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Nonalcoholic fatty liver disease and use of folate
CHAPTER 17
Nonalcoholic fatty liver disease and use of folate O Karmin1,2,3, Connie W.H. Woo4, Victoria Sid2,3 and Yaw L. Siow2,5,6 1
Department of Animal Science, University of Manitoba, Winnipeg, MB, Canada Department of Physiology and Pathophysiology, University of Manitoba, Winnipeg, MB, Canada Laboratory of Integrative Biology, St. Boniface Hospital Research Centre, Winnipeg, MB, Canada 4 Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, SAR, China 5 Agriculture and Agri-Food Canada, Canadian Centre for Agri-Food Research in Health and Medicine, Winnipeg, MB, Canada 6 Innovative Therapy Research Laboratory, St. Boniface Hospital Research Centre, Winnipeg, MB, Canada 2 3
Contents Key facts of Fig. 17.1 Key facts of Fig. 17.2 Key facts of Fig. 17.3 Summary points 17.1 Introduction 17.2 Folate and folic acid 17.2.1 Folate and folic acid in diet—food and supplements 17.2.2 Folate absorption and metabolism 17.2.3 Folate-mediated one-carbon transfer reactions 17.2.4 Metabolic interconnection of B vitamins 17.2.5 Folate deficiency 17.2.6 Folate and metabolic disease 17.2.7 Folate status and NAFLD 17.3 Nonalcoholic fatty liver disease 17.3.1 Prevalence and pathogenesis of NAFLD 17.3.2 Current treatment for nonalcoholic fatty liver disease 17.3.3 Role of folic acid supplementation in nonalcoholic fatty liver disease 17.4 Conclusions Acknowledgements References
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Molecular Nutrition DOI: https://doi.org/10.1016/B978-0-12-811907-5.00028-2
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Key facts of Fig. 17.1 • • • • • •
Absorption of dietary folate and folic acid in the small intestine is mediated through folate transporters: reduced folate carrier and proton-coupled folate transporter (PCFT). Dietary folate is mainly in the form of polyglutamate that is hydrolyzed to monoglutamate [10-formyl-tetrahydrofolate (10-formyl-THF) and 5-methyl tetrahydrofolate (5-MTHF)] prior to absorption. Folic acid is a monoglutamate that is readily transported into intestinal epithelial cells, where it is slowly converted to 5-MTHF. PCFT is the primary transporter for dietary folate/folic acid absorption in the small intestine with a more favorable acidic microenvironment. Upon intestinal absorption, monoglutamate forms of folic acid are delivered via the hepatic portal system to the liver where they undergo intracellular polyglutamylation. Liver is the primary organ for folate storage, metabolism, and redistribution to the circulation and bile.
Key facts of Fig. 17.2 • •
Folate-dependent one-carbon transfer reactions can occur in the cytoplasm, nucleus, and mitochondria. 10-Formyl-tetrahydrofolate (10-formyl-THF) and 5-methyl tetrahydrofolate (5-MTHF) are the major coenzymes in these pathways: purine nucleotide synthesis, interconversion of glycine and serine, and homocysteine remethylation to methionine.
Key facts of Fig. 17.3 • • • •
Folate regulates sulfur-containing amino acid metabolism through the interaction between the methionine cycle, transsulfuration, and desulfuration reactions. Homocysteine is an intermediate amino acid formed during the metabolism of methionine to cysteine. It can be metabolized via the remethylation pathway and the transsulfuration pathway. Remethylation of homocysteine to methionine requires 5-methyl tetrahydrofolate (5-MTHF) and vitamin B12. Metabolism of homocysteine to cysteine through the transsulfuration pathway is catalyzed by cystathionine-β-synthase (CBS) and
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cystathionine-γ-lyase (CSE); both of which require vitamin B6 as a coenzyme in their activities. Cysteine serves as a precursor for the biosynthesis of glutathione, a major endogenous antioxidant. CBS and CSE are also responsible for the biosynthesis of hydrogen sulfide through desulfuration reactions.
Summary points • • • • •
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This chapter focuses on the role of folate in nonalcoholic fatty liver disease. Folate is vitamin B9 and is naturally occurring, while folic acid is the synthetic form. Folate plays a key role in one-carbon transfer reactions that are essential for nucleic acid biosynthesis, methylation reaction, and sulfurcontaining amino acid metabolism. Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease that is associated with obesity and type 2 diabetes. Folate deficiency is uncommon in the general population in countries with mandatory folate fortification policies. However, low serum folate levels are detected in NAFLD patients with adequate dietary intake of folate. Chronic high-fat diet feeding induces fatty liver and impairs hepatic metabolism in rodents. Serum and liver folate levels are decreased in high-fat diet-induced obese mice. In a rodent model of NAFLD, folic acid supplementation (1) protects against oxidative stress and increased inflammatory cytokine production in the liver, and (2) improves hepatic lipid and glucose metabolism through restoration of AMP-activated protein kinase. Clinical studies of folate supplementation in NAFLD patients are warranted.
17.1 Introduction Folate (vitamin B9) is an important micronutrient that plays a key role in one-carbon transfer reactions that are essential for nucleic acid biosynthesis, methylation reactions, and sulfur-containing amino acid metabolism (Tibbetts and Appling, 2010; Stover and Field, 2011). Dysregulation of folate-dependent one-carbon metabolism has been implicated in
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metabolic diseases such as obesity, type 2 diabetes mellitus, hyperhomocysteinemia, and nonalcoholic fatty liver disease (NAFLD) (Da Silva et al., 2014; Nilsson et al., 2015; Sid et al., 2017). NAFLD represents a broad spectrum of liver disorders ranging from steatosis (hepatic lipid accumulation) to its advanced forms such as nonalcoholic steatohepatitis (NASH) and cirrhosis, the latter can further progress to hepatocellular carcinoma (HCC) (Cohen et al., 2011). NAFLD is often found in patients with obesity, hyperlipidemia, hyperglycemia, insulin resistance, or hypertension (Farrell and Larter, 2006). The prevalence of NAFLD is in parallel with a global increase in obesity and type 2 diabetes (Loomba and Sanyal, 2013). Currently, there are no pharmacological agents approved for NAFLD, and only supportive therapies are available to prevent the progression to the advanced conditions (Chalasani et al., 2012). Nutritional approaches are emerging as a promising management strategy for obesity and NAFLD (Veena et al., 2014). Vitamins are essential micronutrients that play important roles in growth and metabolism. Low serum folate levels are associated with obesity, NAFLD, or type 2 diabetes (Mahabir et al., 2008; Nilsson et al., 2015; Xia et al., 2018). The role of folate in NAFLD is currently being evaluated in animal studies and clinical trials. This chapter will focus on the current knowledge regarding the role of folate in NAFLD.
17.2 Folate and folic acid 17.2.1 Folate and folic acid in diet—food and supplements Folic acid is a synthetic form of natural folate and is used in supplements, fortified foods, and feeds. The basic structure of folate comprises three components: (1) a pteridine ring that is attached via a methylene group to (2) p-amino benzoic acid to form pteroic acid, and (3) L-glutamic acid. Folate exists in many forms that differ in the oxidation state of the pteridine ring at the N5 and N10 position and/or in the number of glutamic acid residues conjugated by γ-glutamyl bonds at the end of the molecule (Stover, 2004; Zhao et al., 2009). Mammals lack the enzymatic capacity to synthesize folate; therefore, the intake of dietary folate is necessary to meet their physiological requirements (Zhao et al., 2009). The recommended daily allowance (RDA) for folic acid in healthy individuals is 400 μg of dietary folate equivalents (DFEs) per day. In pregnant women, the RDA increases to 600 μg of DFEs per day to ensure both maternal well-being and healthy fetal development (Institute of Medicine
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Standing Committee on the Scientific Evaluation of Dietary Reference Intakes and Its Panel on Folate, Others B Vitamins and Choline, 1998). Folate is widely distributed in foods. Dark green leafy vegetables, animal liver, and citrus fruits are the most abundant source of naturally occurring folate. Dietary folate often exists in reduced and polyglutamated form. Folic acid is the synthetic (stable) form of folate that is used for dietary supplementation and fortification (Iyer and Tomar, 2009). It is an oxidized monoglutamate with higher bioavailability than its natural counterpart. In North America, cereals and grain products are also important sources of folate due to the mandatory fortification in these foods. In addition to dietary folate and folic acid, folate-producing bacteria in the intestine may serve as an endogenous source of folate (Rong et al., 1991). However, the contribution of intestinal bacteria to whole body folate homeostasis in mammals is significantly less than the dietary source of folate (Visentin et al., 2014).
