 23 (TIM23) and S-adenosylmethionine carrier (SAM) complexes. The association of phospholipids-proteins in specific domains displays a central task in the biogenesis and function of mitochondria [63, 64]. Modifications in the content of lipids within the organelle induce expanded, fragmented, and dysfunctional mitochondria, which is associated with diverse diseases [65].

MASLD is a multifaceted spectrum of liver alterations linked to genetic, epigenetic, and environmental risk factors that affect lipid homeostasis, altering the generation, oxidation, and release of free fatty acids (FFA), cholesterol, triglycerides (TG), and bile acids (BAs). First considered as a consequence of “two-hits” in a simplistic initial hypothesis, the pathogenesis of this complex liver disease was later recognized in the “multiple hits” hypothesis in which the accumulation of lipids in hepatocytes in the onset of MASLD sensitizes the liver to the action of several hits [91–93]. Therefore, steatosis can progress to advanced forms, such as metabolic-associated steatohepatitis (MASH), where lipid deposition coexists with inflammation, liver injury, and fibrosis. Then, it can progress to cirrhosis and culminate in hepatocellular carcinoma (HCC) [94, 95]. MASLD is recognized as the most prevalent chronic liver disease worldwide (25% occurrence of normal population) and more than 50% of type 2 diabetic and overweight patients suffer it [96]. Hence, the current definition of MASLD includes steatotic, overweight, type 2 diabetes, or metabolic dysregulated patients [97, 98].

During steatosis, impaired lipid and glucose metabolism arises principally from high-fat diet (HFD) consumption, resulting in diverse lipid accumulation (FFAs, DAG, TG, ceramides, and cholesterol). The complex process associated with mitochondrial proteome alteration and mitochondrial dysfunction is considered a major player in the transition from MASLD to MASH [99]. Mitochondria play a central role in the regulation of lipid metabolism and hence disruption of mitochondrial function contributes to hepatic steatosis [100].

Mitochondrial lipid variation has been described in patients at different stages of the MASLD continuum, with increased CL and ubiquinone in early MASLD but increased acylcarnitine in MASH [101]. The impact of these changes on mitochondrial function during MASLD is not well established. Patients with MASH display reduced activities of the respiratory chain complexes, which correlated with serum tumor necrotic factor-alpha (TNFα), and insulin resistance (IR) [101]. There are findings either indicating impaired respiration or increased mitochondrial mass but lower maximal respiration in obese subjects with MASH, which contrasts with reports of increased hepatic mitochondrial function in the same category of patients [102]. In line with the impaired mitochondrial performance, increased hepatic oxidative stress and oxidative DNA damage have been reported in parallel with reduced antioxidant defense [74, 103].

MASH patients also accumulate microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and sequestosome-1 (p62), a marker of Mallory-Denk bodies, which are associated with disease complications [104]. As mitochondrial quality control is regulated by mitophagy, intervention in this process modulates the implication of mitochondria in MASLD progression. In this regard, knocking down the cell survival regulator macrophage stimulating 1 (MST1), promoted PTEN-induced kinase 1 (PINK1)/Parkin-related mitophagy and reduced HFD-associated liver damage [105]. Besides, optic atrophy type 1 protein (OPA1) inhibition prevented mitophagy intermediates overload and mitigated methionine-choline-deficient (MCD) diet-promoted liver damage [106].

