NAD⁺ serves as a critical coenzyme in the conversion of carbohydrates into lipids and fuel oxidation (Belenky et al., 2007). The reduction of NAD⁺ levels potentially represents a pivotal mechanism underlying NNMT’s implication in MetS development. The competitive relationship between NAM methylation and NAD⁺ recycling suggests that NNMT could impede energy expenditure while promoting lipid accumulation. This is attributed to the heightened levels of NNMT potentially rendering NAM irretrievable, thereby constraining NAD⁺-dependent processes (Trammell and Brenner, 2015).

Figure 1 shows the three primary synthetic pathways for NAD⁺: the de novo pathway, the Preiss Handler pathway, and the salvage pathway. Among these pathways, the salvage pathway predominates as the principal route for NAD⁺ biosynthesis in mammals (Collins and Chaykin, 1972; Revollo et al., 2007; Mori et al., 2014), contributing to more than 85% of NAD⁺ synthesis (Frederick et al., 2016). Conversely, the de novo pathway and Preiss Handler pathway synthesize NAD⁺ using nicotinic acid (NA) and tryptophan as substrates, respectively, albeit in smaller quantities. Notably, NAM serves as the substrate for NAD⁺ synthesis in the salvage pathway (Pissios, 2017). However, NNMT can methylate NAM, converting it to MNAM. Once NAM undergoes methylation by NNMT, it becomes unavailable for NAD⁺ generation via the salvage pathway. Consequently, NNMT activity can directly influence the primary pathway of NAD⁺ production (i.e., the salvage pathway), culminating in diminished NAD⁺ levels.

Studies have consistently reported that inhibiting NNMT activity or downregulating NNMT expression leads to significant increases in NAD⁺ levels in mouse adipocytes (Kraus et al., 2014; Neelakantan et al., 2018) and hepatocytes (Song et al., 2020). In contrast, it has been demonstrated that overexpression of NNMT in mouse liver leads to a decrease in NAD⁺ levels (Komatsu et al., 2018). Regulation of NNMT may affect cancer-related pathways, Huang et al. (Huang et al., 2024) found through in vitro experiments that NNMT promotes the migration and invasive ability of esophageal squamous cell carcinoma (ESCC), and NNMT also promotes the process of epithelial mesenchymal transition (EMT) by affecting the post-transcriptional modification of E-calmodulin. Numakura et al. (Numakura and Uozaki, 2024) found that NNMT-positive gastric cancer stromal cells, especially fibroblasts, may promote tumor growth and progression by altering their surrounding epigenetic environment. In human colon cancer cells, silencing NNMT resulted in approximately a 30% increase in NAD⁺ levels (Xie et al., 2014), whereas NNMT overexpression decreased NAD⁺ levels by 30% (Xie et al., 2014). Moreover, Eckert et al. (Eckert et al., 2019) reported that the suppression of NNMT resulted in increased NAD⁺ levels in cancer-associated fibroblasts (CAFs). In a study of a mouse unilateral ureteral obstruction (UUO) model, Takahashi et al. (Takahashi et al., 2022) observed an elevation in renal NNMT expression and a reduction in NAD⁺ levels. Additionally, Sun et al. (Sun et al., 2022) illustrated that NNMT knockdown in a low-glucose environment enhanced the NAD⁺/NADH ratio in U251 glioma cells.

Hyperhomocysteinemia (HHcy) stands as a well-established independent risk factor for cardiovascular and cerebrovascular diseases (Dubchenko et al., 2019). Hence, understanding the potential pathophysiological interplay between Hcy levels and MetS is of paramount’ importance. Moreover, there exists evidence linking elevated Hcy levels to an increased risk of MetS (Sreckovic et al., 2017). Esteghamati et al. (Esteghamati et al., 2014) documented a significantly relationship among elevated Hcy levels and major manifestations of MetS, including impaired glucose tolerance (IGT), abdominal obesity, elevated blood pressure, and hypertriglyceridemia. Consequently, the elevation in Hcy levels induced during NAM methylation emerges as another crucial mechanism underlying the involvement of NNMT in MetS development. Figure 1 illustrates NNMT utilizing SAM as a methyl donor to methylate NAM, yielding MNAM and SAH. Subsequently, SAH is converted to Hcy by SAH hydrolase (SAHase) (Aksoy et al., 1994; Okamoto et al., 2003; Souto et al., 2005). Therefore, heightened NNMT activity leads to elevated blood Hcy levels. A genome-wide scanning study conducted by Souto et al. (Souto et al., 2005) on Spanish families corroborated NNMT as the principal genetic determinant of plasma Hcy levels.