17.2.2 Folate absorption and metabolism Dietary folate absorption is mediated through folate transporters in the small intestine (Visentin et al., 2014; Sid et al., 2017) (Fig. 17.1). The reduced folate carrier (RFC/SLC19A1) and proton-coupled folate transporter (PCFT/SLC46A1) are abundantly expressed along the apical membrane of enterocytes (Visentin et al., 2014; Zhao et al., 2011). PCFT has been identified as the primary transporter for folate/folic acid absorption in the proximal intestine, which has a more favorable acidic microenvironment (Visentin et al., 2014). Dietary folate is mainly in the reduced form as formyl- or methyl-polyglutamate (Wright et al., 2007), and requires hydrolysis to monoglutamate by either glutamate carboxypeptidase II (in human intestine) or γ-glutamyl hydrolases (in rodent intestine) prior to the absorption at the intestinal brush border membrane. Conversely, synthetic folic acid is a monoglutamate and therefore does not require hydrolysis for intestinal absorption (Hu et al., 2016). Folic acid approaches 100% bioavailability when it is consumed in an empty stomach. However, only 85% of folic acid is absorbed when taken with food, possibly due to its adsorption or chelation with food matrix (Caudill, 2010). Dietary folate is heat labile and susceptible to destruction in the gastrointestinal tract. In addition, incomplete hydrolysis of the polyglutamate chain of folate may hinder the absorption of food folate. Therefore only 50% of naturally occurring food folate is bioavailable
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Figure 17.1 Folate absorption and metabolism in the intestine and liver. The major routes of folate absorption and metabolism in the intestine and liver are illustrated. The key forms of folate that enter these organs are shown in bold. PCFT, Protoncoupled folate transporter; RFC, reduced folate transporter. Horizontal ovals denote enzymes and overlap the pathways they catalyze. DHF, dihydrofolate; THF, Tetrahydrofolate; 5-MTHF, 5-methyltetrahydrofolate; DHFR, dihydrofolate reductase; SHMT, serine hydroxymethyltransferase; MTHFR, methylenetetrahydrofolate reductase; MTHFD, methylenetetrahydrofolate dehydrogenase; MTHFC, methylenetetrahydrofolate cyclohydrolase; MS, methionine synthase; GCPII, glutamate carboxypeptidase II; FPG synthase, folylpolyglutamate synthase. Modified image based on Sid, V., Siow, Y.L., O, K., 2017. Role of folate in nonalcoholic fatty liver disease. Can. J. Physiol. Pharmacol. 95, 11411148.
(Institute of Medicine Standing Committee on the Scientific Evaluation of Dietary Reference Intakes and Its Panel on Folate, Others B Vitamins and Choline, 1998; Caudill, 2010). Upon absorption by enterocytes, folic acid is reduced to dihydrofolate (DHF) that is subsequently reduced to tetrahydrofolate (THF), the biologically active form of folate (Wright et al., 2007). Because both reactions are catalyzed by DHF reductase (DHFR), the first enzymatic reduction of folic acid to DHF becomes the rate-limiting step. The addition of a one-carbon moiety to THF by serine hydroxymethyltransferase (SHMT) generates 5,10-methylene-tetrahydrofolate (5,10-methylene-THF). Pyridoxal-50 -phosphate (PLP; active form of vitamin B6) is an essential cofactor for SHMT activity. The 5,10-methylene-THF is further reduced to 5-methyl THF (5-MTHF) by 5,10-methylene-THF reductase (Tibbetts and Appling, 2010). The 5-MTHF is the main form of folate
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that is transported across the basolateral membrane of enterocytes and enters the portal circulation (Wright et al., 2007). However, a high intake of folic acid may exceed the metabolic capacity of enterocytes, leading to its accumulation in the portal circulation (Pietrzik et al., 2010; Hu et al., 2016). The folic acid and 5-MTHF derived from intestine are subsequently delivered to liver via the portal vein (Wright et al., 2007; Hu et al., 2016; Sid et al., 2017) (Fig. 17.1). Liver is the major organ for folate storage and metabolism (Wright et al., 2007). It plays an important role in maintaining the folate homeostasis (Steinberg et al., 1979). Both RFC and PCFT are expressed on the basolateral membrane of hepatocytes (Zhao et al., 2011). Once inside hepatocytes, folic acid is metabolized to 5-MTHF that undergoes polyglutamation by folylpolyglutamate synthase. The polyglutamate form is the preferential form for storage in hepatocytes and is the reactive form for folate-dependent enzymatic reactions relative to the monoglutamate form (Zhao et al., 2009). While up to 20% of 5-MTHF is retained in the liver, the rest is delivered to the extrahepatic tissues through the systemic circulation or secreted into the bile via the bile duct. Bile folate can be reabsorbed into the liver for storage and redistribution to other tissues (Steinberg et al., 1979).
17.2.3 Folate-mediated one-carbon transfer reactions Folate serves as a cofactor to facilitate the transfer of one-carbon units. Sarcosine, N,N-dimethylglycine (derived from choline), glycine, histidine, and serine (mainly through its conversion to formate in the mitochondria) are the common sources of one-carbon units. The folate-mediated onecarbon transfer reactions take place in various cellular compartments including the cytoplasm, nucleus, and mitochondria (Tibbetts and Appling, 2010; Sid et al., 2017). Intracellular one-carbon transfers are mediated by coenzymatic forms of THF, which carries a one-carbon unit for amino acid metabolism, nucleotide biosynthesis, and methylation reactions (Stover and Field, 2011; Sid et al., 2017) (Fig. 17.2). In the cytoplasm and the nucleus, 5,10-methylene-THF serves as a substrate for the biosynthesis of deoxythymidylate (dTMP) from deoxyuridylate (dUMP) and for the interconversion of glycine and serine (Tibbetts and Appling, 2010). These reactions may also occur in the mitochondria. The de novo purine nucleotide biosynthesis and the remethylation of homocysteine to methionine are folate-dependent reactions that take place in the cytoplasm
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Figure 17.2 Compartmentalization of folate-dependent one-carbon metabolism. One-carbon metabolism for nucleotide biosynthesis, amino acid metabolism, and methylation reactions are distributed within intracellular compartments such as the cytoplasm, nucleus, and mitochondria. The box with solid lines denotes reactions that only occur in the cytoplasm, the box with dotted lines denotes reactions that mainly occur in the mitochondria, and the box with double dotted lines denotes reactions that occur in both the cytoplasm and nucleus. The other one-carbon metabolism reactions may occur in all three cellular compartments. DHF, Dihydrofolate; THF, tetrahydrofolate; 5-MTHF, 5-methyltetrahydrofolate; DHFR, dihydrofolate reductase; SHMT, serine hydroxymethyltransferase; MTHFR, methylenetetrahydrofolate reductase; MTHFD, methylenetetrahydrofolate dehydrogenase; MTHFC, methylenetetrahydrofolate cyclohydrolase; FTHFS, formyltetrahydrofolate synthetase; TS, thymidylate synthase; dTMP, deoxythymidylate; dUMP, deoxyuridylate; MS, methionine synthase; MAT, methionine adenosyltransferase; CBS, cystathionine-β-synthase; CSE, cystathionine-Y-lyase; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; SAHH, adenosylhomocysteine hydrolase; fmet-tRNA, formylmethionine-tRNA; FT, formyltransferase. Reproduced with permission from Sid, V., Siow, Y.L., O, K., 2017. Role of folate in nonalcoholic fatty liver disease. Can. J. Physiol. Pharmacol. 95, 11411148.
