Diabetes mellitus (DM) affects approximately 9.3% (463 million people) of the population worldwide in 2019 with its prevalence projected to reach 10.2% (578 million) by 2030 and 10.9% (700 million) by 2045 [1]. DM is associated with increased risk of numerous chronic diseases, such as cardiovascular diseases (CVD) and diabetic retinopathy [2,3]. Thus, identifying potentially modifiable risk factors for DM may help develop more effective strategies for its prevention.

Hyperhomocysteinemia (HHcy) has emerged as a risk biomarker for type 2 diabetes mellitus (T2DM) [4]. HHcy was evidenced to promote oxidative stress, β-cell dysfunction, and insulin resistance (IR), which contributes to DM pathology [4-6]. Growing evidence suggests that HHcy can be due to the low status of one-carbon metabolism (OCM) nutrients [7-9]. OCM is a metabolic network that involves biochemical compounds to regulate nucleic acid synthesis and methylation reactions. Homocysteine (Hcy) in this OCM network can be either metabolized into cysteine or recycled into methionine with the aid of a group of OCM nutrients, which act as prerequisite substrate donors (folate, choline, betaine, and methionine) or essential coenzymes (vitamin B2, B6, B12, and zinc) [8,9]. Data directly linking the intake or circulating levels of these OCM nutrients to incident DM are sparse and results remain inconsistent [10-22]. For example, higher choline intake was associated with lower T2DM risk among men in eastern Finland [14], whereas dietary choline or betaine intake was not associated with risk of T2DM among the United States Black or White men [15]. Moreover, the etiology of HHcy may involve genetic variation encoding key enzymes in OCM, which may contribute to DM development [5,6]. A meta-analysis incorporating 68 studies indicated that methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism was correlated with T2DM in Asian populations, but not in White and Black populations [8]. Nevertheless, knowledge regarding the role of OCM in DM development remains in its infancy. Further investigations are warranted to better understand the role of OCM nutrients in the etiology of DM prevention and treatment.

The present review aims to provide an overview of the existing literature assessing the relationships between OCM nutrients status as well as related genetic variation and risk of DM in the context of DM pathology. We will focus on understanding the hypothesized mechanism of OCM nutrients action on DM, limitations of the previous studies, and implications for dietetic practice and future research.

OCM is a metabolic network driven by three interrelated metabolic pathways, which include the folate cycle, the Hcy-methionine cycle, and the transsulfuration pathway [2]. The complex set of biochemical reactions of OCM contributes to the generation or utilization of methyl groups (CH3) [2,3].

As shown in Fig. 1, folic acid (FA) from dietary intake can serve as a precursor to dihydrofolate (DHF) that is converted to tetrahydrofolate (THF) via dihydrofolate reductase (DHFR). DHF can also be converted to 5,10-methylenetetrahydrofolate (5,10-MTHF) via thymidylate synthase (TS), which is used for thymidylate synthesis. Conversion to 5,10-MTHF from THF requires serine via serine hydroxymethyltransferase (SHMT) with vitamin B6 as a cofactor. Methylenetetrahydrofolate dehydrogenase (MTHFD) catalyzes 5,10-MTHF to 10-formyltetrahydrofolate (10-formyl THF), which is used for purine synthesis. Conversion from 5,10-MTHF to 5-methyltetrahydrofolate (5-MTHF) requires MTHFR with vitamin B2 as a cofactor. Folate, the natural form from diet, can also contribute to 5-MTHF donating the methyl groups to Hcy to generate methionine and THF via methionine synthase (MS) with vitamin B12 and zinc as a cofactor [2-6].