Obesity stands as a pathological state characterized by the excessive accumulation of body fat and constitutes a significant contributor to MetS. Emerging evidence suggests a pivotal role of NNMT in the context of obesity. Initial metabolomics investigations unveiled a noteworthy correlation between urinary MNAM levels and BMI (Salek et al., 2007; Li and Wang, 2011). Subsequent studies by Liu et al. (Liu et al., 2015) extended these findings, revealing a positive association between serum MNAM levels and obesity. These observations hint at a potential linkage between obesity and augmented NNMT activity, given MNAM’s production during NNMT enzymatic reactions. This notion gains further support from recent experimental endeavors. Multiple studies have reported a conspicuous upregulation of NNMT expression in WAT among mice prone to obesity compared to their obesity-resistant counterparts (Alexander et al., 2006; Svenson et al., 2007; Wu et al., 2009). Notably, Lee et al. (Lee et al., 2005) documented significantly elevated NNMT expression in abdominal subcutaneous adipocytes of obese Pima Indians relative to non-obese counterparts. Furthermore, Kraus et al. (Kraus et al., 2014) elucidated that NNMT silencing significantly reduced relative adiposity in mice by approximately 47%. They proposed that heightened NNMT expression in WATs and/or liver could potentially serve as a contributory factor to obesity. Additionally, it has been documented that NNMT knockdown significantly mitigated both body weight and adiposity in female mice subjected to a Western diet, characterized by a composition of 47% kcal of fat and 34% kcal of carbohydrate (Brachs et al., 2019). Moreover, Roberti et al. (Roberti et al., 2023) highlighted the preventive effects of NNMT blockade during the initial phases of adipogenesis, which inhibited preadipocyte differentiation into adipocytes in response to glucocorticoids. Furthermore, NNMT gene knockout resulted in a significant reduction in adipogenesis. Dong et al. demonstrated that knockdown of the Nmnat1 gene in hepatic cells leads to depletion of nuclear NAD⁺, thereby causing mitochondrial dysfunction (Dong et al., 2024). NMNAT is an essential key enzyme in the NAD⁺ salvage pathway. Its deficiency results in reduced NAD⁺ synthesis and accumulation of its substrate, NAM, leading to increased NNMT activity and further decline in NAD⁺ levels. NAD⁺ plays a crucial role in maintaining mitochondrial function and energy metabolism. Decreased NAD⁺ levels can impair the efficiency of the mitochondrial respiratory chain, resulting in reduced energy production, which may exacerbate obesity and related metabolic disorders (Amjad et al., 2021). Our previous research investigated the association between NNMT gene sequence variants and obesity, revealing significant findings. Specifically, investigations on the Chinese Han population identified the rs10891644 variant as significantly associated with obesity susceptibility, with heterozygous individuals (GT type) exhibiting heightened vulnerability (Zhou et al., 2017). Analogously, Bañales-Luna et al (Bañales-Luna et al., 2020). Reported a significantly correlation of the variant rs694539 of the NNMT gene sequence with BMI values in the Mexican population, with carriers of the GG and AG genotypes having an increased susceptibility to obesity.

Obesity arises from an imbalance in energy metabolism, wherein energy intake surpasses energy expenditure. As shown in Figure 2, NNMT’s influence on obesity predominantly stems from its regulatory role in energy metabolism, particularly through NAM methylation, which significantly impacts energy modulation. Upon methylation, NAM becomes irretrievable for NAD⁺ synthesis (Trammell and Brenner, 2015). NAD⁺ is a vital coenzyme in fuel metabolism and the transformation of carbohydrate into lipid (Houtkooper et al., 2010). The relationship between NAM metabolism and NAD⁺ utilization indicates that elevated NNMT expression may inhibit energy expenditure and promote fat deposition by limiting NAD⁺-dependent processes (Trammell and Brenner, 2015). Our prior investigations have unveiled substantial correlations between NNMT genetic variants and resting energy expenditure (Zhu et al., 2016), as well as maximal oxygen uptake (Teperino et al., 2010) in the Chinese populace. Bañales-Luna et al. (Bañales-Luna et al., 2020) recently corroborated these findings, discovering a significant link between NNMT genetic variants and resting energy expenditure in Mexican individuals. Additionally, Kraus et al. (Kraus et al., 2014) shed light on NNMT’s regulation of energy metabolism and associated mechanisms through laboratory experiments. In vitro studies revealed that diminishing NNMT expression via RNA interference (RNAi) and inhibiting NNMT with MNAM substantially augmented oxygen consumption (OC) in adipocytes. Conversely, adipocytes exhibited a considerable decrease in oxygen consumption upon NNMT overexpression (Kraus et al., 2014). In animal models, mice with decreased NNMT levels exhibited heightened energy expenditures compared to control mice of similar weights (Kraus et al., 2014). NNMT modulates energy expenditure by regulating SAM and NAD⁺ during NAM methylation. SAM and NAD⁺ serve as critical cofactors in cellular energy metabolism and redox states (Jell et al., 2007). SAM facilitates polyamine synthesis by providing propylamine and adding a methyl group for histone methylation (Doench et al., 2003). Enhanced polyamine flux activation leads to elevated energy expenditure via polyamine acetylation, utilizing Acetyl-coenzyme A (acetyl-CoA) to generate acetylpolyamines (Jackson et al., 2003). Inhibition of NNMT augments SAM and NAD⁺ levels, resulting in the upregulation of polyamine flux activation and increased energy expenditure (Kraus et al., 2014).