Mitochondrial morphology changes during MASLD in experimental models, with the appearance of fragmented morphology in parallel with Ca²⁺ increment, reduced OXPHOS activity, and increased ROS generation. Mitochondrial ROS generation has detrimental effects on the development of MASLD [107]. For instance, elevated ROS levels induce the production of cytokines and c-Jun N-terminal kinase (JNK) activation, which plays a feed-forward loop in mitochondrial dysfunction. This scenario leads to impaired β-oxidation, promoting FA accumulation and ATP depletion in hepatocytes while worsening insulin signaling. Damaged mitochondria accumulation within hepatocytes drives necrotic cell death and the leakage of DNA-enhanced mitochondria-induced danger-associated molecular patterns (DAMPs), such as mtDNA, N-formyl peptides, and ATP, which promote the inflammasomes NLR family pyrin domain containing 3 (NLRP3) and absent in melanoma 2 (AIM2) through toll-like receptors (TLRs) recognition [108–110]. It is also important to mention that the translocation of CL to the OMM acts as a docking site to recruit and bind inflammasome components like NLRP3. Furthermore, these signals promote TLR9 activation on Kupffer cells (KCs) and hepatic stellate cells (HSCs), and formyl peptide receptor 1 (FPR1) stimulation, which induces interferon regulatory factor (IRF) and nuclear factor kappa β (NFκβ) action [111]. In turn, the generation of inflammatory cytokines and activation of fibrogenic players establish a chronic inflammatory environment that participates in the progression of MASLD towards MASH and fibrosis (Figure 2). DAMPs have been detected in the plasma of MASH patients, linking mitochondrial dysfunction and inflammation during MASH [112]. Mitochondrial injury also promotes the depletion of nicotinamide adenine dinucleotide (NAD⁺/NADH) levels, which regulate an adaptive reaction to increased FFA hepatic levels [113] together with an augmented synthesis of mitochondrial free cholesterol and the JNK-dependent proinflammatory routes [114, 115]. The last promotes leakage of mitochondrial components via the mitochondrial permeability transition pore (mPTP) induction and hepatocyte death [116].

Concerning the transition from steatosis to MASH, pioneering observations pointed at free cholesterol increment. Its putative trafficking to mitochondria has emerged as an important player as shown in a cohort of patients with established MASH, which exhibited increased free cholesterol content and high expression of the steroidogenic acute regulatory protein (STARD1) compared to patients with simple steatosis [87]. These findings have been identified in the progression of MASLD towards advanced stages like HCC [122–124]. Thus, the cholesterol deposition at IMM by STARD1 has important negative consequences for the mitochondrial status, including the depletion of a crucial antioxidant defense like GSH, which reflects the defect in the activity of the 2-oxoglutarate carrier (2-OGC; SLC25A11) due to its sensitivity to cholesterol-promoted modifications in membrane fluidity [125, 126], and the impairment in the assembly of respiratory supercomplexes and thus OXPHOS [117, 127]. Hence, STARD1 activation reflecting increased mitochondrial cholesterol (mChol) levels [128], account for the mitochondrial GSH (mGSH) depletion found in MASH patients [129, 130], who also presented mitochondria ultrastructural abnormalities [131] and mitochondrial dysfunction [132]. In addition, the accumulation of cholesterol in KCs and HSCs accounts for stimulated inflammation and fibrosis, characteristic of the advanced stages of MASLD [87, 121, 122, 133]. Overall, the evidences in patients and experimental models indicate that the type rather than the amount of fat is a key player in MASLD progression, with the increase in cholesterol and in particular in mitochondrial membrane emerging as a putative new target for intervention to prevent progression of MASLD.

HCC is the main type of liver cancer and the second principal cause of cancer-related demise in the world due to the delay in diagnosis and modest therapeutic strategies, becoming a global health concern [134, 135]. HCC is the final stage of chronic liver diseases caused by several etiologies, including viral hepatitis, ARLD, or MASH [136, 137]. The pathogenesis of HCC is complex and multifactorial and, besides genetic factors and DNA damage, a plethora of players, including lipotoxicity, mitochondrial dysfunction, oxidative stress, inflammation, ER stress, and the disruption in Ca²⁺ homeostasis induce the perfect milieu for tumor development [136] (Figure 3).