In addition, choline and betaine act as other methyl-donors that can be supplied from diet and endogenously synthesized. Choline as an essential nutrient can be acetylated by choline acetyltransferase to produce acetylcholine, a pivotal neurotransmitter [4]. Also, choline can be phosphorylated by choline kinase to be converted to phosphocholine, and then to cytidine diphosphate-choline and phosphatidylcholine (PC) by cytidylyltransferase (CT) and cholinephosphotransferase, sequentially. PC serves as an essential constituent of cell and mitochondrial membranes as well as a major component of lipoproteins [2-4]. In addition, choline can be endogenously synthesized through the conversion of phosphatidylethanolamine to PC, which is catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT) [3]. Moreover, choline dehydrogenase catalyzes choline to synthesize betaine that can be oxidized by betaine-homocysteine S-methyltransferase with zinc as a cofactor to methylate Hcy to methionine. Then, methionine passes the methyl group to S-adenosylmethionine (SAM), which serves as a universal methyl-donor contributing to methylation modification of DNA, RNA, and protein [2-4]; After that, SAM is converted to S-adenosylhomocysteine and deadenosylated to produce Hcy [20]. If there is abundant methionine, the transsulfuration pathway will become active, by which Hcy reacts with serine to form cystathionine by cystathionine β-synthase with vitamin B6 as a cofactor. Cystathionine is further processed by cystathionine γ-lyase with vitamin B6 as a cofactor to generate cysteine, which is used to produce taurine, glutathione (GSH), and other protein [2-4]. It has been evidenced that deficiency of the OCM nutrients (e.g., folate, choline, betaine, vitamin B6 and B12) and reduced activity of the key enzymes in OCM due to genetic variation (e.g., MS 2756 G>A, MTHFR 677C>T) contribute to HHcy, thus impairing the remethylation and/or transsulfuration pathways [5-9,20].

Observational studies that directly related OCM nutrients to incident DM were limited and results remained contradictory (Table 1). One cross-sectional study reported an inverse relationship between serum folate level and DM prevalence among Chinese adults [20]. Consistently, a cross-sectional study demonstrated that serum choline or betaine was inversely correlated with fasting glucose levels and IR index in Canadian adults [18]. Another cross-sectional study conducted in Chinese adults showed that higher dietary consumptions of vitamin B6 and choline, but not folate, vitamin B12, methionine and betaine, were related to lower incident hyperglycemia [21]. However, a positive correlation between dietary methionine and the rate of DM was observed among Chinese adults [22].

Two case-control studies investigated the relationship between OCM nutrients and DM development. Al-Maskari et al. [10] observed that both dietary intake and serum level of folate and vitamin B12 were lower in Omani adult patients with T2DM, compared to the healthy controls. However, Nie et al. [19] reported a positive correlation between plasma zinc and the odds of DM in Chinese adults.

The inverse associations were reported between dietary folate intake and the rate of DM among Korean women aged ≥40 years with an average 4-year follow-up [11], and in Japanese women aged 40 to 79 years within a 5-year study period [12]. Similarly, our previous study found that higher intake of folate, but not vitamin B6 or vitamin B12, in young adulthood was associated with lower diabetes incidence in midlife among White and Black Americans over 30 years of follow-up [13]. Regarding other B vitamins in OCM, dietary vitamin B2 intake was inversely associated with risk of T2DM in Japanese women aged 40 to 79 years [12]. In addition, higher choline intake was inversely associated with lower T2DM risk among men aged 42 to 60 years in eastern Finland [14], while dietary choline or betaine intake was not significantly associated with risk of T2DM among the United States Black or White male participants aged 45 to 64 years [15]. In contrast, a study reported a positive trend for the association between choline consumption and DM risk among postmenopausal United States women aged 50 to 79 years [15]. As for the biomarkers of OCM nutrients, higher serum betaine was associated with lower T2DM risk in Chinese adults aged 40 to 75 years [17]. Presumably, the inconsistent findings from the aforementioned observational studies are mainly due to heterogeneities in exposure measures, time windows of exposure, and diverse study populations.