Diabetes is caused by a fundamental disturbance in the metabolism of glucose and is characterised by IGT, elevated fasting blood glucose, and inadequate insulin secretion or IR (Tripathi and Srivastava, 2006; Goldberg and Mather, 2012). MetS is a multifaceted condition that is defined as IR, IGT, T2D, or impaired fasting glucose (IFG) along with the existence of two or more of the followings: hypertension, microalbuminuria, hyperlipidemia, and obesity (Giuliante et al., 2015). It is evident that disorders of glucose metabolism in the form of diabetes and IR are the core elements of the MetS.

Multiple trials have robustly shown a strong association of NNMT with IR and T2D (Kraus et al., 2014; Kannt et al., 2015; Liu et al., 2015). Our previous study also identified a significant association between the NNMT genetic variant at rs1941404 and T2D in the Chinese Han population (Li et al., 2018). Additionally, NNMT has been shown to play a causal role in the development of T2D through quantitative trait locus mapping in mice (Yaguchi et al., 2005). Elevated levels of MNAM were detected in the urine (Li et al., 2018) and serum (Kannt et al., 2015) of individuals with T2D, suggesting an increase in NNMT activity during T2D development. Further direct evidence supporting this relationship is primarily derived from the reports outlined below. Kannt et al. (Souto et al., 2005) demonstrated an upregulation of NNMT expression in the WAT of individuals with IR or T2D. Additionally, a noteworthy correlation was found among plasma levels of MNAM and both the degree of IR and the expression levels of NNMT in WAT. Kraus et al. (Kraus et al., 2014) reported an increase in NNMT expression levels in the livers and WATs of T2D mice. They also found that knockdown of NNMT improved insulin sensitivity and glucose tolerance in the T2D mice. The role of NNMT in the development of T2D was further confirmed by Hong et al. (Hong et al., 2015). Their study revealed that hepatocyte glucose output decreased by 50% with NNMT knockdown, while NNMT overexpression increased it by 1.4-fold. Meanwhile, they observed a significantly lower overnight fasted glucose level in the livers of C57BL6/J mice with NNMT knockdown (Hong et al., 2015).

The potential mechanisms underlying NNMT’s regulation of glucose metabolism are primarily derived from two key reports. Kraus et al. (Kraus et al., 2014) demonstrated that reduced NNMT expression enhances glucose tolerance and improves insulin sensitivity in DIO mice. The role of NNMT in gluconeogenesis was further elucidated by Hong et al. (Hong et al., 2015). Their results revealed that decreasing NNMT expression in primary hepatocytes led to a reduction in the expression of phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase (G6pc), resulting in decreased glucose output. Conversely, increasing NNMT expression in primary hepatocytes resulted in elevated glucose output and increased expression of Pck1 and G6pc. Furthermore, Hong et al. (Hong et al., 2015) observed that mice subjected to NNMT knockdown exhibited reduced fasting glucose levels and diminished conversion of pyruvate to glucose. Such observations indicate that NNMT plays a beneficial role in regulating gluconeogenesis in hepatocytes.

