Hepatocytes account for 60–80% of the liver mass and play essential roles in liver function [18], including nutrient metabolism, storage of vitamins, detoxification, bile acid production and secretion, and protein synthesis. Intracellular mitochondria occupy about 20% of the cytoplasmic area in hepatocytes [19] and display diverse functions, such as maintaining metabolic (carbohydrate, lipid, and protein metabolism) and ROS homeostasis [20]. Beta-oxidation is processed to break down fatty acids in mitochondria, the important energy generators, and it can impact lipid accumulation in hepatocytes, as well as the subsequent production of ROS and ER stress [21]. Abnormal lipid metabolism causes mitochondrial dysfunction and ER stress in the pathogenesis of NAFLD. For instance, free cholesterol accumulation in the liver induces mitochondrial dysfunction, ROS production, and triggers UPR in the ER, resulting in ER stress and cell apoptosis. ER stress and mitochondrial dysfunction ultimately promote hepatocyte death and ongoing liver injury [22].

Mitochondria and adjacent ER are connected by mitochondria-associated membranes (MAMs) that regulate cell death and the exchange of lipids and lipid-derived molecules [23, 24]. Interruption of the integrity of MAMs happens during mitochondrial dysfunction and ER stress. Furthermore, persistent ER stress causes hepatocyte apoptosis and liver injury by impacting calcium (Ca²⁺) homeostasis [25] and accelerating the accumulation of toxic levels of ROS [26], leading to NAFLD progression. A schematic figure shows an example of how mitochondrial dysfunction and ER stress in hepatocytes function in the pathogenesis of NAFLD (Figure 1).

Liver non-parenchymal cells (NPCs) consist of Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), hepatic stellate cells (HSCs), and other lymphocytes. All of them are implicated in the pathogenesis of NAFLD [27]. As large abundant NPCs, LSECs as liver gatekeeping cells protect liver injury and filter the transport of nutrients, including lipids and lipoproteins [28]. Under the pathological condition, liver injury causes LSEC defenestration, inflammation, and impaired lipid transport. Meanwhile, inflammation also can increase ROS production and cytokines (e.g., TNF-α) in LSECs accompanying with alteration of mitochondrial permeability, resulting in the activation of KCs and HSCs [29, 30]. Meanwhile, lipid metabolism is critical for the survival and function of T cells, such as regulatory T cells (Tregs). For example, fatty acid-binding protein 5 (FABP5), a lipid chaperone, is required for the uptake and intracellular trafficking of lipid. Inhibition of FABP5 expression in Tregs caused impaired lipid metabolism and mitochondrial integrity, and induced loss of cristae structure [31]. ER-mitochondria communication in macrophages modulates fatty acid metabolism that is associated with inflammation and ER stress [32]. ER stress can further induce the activation of inflammasomes and production of proinflammatory cytokine IL-1β in KCs by triggering UPR, leading to hepatic inflammation and steatosis [33]. Furthermore, ER stress plays an important role in HSC activation and hepatic fibrosis [34, 35]. Overall, ER and mitochondria play pivotal roles in hepatocytes and liver NPCs and are involved in the pathogenesis of NAFLD.

Recent studies show that intracellular Ca²⁺ signaling plays a key role in the regulation of lipid and carbohydrate metabolism in hepatocytes [40], such as modulating the phosphorylation and dephosphorylation of enzymes in glucose metabolism [41]. Disrupting Ca²⁺ signaling can increase lipid accumulation via inhibiting lipolysis and activating lipogenesis, resulting in HCC progression [42]. These changes are associated with ER stress, generation of ROS, and mitochondrial dysfunction. Besides, Ca²⁺ signaling is implicated in the MAM [43]. Proteins such as sarcoendoplasmic reticulum calcium ATPase (SERCA) located in the MAM control the influx of cytosolic free Ca²⁺ into mitochondria to prevent overload of Ca²⁺ that can induce mitochondrial damage, ER stress, and overexpression of ROS [44]. Another study also showed that matrine treatment can significantly suppress ER stress and restore mitochondrial function via directly inhibiting SERCA to regulate Ca²⁺ level [45]. Therefore, agents targeting Ca²⁺ transporters, channels, or binding proteins are potential options for NAFLD treatment.