Randomized clinical trials (RCT) investigating the effects of OCM nutrients supplementation on incident DM were scanty (Table 2). A double-blind RCT reported that Hcy-lowering intervention by daily B vitamins supplementation (FA [2.5 mg], vitamin B6 [50 mg], and vitamin B12 [1 mg]) for 4.5 years failed to decrease incident T2DM among 5,442 United States female health professionals at high risk for CVD [7]. Likewise, another double-blind RCT reported that daily supplementation of FA (0.8 mg) with enalapril (10 mg) for 7.3 years exerted no significant impact on risk of new-onset diabetes among 20,702 Chinese hypertensive adults [8]. Notably, a double-blind RCT involving 200 Sri Lankan participants with prediabetes had noted that 12-month zinc supplementation (20 mg zinc daily) decreased T2DM incidence [9]. These discrepant results are probably explained by different supplementation formulas and duration, and various health statuses across different study populations. Moreover, the participants in these studies were those who were >40 years old and at high risk for metabolic disorders. Thus, the generalizability of these findings is limited.

Although it is insufficient to draw a firm conclusion as to whether OCM nutrients can prevent incident DM, a growing body of interventional trials have demonstrated beneficial effects of OCM nutrients consumption on glucose metabolism indices (Table 2).

Fasting blood glucose (FBG) is a common glucose metabolism index and the easiest way to monitor blood glucose levels for DM diagnosis. The present review identified 26 previous studies that explored the effects of OCM nutrients supplementation on FBG and the results remain elusive (Table 2). Single administration of FA (400 µg/day to 15 mg/day), betaine (100 mg/kg/day), and zinc (30 mg/day) failed to result in significant changes in FBG values [23-36]. In addition, no remarkable alterations in FBG values were reported following the joint supplementation of FA with Fe²⁺ [37], FA with other B vitamins [38-40], FA with metformin [41], B vitamins complex with metformin [42]. However, FA (5 mg/day) administration alone or joint supplementation of FA (0.4 or 0.8 mg/day) and enalapril (10 mg/day) decreased fasting plasma glucose (FPG) among Iranian patients with metabolic syndrome (MetS) [43] and Iranian women with endometrial hyperplasia [44]. Likewise, administration of zinc (20 or 233 mg/day) reduced FPG among Sri Lankan participants with prediabetes [9] and Iranian women with gestational diabetes [45].

Glycosylated hemoglobin (HbA1c) is commonly assayed to indicate average blood glucose level over the past 3 months, which is also used for DM diagnosis [25,37,39,46]. Among five studies identified in the present review, only one reported that FA (5 mg/day) supplementation led to serum HbA1c reduction in overweight and obese Iranian men with T2DM [33]. No significant changes in HbA1c values were observed following administration of FA in T2DM patients [25], supplementation of zinc in prediabetic patients [46], the joint supplementation of FA with Fe²⁺ [37] in diabetic patients, or FA administration with other B vitamins in patients with history of stroke [39].

The homeostasis model assessment of insulin resistance (HOMA-IR) value is calculated by an equation derived from FBG and insulin levels. Higher HOMA-IR was independently associated with an increased DM risk [47]. Single FA administrations at high doses (ranging from 2.5 to 15 mg/day) were inversely associated with HOMA-IR among obese women in Taiwan [26] and overweight adults [29] as well as postmenopausal women in Italy [32]. Similar inverse associations were reported in overweight and obese men with T2DM [33], overweight or obese women with polycystic ovarian syndrome (PCOS) [35], men and women with MetS [43], and women with endometrial hyperplasia in Iran [44]. Likewise, a single administration of zinc (20 mg/day or 233 mg zinc gluconate/ day) reduced HOMA-IR in prediabetic patients [9] and women with gestational diabetes mellitus (GDM) [45]. In addition, concomitant administration of FA (5 mg/day) with vitamin B12 (500 µg/day) or FA (400 µg/day) with metformin (1,700 mg/day) decreased HOMA-IR in patients with MetS and hyperinsulinemia [38], and elderly adults with vitamin B12 deficiency [40]. However, two other studies reported no effect of joint supplementation of B vitamins with metformin on HOMA-IR among Turkish women with PCOS [48] and Israeli patients with T2DM [42].

