Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is a complex chronic metabolic dysfunction of the liver influenced by both genetic and non-genetic factors [1]. The recent renaming to MASLD expands the definition of NAFLD by requiring the presence of at least one of the components of metabolic syndrome, i.e., obesity, fasting hyperglycemia, elevated triglycerides, reduced HDL cholesterol, and hypertension, alongside hepatic steatosis [1].

The global prevalence of MASLD continues to rise steadily, paralleling the epidemics of obesity [2] and type 2 diabetes mellitus [3]. MASLD is primarily characterized by abnormal accumulation of triglycerides in the liver (hepatic steatosis). It can progress to metabolic dysfunction-associated steatohepatitis (MASH), which is mainly characterized by liver inflammation and injury, and in severe cases, hepatic fibrosis [4]. Both MASH and liver fibrosis are significant risk factors for the onset of cirrhosis and hepatocellular carcinoma, leading causes of liver transplantation [4, 5] and responsible for over 800,000 deaths worldwide [6]. Despite these serious implications, MASH is considered a silent disease, frequently diagnosed incidentally [7]. Therefore, early and accurate diagnosis of MASH along with the assessment of liver fibrosis, is crucial for effective disease management to prevent progression to more advanced stages of liver disease [8].

Mitochondrial performance may fail in target tissues under metabolically adverse conditions such as type 2 diabetes, obesity, dyslipidemia, and cardiovascular diseases [9]. Therefore, mitochondria have emerged as sentinels of cellular metabolic stress [10]. In this regard, mitochondrial dysfunction is increasingly recognized as a key feature of MASLD [11–17]. Accordingly, resmetirom, a recently FDA- and EMA-approved, oral, liver-directed, thyroid hormone receptor beta-selective agonist for treating biopsy-proven MASH with advanced fibrosis, has been shown to restore mitochondrial performance [18].

Accumulating data suggests that the cellular content of oxidized nicotinamide adenine dinucleotide (NAD⁺) content is frequently reduced in target tissues due to enhanced NAD⁺ consumption under metabolically adverse scenarios related to impaired insulin signaling conditions, i.e., diabetes, and obesity [19]. Because NAD⁺ reduction is frequently accompanied by impaired mitochondrial performance concomitant to tissue damage and dysfunction [20, 21], tissue NAD⁺ restoration has been proposed as a promising therapeutic target in mitochondria-driven strategies to treat metabolic diseases [19]. Supporting this, tissue NAD⁺ replenishment has been associated with improved mitochondrial function and protection against metabolic dysfunctions [19]. Particularly, hepatic NAD⁺ decline has been frequently related to MASLD [22], hepatic inflammation [23], and fibrosis [24–27] in experimental models, while its restoration is emerging as a therapeutic approach for MASLD treatment [23, 27–30].

Despite significant advances in MASLD diagnosis [31, 32], histological assessment through a liver biopsy remains the ‘gold standard’ for diagnosing, staging, and prognosing MASLD/MASH, as well as for accurately monitoring fibrosis progression [33]. However, its use is limited by factors such as invasiveness, rare but potentially fatal complications, sample variability, and high costs for the healthcare system [34]. Furthermore, due to the high prevalence of MASH in the general population, implementing a large-scale evaluation plan using liver biopsy is neither feasible nor advised in current clinical practice [35]. Therefore, it is crucial to identify new non-invasive and reliable biomarkers that can act as easily accessible diagnostic alternatives to liver biopsy for the regular monitoring of individuals at high risk of MASLD and its progression to MASH.

Notably, recent studies suggest that circulating peripheral blood mononuclear cells (PBMCs), obtained through minimally invasive approaches such as venipuncture, can serve as sensors of changes in the metabolic environment [36–42]. In the context of MASLD, PBMCs may reflect adaptive characteristics of internal target tissues, which are often challenging to obtain in response to disease states or therapeutic interventions [43, 44]. Supporting this notion, mitochondrial homeostasis is profoundly distorted in human PBMCs under various conditions frequently associated with chronic metabolic stress [36–38, 40], including MASLD [39, 41, 42]. Interestingly, mitochondrial dysfunction in PBMCs has also been recently related to another chronic manifestation of liver disease. Specifically, the degree of hepatic fibrosis has been linked to impaired mitochondrial performance and enhanced oxidative stress in PBMCs in postoperatively Fontan patients [45].