Mfn proteins include two homologous transmembrane proteins, Mfn1 and Mfn2. Both Mfn1 and Mfn2 play important roles in mitochondrial fusion, but they also have their distinct functions [46]. Studies have shown that Mfn2 is implicated in ER-mitochondria crosstalk. For example, acute Mfn2 depletion can inhibit mitochondrial uptake of Ca²⁺ released from the ER in mouse embryo fibroblasts and enlarge the distance between ER and mitochondria [47]; however, this phenomenon was not shown in Mfn1-depleted cells. Hepatic Mfn2 protein expression was decreased in mice fed with a high fat-high sugar diet compared to that in mice fed with a normal chow diet [39]. Meanwhile, this study further showed that silencing Mfn2 expression mediated by small interfering RNAs (siRNAs) in human liver cancer cell line Huh7 cells can decrease ER-mitochondria interactions and mitochondrial oxidative capacity, resulting in triglyceride (TG) storage [39]. Furthermore, Mfn2 expression was reduced in the livers of human patients and mice with NASH, and Mfn2-deficient mice showed increased liver inflammation, fat accumulation, fibrosis, and HCC compared to wild-type controls [17]. A molecular mechanism study revealed that Mfn2 was implicated in mitochondrial PS and phosphatidylethanolamine (PE) synthesis [17].

C-X-C motif chemokine receptor 3 (CXCR3) has been found to be upregulated in the liver tissues of human NAFLD patients and mice with diet-induced NASH models [48]. Overexpression of CXCR3 in steatohepatitis was positively associated with liver inflammation and infiltration of macrophages and T cells, as well as ER stress. The expression of Mfn1 was reduced in CXCR3 knockout (CXCR3^−/−) mice, which was associated with improved mitochondrial integrity compared to wild-type mice while they were fed either methionine and choline-deficient diet for 4 weeks or high fat-high carbohydrate-high cholesterol diet for 12 weeks [48].

Mitochondrial fission, an effector stimulating ROS production in the metabolic process, plays an important role in NAFLD progression [49]. Dynamin-related proteins (DRPs) and cofactors regulate mitochondrial fission [50]. DRP1 (also known as DLP1) is a guanosine triphosphatase (GTPase) that controls mitochondrial and peroxisomal fission [51]. Upregulation of DRP1 has been shown to be positively associated with liver metabolic dysfunction in animal models, accompanying an increased expression of peroxisome proliferator-activated receptor-gamma (PPARγ) and sterol regulatory element-binding protein 1 (SRBEP1) [52]. Given the decreased mitochondrial fission, hepatic steatosis and oxidative stress were suppressed in DLP1-K38A mutant mice while feeding a high-fat diet containing 60% of daily caloric intake compared to that in wild-type mice [49].

GRP75 is a tethering protein of the MAM interface, which regulates Ca²⁺ transfer between mitochondria and ER [53]. GRP75 has a similar function with Mfn2, silencing GRP75 can alter MAM integrity and promote TG accumulation in Huh7 cells [39]. GRP78 is mainly located in ER but also shown in mitochondria and other locations, which plays multiple roles in maintaining cellular homeostasis [54], such as control of UPR and macroautophagy. One study showed that hepatic overexpression of GRP78 in ob/ob mice with infection of adenoviruses can inhibit liver triglyceride and cholesterol content and improve insulin resistance [55]. Molecular studies showed that overexpression of GRP78 decreased the expression of sterol regulatory element-binding protein-1c (SREBP-1c) and reduced ER stress markers, such as activating transcription factor 6α (ATF6α) and X box-binding protein-1 (XBP-1) [55].

Voltage-dependent anion channel 1 (VDAC1), an outer mitochondrial membrane protein, plays a pivotal role in cellular homeostasis of energy metabolism and crosstalk of mitochondria and the rest of the cell [56, 57], including Ca²⁺ homeostasis. Multi-omics analysis showed that VDAC1 was associated with cardiolipin composition shift in mitochondria membrane lipids and mitochondrial dysfunction in NAFLD [58]. Overdose of acetaminophen can cause hepatocyte ferroptosis, associated with mitochondrial dysfunction and suppression of tricarboxylic acid cycle (TCA) and fatty acid β-oxidation, which may be driven by VDAC1 oligomerization [59]. Therefore, VDAC1 is a therapeutic target for NAFLD by suppressing mitochondrial dysfunction.