(Stover and Field, 2011). The 5,10-methylene-THF is reversibly converted to 5,10-methenyl-THF, a precursor for 10-formyl-tetrahydrofolate (10-formyl-THF) by methylenetetrahydrofolate dehydrogenase (Herbig et al., 2002). The 10-formyl-THF provides one-carbon moieties for the biosynthesis of purine nucleotides while 5-MTHF donates carbon units for the biosynthesis of methionine (Tibbetts and Appling, 2010). Methionine is an essential precursor for the biosynthesis of S-adenosylmethionine (SAM), a principal methyl donor that regulates a number of fundamental cellular
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processes involved in cell signaling, protein localization, degradation of molecules, and gene expression. Folate deficiency impairs dTMP synthesis and homocysteine remethylation. Accumulation of homocysteine is associated with increased S-adenosylhomocysteine (SAH), a potent inhibitor of SAM-dependent methylation reactions (Stover, 2004).
17.2.4 Metabolic interconnection of B vitamins Folate and several other B vitamins (i.e., vitamin B6 and B12) are essential cofactors in one-carbon transfer reactions and sulfur-containing amino acid metabolism. Deficiency in folate and vitamin B12 leads to impaired DNA/protein methylation by SAM, which has an important implication in the development of cognitive and metabolic disorders. One of the key products generated during one-carbon metabolism is homocysteine (Selhub, 2002; Stover, 2004). Homocysteine is an intermediate amino acid formed during the metabolism of methionine to cysteine. It can be metabolized via the remethylation pathway and the transsulfuration pathway (Sarna et al., 2015) (Fig. 17.3). Under physiological conditions, remethylation of homocysteine to methionine is catalyzed by methionine synthase that utilizes 5-MTHF as a substrate and vitamin B12 as a cofactor. Vitamin B12 deficiency can result in an accumulation of 5-MTHF, which is often known as a “methyl folate trap.” Too much 5-MTHF depletes the other forms of folate, leading to the inhibition of purine, thymidylate, and methionine synthesis. Another major source of methyl groups is choline, which is primarily obtained from the diet. Choline is oxidized to betaine that serves as a methyl donor for remethylation of homocysteine to methionine via betaine hydroxymethyltransferase (BHMT) (Tibbetts and Appling, 2010) (Fig. 17.3). This reaction occurs when the activity of methionine synthase is compromised. In the transsulfuration pathway, homocysteine is metabolized to cysteine through enzymatic reactions catalyzed by cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) (Sarna et al., 2015) (Fig. 17.3). Metabolism of homocysteine through the transsulfuration pathway is increased under oxidative stress. Cysteine is the precursor for the synthesis of glutathione, a potent antioxidant tripeptide (Mosharov et al., 2000; Sarna et al., 2015). Approximately 50% of the glutathione pool in hepatocytes is derived from cysteine that is synthesized through the transsulfuration pathway (Mosharov et al., 2000). Homocysteine and cysteine also serve as substrates for CBS and CSE-mediated desulfuration reactions to
Figure 17.3 The hepatic cystathionine-β-synthase/cystathionine-γ-lyase (CBS/CSE) system. Through regulation of the transsulfuration pathway and performance of alternative desulfuration reactions, the hepatic CBS/CSE system converges homocysteine and cysteine metabolism with H2S biosynthesis. The transsulfuration pathway is linked to the methionine cycle via the intermediate sulfur-containing amino acid, homocysteine. Homocysteine may be remethylated to regenerate methionine, primarily via a folate-dependent reaction, or homocysteine may be irreversibly metabolized by the transsulfuration pathway. CBS and CSE regulate the transsulfuration pathway, consecutively catabolizing homocysteine to produce the sulfur-containing amino acid cysteine. Cysteine, in turn, may be used for protein synthesis or may be stored as glutathione following integration into the glutathione biosynthetic pathway. Alternatively, both cysteine and homocysteine may serve as substrate for CBS- and CSE-mediated desulfuration reactions which lead to the endogenous synthesis of H2S. (Boxes denote enzymes). MAT, Methionine adenosyltransferase; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; SAHH, adenosylhomocysteine hydrolase; BHMT, betaine homocysteine methyltransferase; MS, methionine synthase; THF, tetrahydrofolate; SHMT, serine hydroxymethylase; 5,10-MTHF 5,10-methylene-tetrahydrofolate; MTHFR, methylenetetrahydrofolate reductase; 5-MTHF, 5-methyltetrahydrofolate; CBS, cystathionine-β-synthase; CSE, cystathionine-γ-lyase; H2S, hydrogen sulfide; γ-GCL, γ-glutamate-cysteine ligase; GS, glutathione synthetase. Reproduced with permission from Sarna, L.K., Siow, Y.L., O, K., 2015. The CBS/CSE system: a potential therapeutic target in NAFLD? Can. J. Physiol. Pharmacol. 93, 111.
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produce hydrogen sulfide (H2S) (Sarna et al., 2015) (Fig. 17.3). Both the CBS and CSE reactions require PLP (active form of vitamin B6) as an essential cofactor. In addition, vitamin B6 also contributes to the synthesis of 5-MTHF mediated by SHMT. Deficiency of vitamin B6 is correlated with low levels of vitamin B12 and folate (Parra et al., 2018). Therefore depletion of vitamin B6, B12, and/or folate can impair one-carbon transfer reactions and sulfur-containing amino acid metabolism.
17.2.5 Folate deficiency In North America mandatory folic acid fortification of cereal grain products was established in 1998 to reduce the incidence of neural tube defects in newborns (CDC, 2010). General populations in Canada and the United States have achieved adequate intakes of folate since the implementation of the folic acid fortification policy (Bailey et al., 2010). However, folate deficiency may still present in some European, African, and Asian countries that do not impose mandatory folic acid fortification policies (Dhonukshe-Rutten et al., 2009). Folate deficiency can also be caused by gene mutations and environmental factors that impair folate absorption or metabolism. DHFR deficiency can compromise folate status in red blood cells and cause megaloblastic anemia, a condition of large abnormal red blood cells (Cario et al., 2011). Chronic alcohol consumption can lead to folate deficiency due to intestinal malabsorption, reduced hepatic storage, and increased urinary excretion of folate (Medici and Halsted, 2013). Folate deficiency has been linked to dysregulation of intracellular metabolic processes (Da Silva et al., 2014). Emerging evidence indicates that low circulating folate levels are associated with metabolic disorders including hyperhomocysteinemia, obesity, and NAFLD. An elevation of homocysteine in the circulation is a common biomarker of folate deficiency.