In this review, we will examine the potential of mitochondrial dysfunction in PBMCs as a potential diagnosis/prognosis marker for MASLD. Additionally, we will compile evidence regarding the effects of NAD⁺-boosting therapies—particularly those involving the administration of NAD⁺ precursors—on PBMC mitochondrial bioenergetics in the context of MASLD.

Mitochondria represent the main energy hub in hepatocytes, profoundly influencing oxidative metabolism and liver physiology [46, 47]. Under conditions of chronic metabolic stress, mitochondrial function becomes compromised in hepatocytes, including impaired mitochondrial fission and fusion dynamics [48–50]. This results in inefficient fatty acid oxidation and an inability to manage the excessive accumulation of reactive oxygen species (ROS) [46]. The generation and release of ROS, along with by-products derived from lipid peroxidation in the damaged hepatocytes, further triggers the release of inflammatory cytokines [46]. This cascade contributes to the inflammation, fibrosis, and necrosis associated with advanced stages of MASLD.

Numerous studies have proposed that PBMCs can act as sensors for tissue mitochondrial dysfunction in chronic diseases [51–54]. In most of these studies, the assessment of PBMCs as potential functional biomarkers of disease primarily relies on transcriptomic [43, 55–60] and metabolomic analyses [56, 61]. In the context of MASLD, assessing anaplerotic perturbations, inflammation, and oxidative stress in circulating PBMCs has been proposed as non-invasive biomarkers of liver fibrosis and MASH. Particularly, both transcriptomic and metabolomics approaches are widely used to decipher differentially expressed genes and patterns of metabolite intermediates in PBMCs, including those with a role in major bioenergetic pathways and mitochondrial homeostasis [59, 62].

Only a limited number of studies have explored the relationship between mitochondrial quantity and function in PBMCs and MASLD along with its related complications [12–14, 39, 41, 42, 59, 62] (Table 1). Various methodological approaches have been used to assess real-time mitochondrial bioenergetics in circulating PBMCs isolated from MASLD patients. These methods involve the use of mitochondrially-targeted compounds (e.g., oligomycin, FCCP) in conjunction with the measurement of oxygen consumption rate to assess mitochondrial respiration parameters (Seahorse XFp analyzer technology) [39, 62, 63], as well as high-resolution respirometry to evaluate mitochondrial oxidative phosphorylation (OXPHOS) capacity with complex I + II-linked substrates (Oroboros O2k-technology) [42]. Some of these studies have combined functional analyses of mitochondrial bioenergetics in PBMCs with either metabolomic studies to identify differentially-expressed mitochondrial-based molecular profiles [62] or genetic signatures [63] as markers for predicting disease progression in MASLD. In other studies, the assessment of mitochondrial dysfunction was limited to metabolomic patterns [56, 61].

Mitochondria can adapt their number, mass, and activity through mitohormetic mechanisms in response to changes in energy demand and availability during the early stages of liver disease [10, 11, 64, 65]. However, at advanced stages of MASLD, this mitochondrial flexibility diminishes, leading to oxidative stress, inflammation, and release of mitochondrial damage-associated molecular patterns (mito-DAMPs), with mitochondrial DNA (mtDNA) fragments being one of their major components [66]. These factors collectively contribute to hepatocellular injury. Unfortunately, conventional methods for assessing mitochondrial function often necessitate a liver biopsy [67–69]. Interestingly, studies have reported a lower mtDNA copy number, which serves as a proxy for mitochondria quantity, in PBMCs isolated from patients with MASLD compared to healthy subjects [41]. While the number of mitochondria may not directly correlate with their quality, this finding highlights the potential of mtDNA as a mitochondrial-based metrics in this metabolic disorder.