Metabolic variations are abundant in cancer pathogenesis. HCC cells subsist in a potently abundant fat milieu [78, 147]. Peroxisome proliferator-activated receptor gamma co-activator 1β (PGC-1β) controls hepatic oxidative metabolism, mitochondria biogenesis, and antioxidant defense mechanisms. It also plays a pivotal function in cancer progression, supporting metabolic modifications by lipogenic enzyme activation, driving tumor growth and anabolism, and activating gene expression in FA and TG production [78]. Elevated PGC-1β levels activate the expression of ROS scavengers, such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX), glutathione reductase (GR), thioredoxin (Trx) and peroxiredoxins (Prx), reducing ROS overload and subsequent apoptosis. In contrast, PGC-1β knockdown protects mice from cancer development [78].

Linked to the progression of MASLD, unphysiological overload of mChol has been found in HCC and has been associated with tumor growth and malignancy [141, 142, 148]. As previously described, mChol promotes a reduction of the mitochondria membrane fluidity [84, 149], increasing the mitochondrial membrane order of HCC cancer cells [143, 150–152]. Paradoxically, this event does not decrease mGSH in cancer cells because of the adaptive overexpression of 2-oxoglutarate transporter (2-OGC or SLC25A11) through hypoxia-inducible factor-1 (HIF-1) stabilization [125, 126, 152]. Consequently, ATP production is supported via OXPHOS and glycolysis [152, 153], which is an intriguing vis-à-vis the reported negative effect of mChol in the assembly of mitochondrial respiratory supercomplexes to support OXPHOS [84]. Therefore, this scenario favors tumor growth by the synergism between protection against mitochondrial OMM permeabilization and defense against oxidative stress [117, 154]. In line with these effects of mChol in cancer cell survival, recent findings indicated that mChol overload promotes sorafenib resistance of HCC cells [155], validating the critical function of mChol in HCC progression.

As alluded to above, CL is vital for mitochondrial ATP production in the respiratory chain and for preserving IMM organization [28, 80]. Oxidative modifications of CL, due to ROS attack to double bonds of its FA constituents, not only influence CI, CIII, and complex IV (CIV) function [156–158] but also regulate programmed cell death by controlling Cyt C leakage and by attaching to the B-cell lymphoma 2 (Bcl-2) family protein Bid to promote Bax and Bak oligomerization and subsequent OMM permeabilization [34, 71, 79, 159, 160]. Thus, the outcome of mChol acting as a proapoptotic versus an antiapoptotic factor in HCC cells depends on the oxidized status of CL, which in turn depends on the mGSH levels. In stages pre-HCC like early MASLD, mChol accumulation causes mGSH depletion and CL peroxidation, which overall contribute to hepatocellular cell death and mitochondrial dysfunction. However, in established HCC, CL is intact due to increased expression of SLC25A11, contributing to apoptosis resistance and tumor growth promotion.

The transport and metabolism of cholesterol in IMM acts as an alternative pathway for the generation of BAs generation in the so-called mitochondrial acidic pathway [161–163]. BAs are synthesized in hepatocytes predominantly by the classic pathway, which is controlled by 7α-hydroxylase (CYP7A1), and to a lesser extent by the mitochondrial alternative pathway, which is determined by the availability of cholesterol in the IMM for its metabolism by sterol 27-hydroxylase (CYP27A1). As the transport of cholesterol to IMM is determined by STARD1, the overexpression of this carrier in MASLD dictates a unique role for STARD1 in stimulating BA synthesis in mitochondria. BAs are not only essential for fat digestion but they also regulate gene expression and promote inflammation. Thus, the expression of STARD1 favors the switch in the synthesis of BAs from the classic to the alternative pathway, and the generation of mitochondrial-derived chenodeoxycholic acid and its taurine forms have been described as a crucial step in the MASLD-driven HCC development [88].

ARLD encompasses a spectrum of hepatic alterations as the result of the ingestion of elevated doses of alcohol that comprises from steatosis, the first stage, to alcoholic hepatitis, cirrhosis, and HCC. Alcohol oxidative metabolism induces several events that are involved in ARLD progression, including the disruption in the balance between fat synthesis and degradation that underlies the onset of steatosis and the alterations in mitochondrial function and morphology.