Mitochondrial uncoupling affects mitochondrial proton influx and ATP generation [60], which is implicated in many chronic diseases such as obesity, cardiovascular diseases, and cancer liver metastasis [60, 61]. Mitochondrial uncoupling proteins play an essential role in NAFLD and NASH [61], and Dr. Goedeke and Dr. Shulman [62] report that mitochondrial uncouplers are therapeutic targets for metabolic-associated fatty liver disease (MAFLD) and NASH. For example, disruption of mitochondria proton flux was shown in the livers of rats fed with a high-fat or high-fat and high-cholesterol diet compared to the livers of control rats fed with a chow diet. This abnormal potential of the mitochondrial membrane was associated with increased expression of uncoupling protein-2 (UCP-2) in the liver [63].

In addition, genetic factors impact the progression of NAFLD and NAFLD-related HCC [64, 65], such as the rs738409 C > G single nucleotide polymorphism (SNP) variant of patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene and the rs58542926 C > T genetic variant of transmembrane-6 superfamily member 2 (TM6SF2) gene. Increasing evidence also shows that NAFLD patients have an increased proportion of liver mitochondrial DNA (mtDNA) mutations compared to those in healthy controls [66], such as gene mutations in oxidative phosphorylation (OXPHOS) chain. For example, feeding a western diet caused liver injury and alteration of liver mtDNA, evidenced by downregulation of mitochondrial OXPHOS, reduction in part of the fusion machinery Mfn1, and upregulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inflammatory singling pathway [67]. Mutations of mtDNA and SNPs could be potential diagnostic markers and treatment targets for NAFLD, which is reviewed by another publication recently [68], which will not be discussed here.

Physical exercise can prevent NAFLD development through modulating mitochondria function by increasing cytochrome c content, enzyme activities, and fatty acid oxidation [69]. Excessive fructose taken from foods and beverages can augment lipogenesis and insulin resistance [70] and lead to mitochondrial dysfunction [71], resulting in the development of NAFLD. Therefore, a change of diet and/or physical activity can prevent NAFLD by maintaining mitochondrial function.

Gut microbiota-derived components and metabolites are involved in the pathogenesis of liver diseases [72]. Alteration of gut microbiota through bariatric surgery [73], such as sleeve gastrectomy and Roux-en-Y gastric bypass (RYGB), has been shown to ameliorate hepatic inflammation, NAFLD, and NASH [74, 75]. However, the underlying mechanisms are not completely clear. Ezquerro et al. [74] reported that bariatric surgery increases the hormone ratio of acylated/desacyl ghrelin, which is associated with diminished hepatic steatosis, apoptotic cells, and inflammation. In addition, surgical treatment can reduce mitochondrial dysfunction, oxidative phosphorylation complexes I and II, and ER stress markers [e.g., ATF4 and C/EBP homologous protein (CHOP), as well as chaperone GRP78]. Moreover, acylated ghrelin ameliorates palmitate-induced hepatocyte steatosis and inflammation, associated with a reduction of ER stress [74]. Another study showed that RYGB can preserve liver mitochondrial respiratory chain complex I activity and reduce OS and TNF-α expression [76].

Some natural products and their derivates with anti-inflammatory and antioxidant activity have been applied to treat NAFLD [77, 78]. For example, a study showed that treatment of aqueous açai extract (AAE) in vitro has a high capacity to inhibit ROS production in HepG2 cells. AAE administration in vivo protected liver damage and inflammation, evidenced by a decrease of alanine aminotransferase (ALT), the number of inflammatory cells, and serum level of TNF-α. In addition, AAE treatment protected mitochondrial function and reduced OS by reducing lipid peroxidation and modulating the expression of antioxidant enzymes (e.g., glutathione reductase and catalase) [79]. Studies in rat primary hepatocytes and NAFLD model identified that isosteviol (ISV), a derivative of stevioside extracted from the plant Stevia Rebaudiana Bertoni, can prevent free fatty acid (FFA)- and high-fat diet (HFD)-induced hepatic injury through modulating protein kinase C-β/Src-homology-2-domain-containing transforming protein 1-mediated OS and ER stress, respectively, and it decreased ER-mitochondrial interaction [80]. Another study showed that intragastrical administration of Polygonatum kingianum (PK), a Chinese herb, significantly inhibited HFD-induced increase of ALT, aspartate aminotransferase (AST), total cholesterol (TC), and low-density lipoprotein (LDL) cholesterol in serum, and liver TC and triglyceride in rats. PK also promoted mitochondrial function by inhibiting HFD-induced increase of malondialdehyde and mitochondrial UCP-2 and HFD-induced decrease of cytochrome c and up-regulating carnitine palmitoyl transferase-1 (CPT-1), which alleviated NAFLD [81].