17.2.6 Folate and metabolic disease A study based on the US National Health and Nutrition Examination Survey (NHANES, 20032006) reported that serum folate levels were significantly reduced in obese patients (12.4 g/L) compared with normalweight individuals (13.1 g/L) (Bird et al., 2015). Regression analysis revealed that the serum folate level is negatively correlated with body mass index (BMI) regardless of adjustment for vitamin intakes and demographic variables such as gender, age, ethnicity, smoking status, and
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alcohol use. However, the BMI values were positively associated with folate levels in red blood cells (Bird et al., 2015). The mechanism and clinical significance of such a discrepancy in the correlations between BMI and folate levels in serum and RBC remain to be investigated. In another study, gastric bypass surgery significantly improved folate status in morbidly obese patients (Updegraff and Neufeld, 1981). It is plausible that obesity may be one of the underlying causes for the imbalance of endogenous folate levels. Low circulating folate levels were detected in patients with type 2 diabetes (Nilsson et al., 2015). The low blood folate levels were correlated to high levels of fasting blood glucose and increased expression of hepatic genes involved in the development of diabetes. Folate depletion also caused epigenetic and transcriptional alterations in the liver, which might contribute to the pathogenesis of type 2 diabetes (Nilsson et al., 2015). The reduction of serum folate levels found in patients with metabolic disease suggests a potential relationship between the abnormal metabolic processes and perturbed folate status.
17.2.7 Folate status and NAFLD Several reports have indicated the association between the circulating folate level and NAFLD. Obese female patients with severe NAFLD showed significantly reduced serum folate levels compared with individuals with normal liver morphology or minimal liver damage (21 vs 27 nmol/L) (Hirsch et al., 2005). Another cohort study showed a correlation of low serum folic acid levels with the presence and severity of steatosis (Xia et al., 2018). Such a correlation was independent of gender, BMI, insulin resistance, and parameters of metabolic syndrome. Low serum folate and vitamin B12 levels were also correlated with increased NASH severity (Mahamid et al., 2018). While clinical evidence suggests that patients with NAFLD may be at a higher risk for folate deficiency, a causal relationship has yet to be confirmed. Further clinical investigation is necessary to establish proper folate intake guidelines for patients with NAFLD. Animal models that mimic the histology and pathology of NAFLD have been established. High-fat diets can induce histopathological changes that resemble the NAFLD features including hepatic lipid accumulation, oxidative stress, and inflammation in rodents (Sarna et al., 2012; Nakamura and Terauchi, 2013; Sid et al., 2018a,b). In a recent study, a high-fat diet feeding caused a significant decrease in serum and liver folate
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levels in mice (Sid et al., 2018b). This was attributed to impaired expression of hepatic folate transporters (PCFT and RFC). It appears that prolonged consumption of diets that are rich in fat may exert a negative impact on folate homeostasis (Sid et al., 2018b). Another study reported that a depletion of dietary folate in rodents was associated with an increased expression of lipid biosynthetic genes, which contributed to abnormal lipid metabolism in the liver (Champier et al., 2012). Hepatic lipid transport via very low density lipoprotein (VLDL) was impaired in folate-deficient mice leading to hepatic lipid accumulation (Christensen et al., 2010). These results suggest that alterations in folate metabolism may have an important implication in NAFLD.
17.3 Nonalcoholic fatty liver disease 17.3.1 Prevalence and pathogenesis of NAFLD The global prevalence of NAFLD is approximately 20%30% and increases to 70%90% in patients with obesity and type 2 diabetes (Younossi et al., 2016, 2018a). NAFLD is considered as the hepatic manifestation of metabolic syndrome. Patients with NAFLD tend to be obese (BMI . 30 kg/m2) and often exhibit comorbidities associated with insulin resistance, dyslipidemia, hypertriglyceridemia, and hypertension (Farrell and Larter, 2006). Although insulin resistance is an independent risk factor for NAFLD, some patients may have normal insulin function (Cohen et al., 2011). Aside from metabolic risk factors, other nonmodifiable risk factors such as age, gender, ethnicity, and certain gene variants may also influence the susceptibility to NAFLD. Men are more likely to accumulate abdominal fat, which increases their risk towards NAFLD development. Moreover, Hispanics and Asians are more likely to develop fatty liver compared to individuals of African descent (Browning et al., 2004). Hispanic individuals often carry a gene variant of patatin-like phospholipase domain-containing protein 3, which results in a twofold increase in hepatic triglyceride content and confers susceptibility towards NAFLD. In addition, mutations in other genes involved in lipid metabolism can also increase the risk of NAFLD (Birkenfeld and Shulman, 2014). Steatosis is the main histological feature of NAFLD which is defined as lipid accumulation in more than 5% of hepatocytes (Tiniakos et al., 2010). Steatosis is often a self-limiting condition; however, it can advance to NASH that is characterized by steatosis, inflammation, and hepatocyte injury (Cohen et al., 2011). Hepatocyte ballooning is a form of
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hepatocellular injury that is characterized by cell swelling, which is an essential feature that distinguishes NASH from steatosis. Liver fibrosis is frequently detected in NASH patients (Yeh and Brunt, 2014). The presence of advanced hepatic fibrosis increases the risk for cirrhosis and HCC. Although the progression of steatosis to NASH is reversible, NASH may irreversibly advance to cirrhosis that is increasingly susceptible to the development of portal hypertension and HCC (Kessoku et al., 2014). The pathogenesis of NAFLD is complex and incompletely understood. The two-hit hypothesis was initially proposed to describe NAFLD pathogenesis. This hypothesis suggests that perturbations in lipid metabolism lead to steatosis (first hit), which sensitizes the liver to the secondary hit, such as inflammation, oxidative stress, and cell injury (Day, 2005). However, steatosis may not always precede inflammation. It is plausible that inflammation could precede lipid accumulation in NASH patients (Tiniakos et al., 2010). Furthermore, the two-hit theory does not account for many other metabolic changes that occur in NAFLD. This has led to the development of the multiple-parallel-hit hypothesis, which suggests that multiple factors such as insulin resistance, lipotoxicity, oxidative stress, inflammation, gut-derived endotoxins, adipokines, or genetic factors may simultaneously induce NAFLD (Tilg and Moschen, 2010). As NAFLD is a multifaceted disease associated with several metabolic abnormalities, there are currently no therapeutic drugs approved for its treatment. Novel therapeutic strategies are urgently required for the management of NAFLD (Younossi et al., 2018b).
17.3.2 Current treatment for nonalcoholic fatty liver disease Lifestyle modifications such as increasing physical activity and healthy diet are considered to be the most safe and effective strategies for NAFLD management. Such modifications can promote weight loss, alleviate steatosis, and improve liver function (Chalasani et al., 2012). Minimal weight loss (3%5%) is sufficient to reduce hepatic lipid accumulation in NAFLD patients. However, greater than 7% weight reduction is required to improve histological features associated with NASH (Glass et al., 2015). Patients who receive bariatric surgery have profound weight loss, which is associated with improved liver histology and insulin sensitivity (Mechanick et al., 2009). However, bariatric surgery is not recommended for cirrhotic patients due to concerns of liver failure after a rapid weight loss. Although gradual weight loss appears to be beneficial for NAFLD
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patients, lifestyle changes and adherence to healthy diets can be a challenge. Several pharmacological agents that target certain metabolic risk factors have been proposed for the treatment of NAFLD (Chalasani et al., 2012). Metformin, a first line antidiabetic agent, reduces insulin resistance and alanine transaminase levels but has limited effects on inflammation and steatosis in NAFLD patients (Bugianesi et al., 2005). Thiazolidinediones are insulin sensitizers and have been shown to improve liver function and histology in patients with NASH (Neuschwander-Tetri et al., 2003). However, their long-term safety and efficacy for NAFLD treatment have not been established (Chalasani et al., 2012). Oxidative stress is a key mediator in liver injury in NAFLD. While treatment with antioxidants such as vitamin E has been shown to ameliorate steatosis, inflammation, and hepatocyte ballooning, further investigation is required to determine the effectiveness of antioxidants in NAFLD treatment (Sanyal et al., 2004). NAFLD patients have a high cardiovascular risk and therefore statins have been used to treat dyslipidemia and improve cardiovascular outcomes in these patients. The use of statins appears to be safe and effective for correcting lipid abnormalities in NAFLD patients (Chalasani et al., 2012). There is currently no single agent that can target all metabolic risk factors associated with NAFLD. Researchers are still searching for therapeutic agents that can target multiple pathways associated with NAFLD pathogenesis (Younossi et al., 2018b).