Hepatic ethanol metabolism initiates with the enzyme alcohol dehydrogenase (ADH) transforming alcohol to acetaldehyde, which is then metabolized to acetate by the acetaldehyde dehydrogenase (ALDH). In addition, alcohol is also metabolized via the microsomal system cytochrome P450, CYP2E1, to acetaldehyde. Although the ADH has a higher affinity for alcohol than CYP2E1, the chronic consumption of alcohol induces the expression of CYP2E1 and becomes the preferential metabolic pathway for the metabolism of alcohol. In turn, acetaldehyde and the derived malonaldehyde can form protein adduct due to their reactivity, which can be enclosed by KCs, endothelial, and HSCs. This activates an inflammatory reaction that contributes to ARLD progression [164]. The molecular mechanisms that promote the deleterious effects of alcohol metabolism to cause ARLD are intricate and multifactorial and include disruption of lipid and methionine metabolism, alterations in mitochondrial dynamics, respiration, membrane structure, mtDNA oxidation, and ROS production [69], whose final impact in ARLD onset is determined by genetic and environmental factors.

Lipid organization influences membrane conformation and modifies the function of proteins embedded in the bilayer. Two major modifications have been documented in the lipid configuration of hepatic mitochondria from rodents fed with ethanol: elevated cholesterol content and depleted CL level. Changes in lipid composition directly affect the physical properties of the bilayer, as exemplified by the relative ratio of PC to PE and particularly by the cholesterol/phospholipid molar ratio, which is a pivotal factor of membrane fluidity. The length and saturation of the fatty acyl chains of the PC/PE molecular species is another factor that regulates physical membrane properties, but especially the increase in cholesterol in a particular bilayer restricts the rotation of acyl chains and determines the fluidity of the bilayer, which in turn can affect the activity of mitochondrial membrane proteins [75]. In this regard, alcohol consumption stimulates cholesterol content in hepatocytes and STARD1 expression, resulting in the trafficking and accumulation of cholesterol in mitochondrial membranes. Interestingly, the increase in the expression of STARD1 occurs in a zonal-dependent fashion with the predominant expression in the perivenous zone of the liver, coinciding with the predominant expression of CYP2E1 and the site of injury as a consequence of alcohol consumption [165]. The zonal-dependent increase in mChol by STARD1 causes mGSH depletion through impairment of the SLC25A11 function and the onset of oxidative stress, thus contributing to the pericentral damage caused by alcohol intake [125, 166]. As mentioned above, although mChol is the precursor for the synthesis of BAs in mitochondria and cholestasis is an accompanying complication of patients with alcoholic hepatitis, it remains to be established whether or not the increase in mChol by alcohol feeding in the pericentral zone contributes to the cholestatic manifestations in ARLD.

Mitochondria are dynamic organelles that relocate in the cytoskeleton and control their morphology and function by a fine-tuned and highly regulated fusion/fission process, which is pivotal for the preservation of mitochondrial tasks and the management of metabolism and cellular signaling [167]. The equilibrium between fission and fusion impacts mitochondrial morphology and adapts their function to diverse stresses. It is also associated with cellular division, apoptosis, and autophagy. Mitochondrial dynamics are regulated by mitochondria-shaping proteins (MSP), of which mitofusin 1 and 2 (MFN1/2) and OPA1 are involved in mitochondrial fusion, while the cytosolic dynamin-related protein 1 (Drp-1) has a crucial role in mitochondrial fission [168–170].