As shown in Figure 3, in elucidating the regulatory mechanism of NNMT on glucose metabolism, Hong et al. (Hong et al., 2015) proposed that NNMT’s product, MNAM, mediates its regulatory impact, with Sirt1 playing a pivotal role in this process. Their findings revealed a significant reduction in glucose production and expression of both G6pc and Pck1 in hepatocytes with NNMT overexpression and concurrent Sirt1 inhibition, compared to hepatocytes with NNMT overexpression alone. Furthermore, the downregulation of G6pc and Pck1 expressed by NNMT knockdown was reversed by the upregulation of Sirt1. These observations underscore the essential role of Sirt1 in mediating NNMT’s regulatory effect on glucose metabolism. Additionally, a significant correlation between Sirt1 protein expression and NNMT expression was noted in hepatocytes. In vitro experiments demonstrated that NNMT overexpression significantly increased Sirt1 protein expression in primary hepatocytes, while NNMT knockdown led to a notable decrease in Sirt1 protein expression. Consistent with these findings, in vivo experiments revealed a significant reduction in Sirt1 protein expression in the livers of mice upon NNMT knockdown. Furthermore, MNAM, a product of NNMT, was found to playing a key part in this process. Similar to the effects of NNMT overexpression, hepatocytes treated with MNAM exhibited significant increases in glucose production and expression of Sirt1, G6pc, and Pck1. Notably, the alterations induced by MNAM treatment were abrogated upon Sirt1 knockdown. These results collectively indicate that Sirt1 is indispensable for NNMT-mediated regulation of glucose metabolism, and both NNMT and MNAM have the ability to upregulate Sirt1 expression.

Hyperlipidemia is a medical condition characterized by elevated levels of one or more plasma lipids, including TG, cholesterol, cholesteryl esters, phospholipids, and LDL, coupled with a reduction in HDL levels (P et al., 2021). It represents one of the complications associated with MetS. Despite limited research on the association between NNMT and hyperlipidemia, compelling evidence suggests its potential role in lipid metabolism. Notably, studies have shown that knocking down NNMT in adipose tissues and livers of mice fed a high-fat diet resulted in decreased serum TG and FFA levels (Kraus et al., 2014). Furthermore, our previous analysis of 19 tagSNPs in the NNMT gene sequences of Chinese Han patients revealed a significant association between the variant at rs1941404 and hyperlipidemia. Specifically, individuals carrying the CC genotype at this locus were identified as the susceptible population. The regulatory effect of the rs1941404 variant on resting energy expenditure offers a plausible explanation for the observed association between NNMT and hyperlipidemia. These findings underscore the potential involvement of NNMT in lipid metabolism regulation and its relevance to the development of hyperlipidemia, a key component of MetS (Zhu et al., 2016).

The etiology of hyperlipidemia remains incompletely elucidated; however, it is widely believed to be linked to aberrations in both sugar and fat metabolism. Epidemiological investigations have underscored a significant correlation between hyperlipidemia and conditions such as obesity or disruptions in energy metabolism (Zhu et al., 2016). Numerous studies have underscored a robust association between obesity and dyslipidemia (Vekic et al., 2019). As shown in Figure 4, Evidence suggests that dyslipidemia predominantly stems from IR and the influence of pro-inflammatory adipokines (Klop et al., 2013). Consequently, it is plausible that NNMT’s involvement in hyperlipidemia is mediated through its impact on both sugar and fat metabolism. Moreover, NNMT might contribute to hyperlipidemia by influencing plasma Hcy levels. Multiple studies have highlighted a pronounced link between plasma Hcy levels and hyperlipidemia (Sreckovic et al., 2017; Hasan et al., 2019). Additionally, Mondal et al. (Mondal et al., 2018) demonstrated that elevated homocysteine levels can directly modulate lipid metabolism and induce hyperlipidemia in rats through Hcy administration (50 mg/kg/d).

Hypertension is another common complication of MetS. Although there are limited reports regarding the association between NNMT and hypertension, our research has identified a significant association between a SNP in the NNMT gene (rs1941404) and hypertension within the Chinese Han population (Guan et al., 2021).

While direct evidence linking NNMT activity to hypertension is currently lacking, numerous studies have established a strong association between NNMT activity and various metabolic disorders, including cardiovascular diseases (Li et al., 2006; Williams et al., 2012). For instance, Liu et al. (Liu et al., 2017) demonstrated a significant association between serum MNAM levels and coronary artery disease, as well as left ventricular systolic dysfunction in Chinese patients (Liu M. et al., 2018). Additionally, Bubenek et al. (Bubenek et al., 2012) reported a robust association between NNMT expression, serum MNAM levels, and the onset and progression of peripheral arterial occlusive disease. Notably, NNMT expression has been found to be positively correlated with LDL levels and negatively correlated with HDL levels, both of which are relevant factors in cardiovascular diseases (Herfindal et al., 2020; Kornev et al., 2023; Rus et al., 2023). As shown in Figure 5, left ventricular contractile dysfunction and arterial vascular disease are significant contributors to the development of hypertension, suggesting a potential pathway through which NNMT may influence hypertension. Moreover, elevated plasma Hcy levels have been significantly associated with hypertension (Van Guldener et al., 2003), particularly with systolic blood pressure (SBP) (Esteghamati et al., 2014). Junedi et al. (Junedi et al., 2022) reported higher Hcy levels in hypertensive cases compared to controls. Moreover, increased plasma Hcy levels were associated with elevated SBP, diastolic blood pressure (DBP), and a higher prevalence of hypertension among middle-aged and elderly individuals of Chinese descen (Hidru et al., 2019). Notably, patients with HHcy exhibit significantly higher blood pressure compared to those without HHcy, and serum Hcy levels have been positively correlated with blood pressure in Wistar rats (Shi et al., 2019). Thus, influencing Hcy levels may represent another important pathway through which NNMT is implicated in the development of hypertension.