17.3.3 Role of folic acid supplementation in nonalcoholic fatty liver disease It was reported that folic acid supplementation during high-fat diet feeding in mice could effectively improve lipid and glucose metabolism in the liver (Sid et al., 2015). Such effects were mediated through the regulation of AMP-activated protein kinase, a key regulator of whole body energy balance and metabolic homeostasis (Sid et al., 2015). Folate and folic acid exhibit antioxidant functions due to their ability to directly scavenge reactive oxygen species (ROS) (Gliszczynska-Swiglo and Muzolf, 2007) and regulate ROS-generating or metabolizing enzymes (Woo et al., 2006; Hwang et al., 2011; Sarna et al., 2012). It was shown in rodents that folic acid supplementation could confer protective effects against oxidative stress in the liver (Woo et al., 2006; Sarna et al., 2012) and kidney (Hwang et al., 2011). Hepatic oxidative stress is associated with increased expression of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in animal models of obesity, metabolic
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syndrome, and NAFLD. Folic acid supplementation effectively inhibited NADPH oxidase-mediated superoxide production, restoring the antioxidant response in hyperhomocysteinemic rats and in high-fat diet-induced obese mice (Woo et al., 2006; Hwang et al., 2011; Sarna et al., 2012). Patients with NAFLD also have elevated levels of proinflammatory cytokines in the circulation, immune cells, liver, and adipose tissue (Day, 2006; Braunersreuther et al., 2012). Macrophages grown in a folatedepleted medium had high expression of proinflammatory cytokines including interleukin-1β, monocyte chemoattractant protein-1, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) (Kolb and Petrie, 2013). Incubation of macrophages with folic acid attenuated homocysteine-induced proinflammatory cytokine expression (Au-Yeung et al., 2006). Folic acid supplementation in high-fat diet-fed mice significantly decreased the expression of hepatic proinflammatory cytokines (IL-6, TNF-α) through inhibition of nuclear factor kappa B, a key transcriptional factor of proinflammatory cytokine genes (Sid et al., 2018a). Folic acid supplementation in overweight individuals or hyperhomocysteinemic patients was associated with a reduction of proinflammatory cytokine levels (Wang et al., 2005; Solini et al., 2006). Those results indicate that folic acid possesses lipid and glucose lowering effects, as well as antioxidant and antiinflammatory functions, which makes it a promising candidate for managing NAFLD. Although folic acid supplementation appears to be beneficial for alleviating metabolic abnormalities, clinical studies are required to further validate the effect of folic acid supplementation in patients with NAFLD.
17.4 Conclusions Vitamins are micronutrients that support physiological functions in the body. Folate (vitamin B9) plays a fundamental role in one-carbon metabolism that is essential for gene expression and sulfur-containing amino acid homeostasis. Although low folate status is observed in patients with malabsorption, kidney dysfunction, or liver disease, dietary folate deficiency is uncommon in generally healthy populations in countries that have implemented mandatory folate fortification policies. However, obese individuals with NAFLD are reported to have low folate levels in the serum despite the adequate intake of folate from diets and/or supplements (Hirsch et al., 2005; Xia et al., 2018; Mahamid et al., 2018). A recent study has revealed that fatty liver is associated with impaired folate
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transporter expression and low folate levels in diet-induced obese mice (Sid et al., 2018b). In animal studies, folic acid supplementation can restore hepatic redox balance, improve lipid and glucose metabolism, and attenuate inflammatory cytokine production in rodents with diet-induced metabolic abnormalities (i.e., overweight, fatty liver, hyperglycemia) (Woo et al., 2006; Sarna et al., 2012; Sid et al., 2015, 2018a). Nutritional therapy including folic acid supplementation appears to be an alternative approach for the management of metabolic diseases, such as NAFLD and diabetes. Future research is warranted to address whether the regulation of folate homeostasis can improve the clinical outcomes in patients with NAFLD or other metabolic diseases. If proven effective, folic acid supplementation may be an economical and effective approach to control this emerging public health threat (NAFLD) globally.
Acknowledgements This work was supported, in part, by grants from the Natural Sciences and Engineering Research Council of Canada and St. Boniface Hospital Foundation.
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Sid, V., Wu, N., Sarna, L.K., Siow, Y.L., House, J.D., O, K., 2015. Folic acid supplementation during high-fat diet feeding restores AMPK activation via an AMP-LKB1dependent mechanism. Am. J. Physiol. Regul. Integr. Comp. Physiol. 309, R1215R1225. Sid, V., Siow, Y.L., O, K., 2017. Role of folate in nonalcoholic fatty liver disease. Can. J. Physiol. Pharmacol. 95, 11411148. Sid, V., Shang, Y., Siow, Y.L., Hewage, S.M., House, J.D., O, K., 2018a. Folic acid supplementation attenuates chronic hepatic inflammation in high-fat diet fed mice. Lipids 53, 709716. Sid, V., Siow, Y.L., Shang, Y., Woo, C.W., O, K., 2018b. High-fat diet consumption reduces hepatic folate transporter expression via nuclear respiratory factor-1. J. Mol. Med. (Berl.) 96 (11), 12031213. Solini, A., Santini, E., Ferrannini, E., 2006. Effect of short-term folic acid supplementation on insulin sensitivity and inflammatory markers in overweight subjects. Int. J. Obes. (Lond.) 30, 11971202. Steinberg, S.E., Campbell, C.L., Hillman, R.S., 1979. Kinetics of the normal folate enterohepatic cycle. J. Clin. Invest. 64, 8388. Stover, P.J., 2004. Physiology of folate and vitamin B12 in health and disease. Nutr. Rev. 62, S312 (Discussion S13). Stover, P.J., Field, M.S., 2011. Trafficking of intracellular folates. Adv. Nutr. 2, 325331. Tibbetts, A.S., Appling, D.R., 2010. Compartmentalization of mammalian folatemediated one-carbon metabolism. Annu. Rev. Nutr. 30, 5781. Tilg, H., Moschen, A.R., 2010. Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis. Hepatology 52, 18361846. Tiniakos, D.G., Vos, M.B., Brunt, E.M., 2010. Nonalcoholic fatty liver disease: pathology and pathogenesis. Annu. Rev. Pathol. 5, 145171. Updegraff, T.A., Neufeld, N.J., 1981. Protein, iron, and folate status of patients prior to and following surgery for morbid obesity. J. Am. Diet. Assoc. 78, 135140. Veena, J., Muragundla, A., Sidgiddi, S., Subramaniam, S., 2014. Non-alcoholic fatty liver disease: need for a balanced nutritional source. Br. J. Nutr. 112, 18581872. Visentin, M., Diop-Bove, N., Zhao, R., Goldman, I.D., 2014. The intestinal absorption of folates. Annu. Rev. Physiol. 76, 251274. Wang, G., Dai, J., Mao, J., Zeng, X., Yang, X., Wang, X., 2005. Folic acid reverses hyper-responsiveness of LPS-induced chemokine secretion from monocytes in patients with hyperhomocysteinemia. Atherosclerosis 179, 395402. Woo, C.W., Prathapasinghe, G.A., Siow, Y.L., O, K., 2006. Hyperhomocysteinemia induces liver injury in rat: protective effect of folic acid supplementation. Biochim. Biophys. Acta 1762, 656665. Wright, A.J., Dainty, J.R., Finglas, P.M., 2007. Folic acid metabolism in human subjects revisited: potential implications for proposed mandatory folic acid fortification in the UK. Br. J. Nutr. 98, 667675. Xia, M.F., Bian, H., Zhu, X.P., Yan, H.M., Chang, X.X., Zhang, L.S., et al., 2018. Serum folic acid levels are associated with the presence and severity of liver steatosis in Chinese adults. Clin. Nutr. 37, 17521758. Yeh, M.M., Brunt, E.M., 2014. Pathological features of fatty liver disease. Gastroenterology 147, 754764. Younossi, Z.M., Koenig, A.B., Abdelatif, D., Fazel, Y., Henry, L., Wymer, M., 2016. Global epidemiology of nonalcoholic fatty liver disease-meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology 64, 7384. Younossi, Z., Anstee, Q.M., Marietti, M., Hardy, T., Henry, L., Eslam, M., et al., 2018a. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nat. Rev. Gastroenterol. Hepatol. 15, 1120.