ARLD patients exhibit alterations in the morphology of hepatic mitochondria, with the pioneering description of the presence of megamitochondria (large and elongated mitochondria) as a result of an elevated mitochondrial fusion activity, which triggers mitochondrial elongation and is related to the induction of OXPHOS activity and mtDNA relocation in the mild stage of the ARLD spectrum [171]. In addition, patients with alcoholic hepatitis displayed an elevated expression of Drp-1 in severe stages of the disease, an event that correlates with the data described in human precision-cut hepatic slices incubated with different ethanol concentrations [169]. Thus, chronic alcohol ingestion induces mitochondrial hyperfragmentation through Drp-1 overexpression, promoting more serious hepatic damage [169], pointing at Drp-1 inhibition as a potential therapy to regenerate the balance between mitochondrial dynamics. In line with this possibility, Drp-1 genetic knockdown protected against alcohol-induced hepatotoxicity in VL-17A cells and abolished the growth impairment induced by ethanol exposure. Besides, mice with Drp-1 liver-specific deletion fed the acute-on-chronic alcohol model exhibited less liver injury and the appearance of megamitochondria, with similar findings observed in MASLD following Drp-1 inhibition, which caused decreased steatosis and oxidative stress [170, 172]. Thus, based on this existing evidence it is clear that the presence of megamitochondria is a positive adaptive reaction to alcohol intake and their presence in liver biopsies is a clinical and histological factor linked to a better prognosis in AH patients [173–176]. However, the elimination of megamitochondria via mitophagy is not an efficient process due to the size of this type of mitochondria, raising the question of whether the presence of megamitochondria is a transient process during the early adaptation to alcohol intake or not, which in case of persistence may contribute to mitochondrial maladaptation and disruption in the innate immune reaction that increases liver injury in the late phase of chronic ARLD [177–179]. The influence that the increase in cholesterol accumulation in mitochondria and subsequent change in membrane fluidity has on mitochondrial dynamics in ARLD remains to be fully established.

In comparison with nuclear DNA, mtDNA is highly susceptible to free radical attack since is not protected by histones, and it is located near the IMM, the main cellular ROS source. Mitochondria display several mtDNA copies, and while some degree of mtDNA oxidation can occur, this may not necessarily compromise mitochondrial function, providing that intact copies are still available [180]. Cells mainly remove damaged mtDNA and replicate intact copies to preserve the global mtDNA pool integrity. Thus, a remarkable transient decline of murine hepatic mtDNA was reported within hours after acute ethanol exposure, which was further prominent in older animals. This is consistent with the appearance of mtDNA fragments in the serum of patients with ARLD [181].

Alcohol intake and its oxidative metabolism induce oxidative stress, reflecting the unbalance between ROS generation and its scavenging by compromised antioxidant defense strategies. This disrupts mitochondrial organization and function, and subsequently drives to ROS formation in a vicious cycle of injury, which is more evident with aging. The “mitochondrial theory of aging” proposes that alterations in mtDNA overload compromise cellular energy metabolism, affecting cellular life span [182]. Besides O₂^•− and H₂O₂ generated by changes in cholesterol homeostasis in mitochondria by ethanol intake, these species can also generate hydroxyl radical (OH^–) by the Fenton reaction, which can attack mtDNA, leading to the release of purine and pyrimidine bases from mtDNA and strand breakdown [183]. Moreover, OH^– directly modifies purine and pyrimidine bases, generating 8-oxoguanine (8-oxoG), which is regularly used as an indicator of free radical damage to DNA. It has been demonstrated that 8-oxoG and mtDNA strand break accumulation were remarkably increased in the livers of long-term ethanol-fed rats [184]. Since mtDNA encodes for 13 constituents of the respiratory chain machinery, ethanol-driven mtDNA harm via ROS production can injure the mitochondrial respiratory complexes [185]. Moreover, alcohol impairs mitochondrial ribosomes leading to a reduction in mitochondrial protein synthesis, and ROS can oxidize intramitochondrial proteins [186], contributing to alcohol-induced liver injury.

Damaged mitochondria can activate the cascade of apoptosis and induce an innate and sterile inflammatory reaction by releasing apoptotic factors and/or mtDNA. In turn, mtDNA triggers cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), which elevates secondary messenger cyclic 2’3’-cGAMP generation that initiates stimulator of interferon genes (STING), leading to IRF3 and IRF7 activation [187, 188]. This cGAS-IRF3 route is activated in experimental ARLD models and is positively associated with disorder severity in ARLD patients [189].