As previously stated, NNMT activity results in reduced NAD⁺ production and increased Hcy production. Studies have shown that knocking down the NNMT gene leads to increased expenditure of energy in adipocytes (Kraus et al., 2014) and decreased output of glucose from hepatocytes (Hong et al., 2015). Additionally, in animal models, NNMT gene knockdown has been found to improve insulin sensitivity (IS), glucose tolerance (GT), and overnight fasting glucose (OFG) levels, while also reducing lipid accumulation and triglyceride content (Kraus et al., 2014; Hong et al., 2015; Xu et al., 2022). These findings suggest that NNMT may represent a promising therapeutic target for the development of RNAi drugs aimed at preventing or treating MetS (Iyamu and Huang, 2020; Van Haren et al., 2021; Harikrishna and Venkitasamy, 2023). Some studies have used endogenous small interfering RNAs (miRNAs) to inhibit NNMT and competitive endogenous RNAs (ceRNAs) as “sponges” of miRNAs to reduce the inhibitory effect of miRNAs on target genes. MiRNAs are small noncoding RNAs that regulate gene expression via recognition of cognate sequences and interference of transcriptional, translational or epigenetic processes (Chen L. et al., 2019), miRNAs control mRNA expression, miRNAs are also capable of targeting non-coding RNAs, including long non-coding RNAs and miRNAs (Hill and Tran, 2021). Recently Yu et al. (Yu et al., 2021) and Wang et al. (Wang et al., 2021) identified miR-29b-3p and miR-378g as potential miRNAs upstream of NNMT by bioinformatics analysis. It was confirmed by dual luciferase reporter assay that miR-29b-3p and miR-378g directly target the 3′UTR of NNMT and negatively regulate NNMT expression. In osteoporosis, both miR-29b-3p and miR-378g regulate osteogenic differentiation of BMSCs by targeting NNMT. Inhibition of miR-29b-3p or miR-378g upregulates NNMT expression and promotes osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), whereas overexpression of miR-29b-3p or miR-378g downregulates NNMT expression and inhibits osteogenic differentiation of BMSCs. Competitive endogenous RNAs (ceRNAs) act as “sponges” for miRNAs, ceRNAs can bind to miRNAs and reduce the abundance of miRNAs in the cell, thus reducing the inhibitory effect of miRNAs on target genes. Yu et al. (Yu et al., 2021) and Wang et al. (Wang et al., 2021) found that XIST and HOTAIR are long non-coding RNAs (lncRNAs) with significantly elevated expression in the serum of patients with osteoporosis, and that XIST and HOTAIR acted as ceRNAs (competing endogenous RNAs) to upregulate NNMT expression by adsorbing miR-29b-respectively. 3p and miR-378g, respectively, to upregulate the expression of NNMT, thereby inhibiting the osteogenic differentiation of BMSCs.

However, RNAi therapeutics still face technical obstacles, such as achieving efficient delivery of RNAi drugs targeting organs or tissues and overcoming the off-target interactions (Doench et al., 2003; Jackson et al., 2003; Persengiev et al., 2004; Scacheri et al., 2004), as well as addressing the issues of sequence-dependent and chemical-dependent toxicity (Grimm et al., 2006; Jackson et al., 2006). While certain RNAi drugs aimed at NNMT have shown efficacy in vitro and animal studies, there are no reports of such products being utilized in clinical trials thus far. It is worth noting, however, that the Food and Drug Administration (FDA) of the U.S. has approved over 10 RNAi drugs (Roberts et al., 2020). The technical barriers to RNAi therapies are gradually being resolved, implying that developing RNAi drugs that target NNMT remains a viable approach to prevent or treat MetS.

In addition to RNAi drugs, the development of small molecule inhibitors targeting NNMT represents a current research focus for the treatment of MetS-related diseases. As shown in Table 1 and Figure 6, the small molecule inhibitors targeting NNMT can be categorized into SAM-competitive inhibitors, NAM-competitive inhibitors, Dual-substrate-competitive inhibitors and isomerization inhibitors.