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Nonalcoholic fatty liver disease and use of folate O Karmin1,2,3, Connie W.H. Woo4, Victoria Sid2,3 and Yaw L. Siow2,5,6 1
Department of Animal Science, University of Manitoba, Winnipeg, MB, Canada Department of Physiology and Pathophysiology, University of Manitoba, Winnipeg, MB, Canada Laboratory of Integrative Biology, St. Boniface Hospital Research Centre, Winnipeg, MB, Canada 4 Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, SAR, China 5 Agriculture and Agri-Food Canada, Canadian Centre for Agri-Food Research in Health and Medicine, Winnipeg, MB, Canada 6 Innovative Therapy Research Laboratory, St. Boniface Hospital Research Centre, Winnipeg, MB, Canada 2 3
Contents Key facts of Fig. 17.1 Key facts of Fig. 17.2 Key facts of Fig. 17.3 Summary points 17.1 Introduction 17.2 Folate and folic acid 17.2.1 Folate and folic acid in diet—food and supplements 17.2.2 Folate absorption and metabolism 17.2.3 Folate-mediated one-carbon transfer reactions 17.2.4 Metabolic interconnection of B vitamins 17.2.5 Folate deficiency 17.2.6 Folate and metabolic disease 17.2.7 Folate status and NAFLD 17.3 Nonalcoholic fatty liver disease 17.3.1 Prevalence and pathogenesis of NAFLD 17.3.2 Current treatment for nonalcoholic fatty liver disease 17.3.3 Role of folic acid supplementation in nonalcoholic fatty liver disease 17.4 Conclusions Acknowledgements References
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Key facts of Fig. 17.1 • • • • • •
Absorption of dietary folate and folic acid in the small intestine is mediated through folate transporters: reduced folate carrier and proton-coupled folate transporter (PCFT). Dietary folate is mainly in the form of polyglutamate that is hydrolyzed to monoglutamate [10-formyl-tetrahydrofolate (10-formyl-THF) and 5-methyl tetrahydrofolate (5-MTHF)] prior to absorption. Folic acid is a monoglutamate that is readily transported into intestinal epithelial cells, where it is slowly converted to 5-MTHF. PCFT is the primary transporter for dietary folate/folic acid absorption in the small intestine with a more favorable acidic microenvironment. Upon intestinal absorption, monoglutamate forms of folic acid are delivered via the hepatic portal system to the liver where they undergo intracellular polyglutamylation. Liver is the primary organ for folate storage, metabolism, and redistribution to the circulation and bile.
Key facts of Fig. 17.2 • •
Folate-dependent one-carbon transfer reactions can occur in the cytoplasm, nucleus, and mitochondria. 10-Formyl-tetrahydrofolate (10-formyl-THF) and 5-methyl tetrahydrofolate (5-MTHF) are the major coenzymes in these pathways: purine nucleotide synthesis, interconversion of glycine and serine, and homocysteine remethylation to methionine.
Key facts of Fig. 17.3 • • • •
Folate regulates sulfur-containing amino acid metabolism through the interaction between the methionine cycle, transsulfuration, and desulfuration reactions. Homocysteine is an intermediate amino acid formed during the metabolism of methionine to cysteine. It can be metabolized via the remethylation pathway and the transsulfuration pathway. Remethylation of homocysteine to methionine requires 5-methyl tetrahydrofolate (5-MTHF) and vitamin B12. Metabolism of homocysteine to cysteine through the transsulfuration pathway is catalyzed by cystathionine-β-synthase (CBS) and
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cystathionine-γ-lyase (CSE); both of which require vitamin B6 as a coenzyme in their activities. Cysteine serves as a precursor for the biosynthesis of glutathione, a major endogenous antioxidant. CBS and CSE are also responsible for the biosynthesis of hydrogen sulfide through desulfuration reactions.
Summary points • • • • •
• •
•
This chapter focuses on the role of folate in nonalcoholic fatty liver disease. Folate is vitamin B9 and is naturally occurring, while folic acid is the synthetic form. Folate plays a key role in one-carbon transfer reactions that are essential for nucleic acid biosynthesis, methylation reaction, and sulfurcontaining amino acid metabolism. Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease that is associated with obesity and type 2 diabetes. Folate deficiency is uncommon in the general population in countries with mandatory folate fortification policies. However, low serum folate levels are detected in NAFLD patients with adequate dietary intake of folate. Chronic high-fat diet feeding induces fatty liver and impairs hepatic metabolism in rodents. Serum and liver folate levels are decreased in high-fat diet-induced obese mice. In a rodent model of NAFLD, folic acid supplementation (1) protects against oxidative stress and increased inflammatory cytokine production in the liver, and (2) improves hepatic lipid and glucose metabolism through restoration of AMP-activated protein kinase. Clinical studies of folate supplementation in NAFLD patients are warranted.
17.1 Introduction Folate (vitamin B9) is an important micronutrient that plays a key role in one-carbon transfer reactions that are essential for nucleic acid biosynthesis, methylation reactions, and sulfur-containing amino acid metabolism (Tibbetts and Appling, 2010; Stover and Field, 2011). Dysregulation of folate-dependent one-carbon metabolism has been implicated in
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metabolic diseases such as obesity, type 2 diabetes mellitus, hyperhomocysteinemia, and nonalcoholic fatty liver disease (NAFLD) (Da Silva et al., 2014; Nilsson et al., 2015; Sid et al., 2017). NAFLD represents a broad spectrum of liver disorders ranging from steatosis (hepatic lipid accumulation) to its advanced forms such as nonalcoholic steatohepatitis (NASH) and cirrhosis, the latter can further progress to hepatocellular carcinoma (HCC) (Cohen et al., 2011). NAFLD is often found in patients with obesity, hyperlipidemia, hyperglycemia, insulin resistance, or hypertension (Farrell and Larter, 2006). The prevalence of NAFLD is in parallel with a global increase in obesity and type 2 diabetes (Loomba and Sanyal, 2013). Currently, there are no pharmacological agents approved for NAFLD, and only supportive therapies are available to prevent the progression to the advanced conditions (Chalasani et al., 2012). Nutritional approaches are emerging as a promising management strategy for obesity and NAFLD (Veena et al., 2014). Vitamins are essential micronutrients that play important roles in growth and metabolism. Low serum folate levels are associated with obesity, NAFLD, or type 2 diabetes (Mahabir et al., 2008; Nilsson et al., 2015; Xia et al., 2018). The role of folate in NAFLD is currently being evaluated in animal studies and clinical trials. This chapter will focus on the current knowledge regarding the role of folate in NAFLD.