NAD⁺ metabolism is complex and multicompartmental [70]. It involves the participation of intracellular and extracellular NAD⁺-synthesizing and -consuming enzymes, as well as specific cell transporters that facilitate the transport and uptake of nicotinamide-related metabolites [70]. Cellular NAD⁺ pools are constantly replaced using various precursors, including tryptophan, nicotinic acid (NA), or nicotinamide, through three distinct pathways (Figure 1): [i] the de novo pathway starting from tryptophan, [ii] the Preiss-Handler pathway from NA, and [iii] the salvage pathway, which uses nicotinamide to synthesize nicotinamide mononucleotide (NMN) in a reaction catalyzed by the action of nicotinamide phosphoribosyl transferase (NAMPT), followed by the conversion of NMN to NAD⁺ via NMN adenylyltransferases [71]. In turn, nicotinamide is produced by various NAD⁺-consuming enzymes, such as poly-adenosine diphosphate-ribose polymerase (PARPs) isoforms, which are involved in DNA repair [72], and sirtuins (SIRTs), also known as NAD⁺-dependent protein deacetylases, that are involved in numerous cellular processes, including inflammation and energy metabolism [73, 74]. As a result of NAD⁺ consumption, nicotinamide is regenerated and either recycled or converted into 1-methylnicotinamide (MeNAM). Additional precursors, such as nicotinamide riboside (NR), are directly converted to NMN, thereby contributing to the replenishment of NAD⁺. Collectively, these pathways support the dynamic regulation of cellular health [75, 76].

NAD⁺ has emerged as an essential molecule in cell physiology. This molecule is used as a key cofactor for hundreds of cellular redox reactions and serves as the mandatory substrate for NAD⁺-consuming enzymes that are critical players in the control of DNA repair (i.e., PARPs) or modulation of cellular energetic status, especially the activation of oxidative metabolism and stress resistance in mitochondria in various physiological or pathological conditions (i.e., SIRTs) [21, 71]. NAD⁺ content is negatively influenced by a wide range of metabolic complications and has recently emerged as a potential biomarker for metabolic diseases [19, 77]. Remarkably, NAD⁺ depletion has been identified as a characteristic feature of MASLD in experimental animals [78, 79], indicating that this molecule could serve as a potential therapeutic target for protecting against hepatic steatosis and further advanced stages such as MASH.

Beyond its role in energy metabolism, NAD⁺ is also involved in regulating the immune response [77, 80, 81] and adaptation to systemic chronic inflammation and oxidative stress [77, 81–85]. In the context of MASH, aberrant immune activation is frequently observed in NAD⁺-deficient states [73]. For instance, the expression and activity of several NAD⁺-consuming enzymes (i.e., PARPs, CD38) in activated immune cells are frequently upregulated during inflammation [20, 21, 38, 82, 86, 87], thereby contributing to further NAD⁺ depletion. Concomitantly, several NAD⁺-dependent mechanisms, such as DNA repair and epigenetic reprogramming, are also upregulated in immune cells, further contributing to decreased cellular NAD⁺ pools [21, 82].

Intracellular NAD⁺ pools can be replenished through the administration of various nicotinamide-related metabolites, including NMN, NR, and nicotinamide, all of which serve as NAD⁺ precursors [71]. In the context of MASLD, the beneficial effects of NAD⁺-boosting strategies have been demonstrated in preclinical models of MASLD [23, 28, 29, 88–90] and MASH/hepatic fibrosis [27, 79] (Table 2).