The quantitative insulin sensitivity check index (QUICKI) is another surrogate biomarker of IR, the higher of which reflects a lower degree of IR [44,45]. In the study by Karamali et al. [45], 6-week zinc supplementation (233 mg/day zinc gluconate) increased QUICKI in women with GDM. Likewise, Bahmani et al. [44] demonstrated that 6-week supplemental FA at 5 mg/day augmented QUICKI in women with endometrial hyperplasia.

Homeostatic model assessment of β-cell function (HOMA-β), derived from fasting plasma insulin and glucose levels, is applied as an index of the insulin secretory function of pancreatic β-cells [47]. Lower HOMA-β was independently associated with an increased DM risk [47]. Increasement of HOMA-β were observed under administration of zinc (20 mg/day) in prediabetic patients [9], whereas zinc (233 mg zinc gluconate/day) or FA (5 mg/day) supplementation decreased HOMA-β among women with GDM [45] or women with cervical intraepithelial neoplasia [49]. However, no significant effect on HOMA-β was found under single supplementation of FA (5 mg/day) in patients with MetS or zinc (30 mg zinc gluconate/day) in prediabetic patients [46].

The heterogeneous effects of either the single or the combined supplementation of OCM nutrients on the above glucose metabolism indices are due to the various dosages and combinations of the OCM nutrients with various durations [23-46,48-50]. In addition, the medication (e.g., enalapril, metformin) that was co-ingested with the OCM nutrients may also counterbalance the beneficial impact of the OCM nutrients on the glucose metabolism indices [41,42,50]. Moreover, most of the studies were conducted among patients with various health problems which contributed to the disputed results [23-46,48-50]. Whether the OCM nutrients intake prevents the public at an early age from developing prediabetic status or incident DM later in life merits further investigation. Furthermore, interventions aimed at optimizing balanced OCM nutrients status and preventing HHcy may help mitigate IR and improve insulin signaling and glucose homeostasis. While further research is warranted, incorporating OCM nutrients into holistic lifestyle strategies may provide a valuable avenue for DM management and prevention.

Emerging evidence indicates that the etiology of HHcy may also involve genetic variation encoding key enzymes of OCM, which may contribute to diabetes development [6,13]. The transformation of the methyl group from these OCM nutrients to Hcy is catalyzed by an array of key enzymes including MTHFR, methionine synthase reductase (MTRR), and MS [51]. Genetic single nucleotide polymorphisms (SNPs) may alter these key enzymes activities, thus changing the enzymes catalytic efficiencies of metabolizing OCM nutrients after dietary intake. SNPs encoding these enzymes such as MS 2756A>G, MTRR 66A>G, MTHFR 677C>T, and 1298A>C, have been evidenced to alter blood Hcy level [51-53]. Therefore, these genetic variants may also be potential genetic markers for incident diabetes development. A meta-analysis incorporating 68 studies indicated that MTHFR 677C>T polymorphism was correlated with T2DM in Asian populations, but not in White and Black populations [54]. Additionally, MTRR 66A>G polymorphism was related to T2DM risk in overweight and obese Chinese individuals [55]. Another study showed that only those Chinese adults who carry MTHFR 1793 GA+AA genotype or MTHFR 1298 AC+CC genotype appeared to have lower T2DM risk [17]. In addition, in the middle-age Han Chinese, those with the genotype CC of MTHFR 1470 A>C had a significantly higher likelihood of T2DM, whereas those with the genotype AA of MTHFD 1958G>A or carrying CT+TT of PEMT (rs4646356) had a significantly lower likelihood of T2DM [53]. Moreover, MTHFR CTCCGA haplotype (rs12121543-rs13306553-rs9651118- rs1801133-rs2274976-rs1801131) was found to be related to decreased risk of T2DM in a Chinese population, compared with CTTTGA haplotype [56]. Nevertheless, the frequencies of these SNPs appear different among people with diverse ethnicities, which may partially explain the inconsistent results from previous studies that a direct relationship between these SNPs and diabetes remains controversial among different study populations. However, previous studies investigating the interplay between OCM nutrients status and genetic variation encoding key enzymes of OCM on DM risk were limited. Lu et al. [17] reported the joint effects of higher serum betaine levels (>47.82 µmol/L) and heterozygous or homozygous variants of MTHFR (G1793A, A1298C) could be found influencing risk of T2DM among Chinese adults aged 40 to 75 years. The biological relevance of these OCM nutrients and genetic variation to the efficiency of the OCM pathway and Hcy homeostasis makes it necessary to consider interrelationships of the OCM nutrients intakes/circulation levels and SNPs with DM risk.