17.2 Folate and folic acid 17.2.1 Folate and folic acid in diet—food and supplements Folic acid is a synthetic form of natural folate and is used in supplements, fortified foods, and feeds. The basic structure of folate comprises three components: (1) a pteridine ring that is attached via a methylene group to (2) p-amino benzoic acid to form pteroic acid, and (3) L-glutamic acid. Folate exists in many forms that differ in the oxidation state of the pteridine ring at the N5 and N10 position and/or in the number of glutamic acid residues conjugated by γ-glutamyl bonds at the end of the molecule (Stover, 2004; Zhao et al., 2009). Mammals lack the enzymatic capacity to synthesize folate; therefore, the intake of dietary folate is necessary to meet their physiological requirements (Zhao et al., 2009). The recommended daily allowance (RDA) for folic acid in healthy individuals is 400 μg of dietary folate equivalents (DFEs) per day. In pregnant women, the RDA increases to 600 μg of DFEs per day to ensure both maternal well-being and healthy fetal development (Institute of Medicine
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Standing Committee on the Scientific Evaluation of Dietary Reference Intakes and Its Panel on Folate, Others B Vitamins and Choline, 1998). Folate is widely distributed in foods. Dark green leafy vegetables, animal liver, and citrus fruits are the most abundant source of naturally occurring folate. Dietary folate often exists in reduced and polyglutamated form. Folic acid is the synthetic (stable) form of folate that is used for dietary supplementation and fortification (Iyer and Tomar, 2009). It is an oxidized monoglutamate with higher bioavailability than its natural counterpart. In North America, cereals and grain products are also important sources of folate due to the mandatory fortification in these foods. In addition to dietary folate and folic acid, folate-producing bacteria in the intestine may serve as an endogenous source of folate (Rong et al., 1991). However, the contribution of intestinal bacteria to whole body folate homeostasis in mammals is significantly less than the dietary source of folate (Visentin et al., 2014).
17.2.2 Folate absorption and metabolism Dietary folate absorption is mediated through folate transporters in the small intestine (Visentin et al., 2014; Sid et al., 2017) (Fig. 17.1). The reduced folate carrier (RFC/SLC19A1) and proton-coupled folate transporter (PCFT/SLC46A1) are abundantly expressed along the apical membrane of enterocytes (Visentin et al., 2014; Zhao et al., 2011). PCFT has been identified as the primary transporter for folate/folic acid absorption in the proximal intestine, which has a more favorable acidic microenvironment (Visentin et al., 2014). Dietary folate is mainly in the reduced form as formyl- or methyl-polyglutamate (Wright et al., 2007), and requires hydrolysis to monoglutamate by either glutamate carboxypeptidase II (in human intestine) or γ-glutamyl hydrolases (in rodent intestine) prior to the absorption at the intestinal brush border membrane. Conversely, synthetic folic acid is a monoglutamate and therefore does not require hydrolysis for intestinal absorption (Hu et al., 2016). Folic acid approaches 100% bioavailability when it is consumed in an empty stomach. However, only 85% of folic acid is absorbed when taken with food, possibly due to its adsorption or chelation with food matrix (Caudill, 2010). Dietary folate is heat labile and susceptible to destruction in the gastrointestinal tract. In addition, incomplete hydrolysis of the polyglutamate chain of folate may hinder the absorption of food folate. Therefore only 50% of naturally occurring food folate is bioavailable
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Figure 17.1 Folate absorption and metabolism in the intestine and liver. The major routes of folate absorption and metabolism in the intestine and liver are illustrated. The key forms of folate that enter these organs are shown in bold. PCFT, Protoncoupled folate transporter; RFC, reduced folate transporter. Horizontal ovals denote enzymes and overlap the pathways they catalyze. DHF, dihydrofolate; THF, Tetrahydrofolate; 5-MTHF, 5-methyltetrahydrofolate; DHFR, dihydrofolate reductase; SHMT, serine hydroxymethyltransferase; MTHFR, methylenetetrahydrofolate reductase; MTHFD, methylenetetrahydrofolate dehydrogenase; MTHFC, methylenetetrahydrofolate cyclohydrolase; MS, methionine synthase; GCPII, glutamate carboxypeptidase II; FPG synthase, folylpolyglutamate synthase. Modified image based on Sid, V., Siow, Y.L., O, K., 2017. Role of folate in nonalcoholic fatty liver disease. Can. J. Physiol. Pharmacol. 95, 11411148.
(Institute of Medicine Standing Committee on the Scientific Evaluation of Dietary Reference Intakes and Its Panel on Folate, Others B Vitamins and Choline, 1998; Caudill, 2010). Upon absorption by enterocytes, folic acid is reduced to dihydrofolate (DHF) that is subsequently reduced to tetrahydrofolate (THF), the biologically active form of folate (Wright et al., 2007). Because both reactions are catalyzed by DHF reductase (DHFR), the first enzymatic reduction of folic acid to DHF becomes the rate-limiting step. The addition of a one-carbon moiety to THF by serine hydroxymethyltransferase (SHMT) generates 5,10-methylene-tetrahydrofolate (5,10-methylene-THF). Pyridoxal-50 -phosphate (PLP; active form of vitamin B6) is an essential cofactor for SHMT activity. The 5,10-methylene-THF is further reduced to 5-methyl THF (5-MTHF) by 5,10-methylene-THF reductase (Tibbetts and Appling, 2010). The 5-MTHF is the main form of folate
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that is transported across the basolateral membrane of enterocytes and enters the portal circulation (Wright et al., 2007). However, a high intake of folic acid may exceed the metabolic capacity of enterocytes, leading to its accumulation in the portal circulation (Pietrzik et al., 2010; Hu et al., 2016). The folic acid and 5-MTHF derived from intestine are subsequently delivered to liver via the portal vein (Wright et al., 2007; Hu et al., 2016; Sid et al., 2017) (Fig. 17.1). Liver is the major organ for folate storage and metabolism (Wright et al., 2007). It plays an important role in maintaining the folate homeostasis (Steinberg et al., 1979). Both RFC and PCFT are expressed on the basolateral membrane of hepatocytes (Zhao et al., 2011). Once inside hepatocytes, folic acid is metabolized to 5-MTHF that undergoes polyglutamation by folylpolyglutamate synthase. The polyglutamate form is the preferential form for storage in hepatocytes and is the reactive form for folate-dependent enzymatic reactions relative to the monoglutamate form (Zhao et al., 2009). While up to 20% of 5-MTHF is retained in the liver, the rest is delivered to the extrahepatic tissues through the systemic circulation or secreted into the bile via the bile duct. Bile folate can be reabsorbed into the liver for storage and redistribution to other tissues (Steinberg et al., 1979).
17.2.3 Folate-mediated one-carbon transfer reactions Folate serves as a cofactor to facilitate the transfer of one-carbon units. Sarcosine, N,N-dimethylglycine (derived from choline), glycine, histidine, and serine (mainly through its conversion to formate in the mitochondria) are the common sources of one-carbon units. The folate-mediated onecarbon transfer reactions take place in various cellular compartments including the cytoplasm, nucleus, and mitochondria (Tibbetts and Appling, 2010; Sid et al., 2017). Intracellular one-carbon transfers are mediated by coenzymatic forms of THF, which carries a one-carbon unit for amino acid metabolism, nucleotide biosynthesis, and methylation reactions (Stover and Field, 2011; Sid et al., 2017) (Fig. 17.2). In the cytoplasm and the nucleus, 5,10-methylene-THF serves as a substrate for the biosynthesis of deoxythymidylate (dTMP) from deoxyuridylate (dUMP) and for the interconversion of glycine and serine (Tibbetts and Appling, 2010). These reactions may also occur in the mitochondria. The de novo purine nucleotide biosynthesis and the remethylation of homocysteine to methionine are folate-dependent reactions that take place in the cytoplasm
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Figure 17.2 Compartmentalization of folate-dependent one-carbon metabolism. One-carbon metabolism for nucleotide biosynthesis, amino acid metabolism, and methylation reactions are distributed within intracellular compartments such as the cytoplasm, nucleus, and mitochondria. The box with solid lines denotes reactions that only occur in the cytoplasm, the box with dotted lines denotes reactions that mainly occur in the mitochondria, and the box with double dotted lines denotes reactions that occur in both the cytoplasm and nucleus. The other one-carbon metabolism reactions may occur in all three cellular compartments. DHF, Dihydrofolate; THF, tetrahydrofolate; 5-MTHF, 5-methyltetrahydrofolate; DHFR, dihydrofolate reductase; SHMT, serine hydroxymethyltransferase; MTHFR, methylenetetrahydrofolate reductase; MTHFD, methylenetetrahydrofolate dehydrogenase; MTHFC, methylenetetrahydrofolate cyclohydrolase; FTHFS, formyltetrahydrofolate synthetase; TS, thymidylate synthase; dTMP, deoxythymidylate; dUMP, deoxyuridylate; MS, methionine synthase; MAT, methionine adenosyltransferase; CBS, cystathionine-β-synthase; CSE, cystathionine-Y-lyase; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; SAHH, adenosylhomocysteine hydrolase; fmet-tRNA, formylmethionine-tRNA; FT, formyltransferase. Reproduced with permission from Sid, V., Siow, Y.L., O, K., 2017. Role of folate in nonalcoholic fatty liver disease. Can. J. Physiol. Pharmacol. 95, 11411148.