Melatonin, a hormone produced primarily by the pineal gland, has been found to protect liver function in obese subjects. In a diet-induced mouse NAFLD model, melatonin supplementation with drinking water ameliorated hepatic steatosis and metabolic dysfunction by suppressing the expression of microRNA (miR)-34a-5p and SREBP-1-dependent on silent information regulator 1 (SIRT1) signaling [82]. In addition, it can reduce ER stress evidenced by decreased GRP78 expression and modulate the change of mitochondrial shape. Another study showed melatonin supplementation can halt DRP1-mediated mitochondrial fission but recover mitophagy via blockade of nuclear receptor subfamily 4 group A member 1 DNA-dependent protein kinase catalytic subunit-p53 (NR4A1-DNA-PKcs-p53) pathway, displaying a protective effect to hepatocytes and mitochondrial function [83]. In ob/ob mice, melatonin treatment significantly reduced glycemia and steatosis and ameliorated the morphology and organization of these organelles. Treatment with melatonin also improved mitochondrial and ER function, including partially restoring ATP synthase β function and decreased GRP78 and CHOP expression in hepatocytes [84].

Azoramide, a small-molecule modulator of UPR with antidiabetic activity [85], has been shown to reverse the fructose-induced increase of body and liver weights, insulin resistance, ER-stress markers (GRP78 and XBP-1), lipid profile, AST, ALT, and histopathological changes in mice [86]. Anti-T2D medicines such as empagliflozin also can reduce the expression of genes involved in hepatic lipogenesis and ER stress (CHOP and ATF4) [87].

Metabolic abnormalities such as dyslipidemia and lipotoxicity contribute to liver injury and inflammation, promoting NAFLD progression to NASH, cirrhosis, and HCC [88]. Metabolic modulation is a potent effective pharmacotherapy for NAFLD. Analysis of hepatic proteomics and plasma lipidomics indicated that 6-month treatment with n-3 polyunsaturated fatty acids (PUFA), consisting of 64% alpha-linolenic, 21% eicosapentaenoic, and 16% docosahexaenoic acid (DHA), significantly ameliorated markers of lipid metabolism, ER stress, and mitochondrial dysfunction in NASH patients [89]. Maresin 1 (MaR1), a DHA-derived metabolite, has anti-inflammatory and anti-OS properties [90]. MaR1 treatment improved insulin sensitivity and attenuated adipose tissue inflammation in ob/ob mice and diet-induced obese mice [91]. Furthermore, MaR1 treatment inhibited hepatic lipid synthesis and steatosis via activating 5’ adenosine monophosphate-activated protein kinase (AMPK)/SERCA2b-mediated suppression of ER stress in HFD-fed mice [92].

Saturated palmitic acid (PA) can induce hepatocyte lipotoxicity to induce NASH. Treatment with monounsaturated oleic acid (OA) markedly improved PA-induced cellular apoptosis, OS, ER stress, mitochondrial dysfunction, as well as inflammation in hepatocytes. In rats, half replacement of HFD with OA producing olive oil decreased NASH symptoms, while full replacement of HFD with olive oil profoundly reversed NASH, reducing hepatocyte ballooning, liver inflammation, and liver fibrosis [93].

Over the past decade, miRs have been recognized to be involved in the pathogenesis of NAFLD and NASH [94]. For instance, in a fast-food diet-induced NASH model, miR-21 ablation inhibited the progression of hepatic steatosis, lipoapoptosis, and fibrosis [95], via modulating the expression of PPARα. Increased expression of the miR-21 signaling pathway was found in liver biopsies of NAFLD patients. In addition, supplementation of farnesoid X receptor (FXR) agonist obeticholic acid was helpful to markedly improve metabolic symptoms of NASH, showing a potential therapy for NAFLD [95]. Similarly, miR-34a levels were significantly upregulated in liver tissues of HFD-fed mice, associated with the downregulation of SIRT1. Suppressing miR-34a expression with inhibitor reduced lipid accumulation and improved the degree of steatosis, ameliorating the development of NAFLD [96]. Overall, potential treatment agents that can function on energy metabolism have effects on ameliorating mitochondrial dysfunction and ER stress (Table 1).