Alcohol-induced structural changes in mitochondria reflect alterations in mitochondrial OXPHOS. Although primary findings in alcohol-fed rat hepatic mitochondria have revealed depleted respiration via the downregulation of CIII and V synthesis [186, 190], recent findings described an opposing effect in mice, in which alcohol intake elevated state III respiration in hepatic mitochondria [66]. Remarkably, this increment in state III was more dramatic in the intragastric ethanol-fed model, which is associated with increased hepatic damage and advanced stage of ARLD, compared to the milder effect observed in oral ethanol consumption models. The impact of alcohol ingestion on mitochondrial performance is linked to the regeneration of NAD⁺ from NADH oxidation to stimulate oxidative alcohol metabolism. The transfer of electrons from NADH to the respiratory machinery to oxidize it to NAD⁺ in murine mitochondria stimulates the generation of ROS from the increased consumption of O₂ in the ETC. Interestingly, the elevated respiration in mice vs rats correlates with the species-dependent susceptibility to ethanol-induced liver damage. That fact matches the mitochondrial regeneration of NAD⁺ from NADH, indicating that augmented respiration links ethanol consumption with elevated metabolism and ROS production. The importance of putative variations of mitochondrial respiration to ARLD patients is not well defined. Indirect evidence indicates a reduced mitochondrial function in ARLD patients, who exhibited a decreased peak exhalation of ¹³CO₂ from 2-keto[1-¹³C]isocaproic acid whereas aminopyrine breath assay and galactose removal capability were not modified [191]. Besides, in patients with high alcohol consumption, ARLD severity was linked to mtDNA fragment expression in hepatic tissue [192, 193]. Hence, these data suggest that ethanol ingestion is related to mitochondrial respiration disturbances, correlating with the severity of ARLD progression.

As alluded to above, a consequence of NAD⁺ regeneration from NADH oxidation, which is required for endured ethanol metabolism, is the increase in the leakage of electrons transported directly to O₂ to produce O₂^•− [72]. In addition to this event, the consumption of ethanol further increased ROS generation via its metabolism through CYP2E1, as besides ER, it is also found in mitochondria and ethanol consumption induces its expression [194, 195]. In mitochondria, the principal defense against O₂^•− production is SOD2, which generates H₂O₂, a powerful oxidant that forms reactive radicals via the Fenton reaction. Mitochondrial H₂O₂ detoxification can occur via the GSH redox cycle and the Prx-III systems. Reduced GSH level is critical for this GSH redox cycle which requires the participation of GPX-4, while the mitochondrial oxidized form of Prx-III, generated after the reduction of H₂O_2, is regenerated by Trx2 [196]. Mitochondria do not generate GSH de novo, therefore, they depend on cytosolic GSH to provide mGSH to the mitochondrial matrix. mGSH acts as a cofactor for GPX4 to detoxify H₂O₂ and other FAs-derived peroxides [190]. As mentioned above, the transport of cytosolic GSH through SLC25A11 is susceptible to changes in cholesterol-dependent modifications in membrane fluidity [190, 196, 197]. Although the relative importance between mGSH/GPX and the Prx-III/Trx2 systems in detoxifying H₂O₂ remains to be established, both antioxidant systems are interconnected, and mGSH depletion determines the efficiency of the Prx-III/Trx2 pair. Thus, alcohol-induced mChol trafficking may result in the perturbation of membrane physical properties, which can alter efficient antioxidant defense mechanisms, leading to alterations in the mechanisms of mitochondrial respiration.