Pioneering studies primarily conducted with healthy participants over short-term periods were designed to assess the dose tolerance and safety of NAD⁺ precursors, particularly NR [76] (Table 2). The effect of various NAD⁺ precursors has been associated with signs of improved liver function in human volunteers [94, 95, 105]. For instance, in one of these studies, while no effects of nicotinamide on MASLD and liver fibrosis were observed, a significant decrease in circulating alanine transaminase levels was reported [105]. In contrast, two other studies reported a significant reduction in hepatic fat content among participants treated with NR compared to those receiving a placebo [94, 95].

Although oral administration of NR did not have a direct impact on liver status, as revealed by the circulating levels of liver enzymes [97], levels of relevant NR-derived metabolites, especially NAD⁺ and nicotinic acid adenine dinucleotide (NAAD), were significantly elevated in circulating PBMCs of treated human participants [76, 97]. Interestingly, concomitant increases in metabolites related to methylated and oxidized waste products of nicotinamide were also observed in PBMCs, especially MeNAM, N-methyl-2-pyridone-5-carboxamide (Me2PY), and N-methyl-4-pyridone-5-carboxamide (Me4PY) [76] (Table 2). Consistent findings were reported in an independent study showing significant elevations of cellular NAD⁺ in isolated PBMCs from subjects treated with NMN [104].

Only a few intervention studies have reported that NAD⁺ precursors, particularly NR, enhance mitochondrial respiration in isolated PBMCs using Seahorse technology [38, 98, 99] (Table 2). This effect was also associated with reduced mitochondrial ROS (mtROS) production and inhibition of the NLRP3 inflammasome, as revealed by decreased IL-1β production from isolated leukocytes obtained from NR-treated human healthy volunteers [99]. However, the impact of this improved bioenergetic and anti-inflammatory profile in PBMC, driven by NR, on MASLD was not explored, as these studies were not focused on MASLD, and liver function was assessed only through routine laboratory tests as part of the safety protocols, with no significant changes reported [99, 104].

MASLD and its related complications can be defined as a complex condition involving both genetic and non-genetic factors, which influence its progression through the MASLD spectrum [106]. Emerging evidence suggests that circulating PBMCs act as sensors of metabolic and immunologic changes associated with the onset of MASLD/MASH [36, 42]. It has been proposed that PBMCs are sensitive to metabolic alterations in internal tissues, including the liver, and can have the capability to modulate their immunomodulatory properties in response to MASLD/MASH progression [43, 44, 107]. Since PBMCs can be obtained through minimally invasive methods, this characteristic has garnered clinical interest in investigating their potential to differentiate between stages of MASLD. However, the contribution of PBMCs to MASLD/MASH diagnosis has been assessed in only a limited number of studies [56, 108, 109].

Mitochondrial performance is tightly linked to the immune response of PBMCs. For instance, recent evidence shows that transcriptomic and metabolomic markers related to mitochondrial dynamics and cell death are differentially expressed in PBMCs from subjects with MASLD/MASH, as demonstrated through transcriptomic [43, 55–59] and metabolomic analyses [61]. Supporting this concept, mitochondrial dysfunction in circulating PBMCs features enhanced immunomodulation in patients with advanced acute-chronic liver diseases [110, 111]. Recent investigations suggest mitochondrial dysfunction in PBMCs could serve as a diagnostic and functional biomarker of MASLD [12–14, 39, 41, 42, 59, 62]. Several studies have used the Seahorse technology to assess mitochondrial function [38, 39, 61, 98, 99], introducing a novel automated methodology to precisely sense ex vivo metabolic alterations associated with MASLD.

The use of NAD⁺ precursors offers potential new avenues for therapeutic intervention approaches targeting activated immune cells; however, the mechanisms by which NAD⁺ and its precursors regulate immune cell function are poorly understood, and the contribution of NAD⁺ metabolizing enzymes to this process remains unclear. Currently, very little is known about the impact of NAD⁺-based interventions on the metabolic reprogramming and improved mitochondrial respiration of PBMCs in MASLD. Emerging data suggests that NAD⁺-increasing therapeutic interventions targeting mitochondrial performance in PBMC may show promise in monitoring MASLD [38]. Further research in this area is therefore warranted.