Emerging evidence from in vivo and in vitro studies suggests that OCM nutrients are essential for facilitating energy and glucose metabolism through multifactorial mechanisms. Low status of the OCM nutrients (e.g., folate, choline, or vitamin B12) has been evidenced to induce HHcy [4,10,57], which has been implicated in the pathogenesis of DM [5,13]. HHcy increases reactive oxygen species (ROS) and C-reactive protein (CRP) levels to promote oxidative stress and systemic inflammation, which have been reported to activate various stress-sensitive signaling pathways and eventually lead to pancreatic β-cells dysfunction [5,10], glucose intolerance [58], and IR [21,59,60]. OCM nutrients can directly scavenge ROS, decrease CRP, and promote production of GSH, the major intracellular antioxidant [10,21,61-64], which can counteract the disturbance in glucose metabolism by HHcy. Moreover, OCM nutrients (e.g., folate, choline, betaine) provide the methyl group to the universal methyl-donor, SAM, the change of which can modify methylation status of genetic loci signals involved in insulin signaling and glucose homeostasis [2,6,10,13,65,66]. These alterations in DNA methylation patterns may generate different gene expression profiles that facilitate the development or progression of DM [2,65,66]. However, the molecular mechanisms by which OCM nutrients contribute to DM pathology are only partially understood. The plausible mechanisms remain to be elucidated in human studies. Thus, more future studies exploring the underlying mechanisms are warranted.

Based on the existing literature, it is premature to infer the causal relationship between OCM nutrients intake and incident DM. However, the beneficial effect of OCM nutrients on the major glucose metabolism indices indicates that consumption of natural food rich in OCM nutrients should be stimulated for maintaining optimal glucose homeostasis and preventing DM development. Co-ingestion of OCM nutrients-enriched foods, such as green leafy vegetables, legumes, fruit, nuts, whole-grain products, eggs, less-processed dairy products, and deep-sea fish, may exert a synergistic beneficial effect on better glucose control and insulin sensitivity. Common foods rich in OCM nutrients are listed in Supplementary Tables 1-7 [67-69]. In addition, there is no sufficient evidence to establish personalized OCM nutrient recommendations for DM prevention, based on the genetic variation information. Lower blood concentrations of these OCM nutrients may be indicators of a higher risk of DM. Therefore, it is worth monitoring these OCM nutrients biomarkers (e.g., plasma/serum levels of OCM nutrients) regularly to adjust their intake for optimal health.

The present review summarizes the existing evidence of whether OCM nutrients status influences the occurrence of DM. Currently, our knowledge of how OCM nutrients play a role in protesting against DM development in humans is in its early stages. Although limited RCTs using treatment with single/ multiple OCM nutrient(s) reported different onsets on DM, the majority of observational studies manifested that intakes or blood biomarkers of OCM nutrients, particularly folate and betaine, were inversely associated with abnormal glucose metabolism indices and affect the progression of DM. In addition, association studies suggest that several SNPs in OCM-related genes may influence the metabolic handling of methyl-donors and presumably the risk of DM. While OCM nutrients interventions displayed promise, current human studies were mainly conducted among participants with different underlying medical conditions or middle-aged and elderly populations who may have already had disease onset. Future well-designed RCTs are warranted to examine whether balanced OCM nutrients intakes at an early age prevent DM later in life among the general population. Moreover, it is essential to examine whether the OCM nutrients intakes/circulating levels interplay with genetic risk factors to influence DM development in multi-ethnic populations, which will inform the personalized recommendations for OCM nutrients intakes in terms of DM prevention and management.