The ER regulates the transport and maturation of membrane and secretory proteins, post-translational protein processing, and Ca²⁺ homeostasis. ER stress leads to lipid overload, inflammation, and cell death [198], and has been also described to play a key role in hepatic steatosis via sterol regulatory element-binding proteins (SREBPs) activation (Figure 4). Disturbances in protein folding or modifications in lipid homeostasis lead to the initiation of the unfolded protein response (UPR) that induces the expression of the transcription of chaperones (GRP78/BiP) and the ER-associated degradation (ERAD) mechanisms [199–201] to reestablish homeostasis. Alcohol ingestion promotes ER stress via diverse mechanisms, such as ceramide production via the acid sphingomyelinase (ASMase) activation, the production of acetaldehyde as a result of protein adducts formation in the ER, and also via oxidative stress [202]. A crucial mechanism of ethanol-promoted ER stress is the disruption of methionine metabolism, followed by a rise in homocysteine levels. In this regard, it has been demonstrated that feeding mice with betaine reduced ethanol-induced ER stress, liver steatosis, and hepatic damage [203]. In addition, ER stress can control mitochondrial function. Thus, Ca²⁺ homeostasis disturbances in the ER affect mitochondria due to the transport of Ca²⁺, which induces mitochondrial mPTP and cellular damage [70]. ER and mitochondria display physical interaction via mitochondrial-associated membranes (MAMs) defining the boundary of ER and mitochondria, which operate as a channel for the transfer of ions and lipids. In addition, ER stress can regulate cholesterol overload in mitochondrial membranes via STARD1 upregulation [86], a pivotal participant in metabolic hepatic disorders such as MASH and ARLD as mentioned above [87, 88]. Moreover, another connection between methionine metabolism and mitochondrial crosstalk has been revealed in ARLD. Methionine adenosyltransferase 1A (MAT1A) is normally located in the cytosol and nucleus, although new data described MAT1A also present in mitochondria, where it protects mitochondrial proteome and regulates mitochondrial performance [204]. Hepatic tissue from ARLD patients and mice fed alcohol displayed a remarkable reduction in MAT1A localization in mitochondria, facilitated by the isomerase peptidyl-prolyl cis/trans isomerase (PIN1) and the casein kinase (CK2). Prevention of PIN1-MAT1A interaction increases MAT1A levels in mitochondria and protects against alcohol-promoted mitochondrial malfunction and fat storage. However, whether the advantageous outcomes of targeting mitochondrial MAT1A results from SAM local generation to induce stronger methylation and elevated mitochondrial proteins remains to be established vis-à-vis the direct transport of cytosolic SAM to mitochondrial fraction by a transport system that is insensitive to changes in mitochondrial membrane fluidity [205].

Inflammasome is known to be activated in ARLD [207] and it consists of a set of intracellular multiprotein oligomers [NLRP3, caspase-1, interleukin (IL)-1β] found in the cytosol. Inflammasome senses the pathogen-associated molecular patterns (PAMPs) and DAMPs, RNA viruses, pore-forming toxins, cholesterol crystals and uric acid [120, 208], and triggers the generation and release of pro-inflammatory cytokines, as IL-1β and IL-18 [119, 209]. Diverse processes can activate NLRP3 inflammasome at MAMs, such as ROS generation by dysfunctional mitochondria [210], impaired mitophagy [118], mtDNA oxidation [120], and disturbances of the intestinal barrier that trigger the translocation of DAMPs and PAMPs. CL, located in MAMs, flips from the IMM to the OMM triggering NLRP3 initiation [211]. Furthermore, in MAMs, the trafficking of lipids and Ca²⁺ between ER and mitochondria takes place via voltage-dependent anion channel (VDAC) [212], which enables the NLRP3 inflammasome assembly [73]. Then, the association between ethanol metabolism and NLRP3 initiation is orchestrated via mitochondrial disruption, since mitochondria participate in the oxidative metabolism of ethanol, the initiation of ROS production, and oxidative stress. Thus, the crosstalk between mitochondria and inflammasome promotes ARLD progression and arises as a possible target for therapeutic intervention.