(Stover and Field, 2011). The 5,10-methylene-THF is reversibly converted to 5,10-methenyl-THF, a precursor for 10-formyl-tetrahydrofolate (10-formyl-THF) by methylenetetrahydrofolate dehydrogenase (Herbig et al., 2002). The 10-formyl-THF provides one-carbon moieties for the biosynthesis of purine nucleotides while 5-MTHF donates carbon units for the biosynthesis of methionine (Tibbetts and Appling, 2010). Methionine is an essential precursor for the biosynthesis of S-adenosylmethionine (SAM), a principal methyl donor that regulates a number of fundamental cellular
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processes involved in cell signaling, protein localization, degradation of molecules, and gene expression. Folate deficiency impairs dTMP synthesis and homocysteine remethylation. Accumulation of homocysteine is associated with increased S-adenosylhomocysteine (SAH), a potent inhibitor of SAM-dependent methylation reactions (Stover, 2004).
17.2.4 Metabolic interconnection of B vitamins Folate and several other B vitamins (i.e., vitamin B6 and B12) are essential cofactors in one-carbon transfer reactions and sulfur-containing amino acid metabolism. Deficiency in folate and vitamin B12 leads to impaired DNA/protein methylation by SAM, which has an important implication in the development of cognitive and metabolic disorders. One of the key products generated during one-carbon metabolism is homocysteine (Selhub, 2002; Stover, 2004). Homocysteine is an intermediate amino acid formed during the metabolism of methionine to cysteine. It can be metabolized via the remethylation pathway and the transsulfuration pathway (Sarna et al., 2015) (Fig. 17.3). Under physiological conditions, remethylation of homocysteine to methionine is catalyzed by methionine synthase that utilizes 5-MTHF as a substrate and vitamin B12 as a cofactor. Vitamin B12 deficiency can result in an accumulation of 5-MTHF, which is often known as a “methyl folate trap.” Too much 5-MTHF depletes the other forms of folate, leading to the inhibition of purine, thymidylate, and methionine synthesis. Another major source of methyl groups is choline, which is primarily obtained from the diet. Choline is oxidized to betaine that serves as a methyl donor for remethylation of homocysteine to methionine via betaine hydroxymethyltransferase (BHMT) (Tibbetts and Appling, 2010) (Fig. 17.3). This reaction occurs when the activity of methionine synthase is compromised. In the transsulfuration pathway, homocysteine is metabolized to cysteine through enzymatic reactions catalyzed by cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) (Sarna et al., 2015) (Fig. 17.3). Metabolism of homocysteine through the transsulfuration pathway is increased under oxidative stress. Cysteine is the precursor for the synthesis of glutathione, a potent antioxidant tripeptide (Mosharov et al., 2000; Sarna et al., 2015). Approximately 50% of the glutathione pool in hepatocytes is derived from cysteine that is synthesized through the transsulfuration pathway (Mosharov et al., 2000). Homocysteine and cysteine also serve as substrates for CBS and CSE-mediated desulfuration reactions to
Figure 17.3 The hepatic cystathionine-β-synthase/cystathionine-γ-lyase (CBS/CSE) system. Through regulation of the transsulfuration pathway and performance of alternative desulfuration reactions, the hepatic CBS/CSE system converges homocysteine and cysteine metabolism with H2S biosynthesis. The transsulfuration pathway is linked to the methionine cycle via the intermediate sulfur-containing amino acid, homocysteine. Homocysteine may be remethylated to regenerate methionine, primarily via a folate-dependent reaction, or homocysteine may be irreversibly metabolized by the transsulfuration pathway. CBS and CSE regulate the transsulfuration pathway, consecutively catabolizing homocysteine to produce the sulfur-containing amino acid cysteine. Cysteine, in turn, may be used for protein synthesis or may be stored as glutathione following integration into the glutathione biosynthetic pathway. Alternatively, both cysteine and homocysteine may serve as substrate for CBS- and CSE-mediated desulfuration reactions which lead to the endogenous synthesis of H2S. (Boxes denote enzymes). MAT, Methionine adenosyltransferase; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; SAHH, adenosylhomocysteine hydrolase; BHMT, betaine homocysteine methyltransferase; MS, methionine synthase; THF, tetrahydrofolate; SHMT, serine hydroxymethylase; 5,10-MTHF 5,10-methylene-tetrahydrofolate; MTHFR, methylenetetrahydrofolate reductase; 5-MTHF, 5-methyltetrahydrofolate; CBS, cystathionine-β-synthase; CSE, cystathionine-γ-lyase; H2S, hydrogen sulfide; γ-GCL, γ-glutamate-cysteine ligase; GS, glutathione synthetase. Reproduced with permission from Sarna, L.K., Siow, Y.L., O, K., 2015. The CBS/CSE system: a potential therapeutic target in NAFLD? Can. J. Physiol. Pharmacol. 93, 111.
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produce hydrogen sulfide (H2S) (Sarna et al., 2015) (Fig. 17.3). Both the CBS and CSE reactions require PLP (active form of vitamin B6) as an essential cofactor. In addition, vitamin B6 also contributes to the synthesis of 5-MTHF mediated by SHMT. Deficiency of vitamin B6 is correlated with low levels of vitamin B12 and folate (Parra et al., 2018). Therefore depletion of vitamin B6, B12, and/or folate can impair one-carbon transfer reactions and sulfur-containing amino acid metabolism.
17.2.5 Folate deficiency In North America mandatory folic acid fortification of cereal grain products was established in 1998 to reduce the incidence of neural tube defects in newborns (CDC, 2010). General populations in Canada and the United States have achieved adequate intakes of folate since the implementation of the folic acid fortification policy (Bailey et al., 2010). However, folate deficiency may still present in some European, African, and Asian countries that do not impose mandatory folic acid fortification policies (Dhonukshe-Rutten et al., 2009). Folate deficiency can also be caused by gene mutations and environmental factors that impair folate absorption or metabolism. DHFR deficiency can compromise folate status in red blood cells and cause megaloblastic anemia, a condition of large abnormal red blood cells (Cario et al., 2011). Chronic alcohol consumption can lead to folate deficiency due to intestinal malabsorption, reduced hepatic storage, and increased urinary excretion of folate (Medici and Halsted, 2013). Folate deficiency has been linked to dysregulation of intracellular metabolic processes (Da Silva et al., 2014). Emerging evidence indicates that low circulating folate levels are associated with metabolic disorders including hyperhomocysteinemia, obesity, and NAFLD. An elevation of homocysteine in the circulation is a common biomarker of folate deficiency.
17.2.6 Folate and metabolic disease A study based on the US National Health and Nutrition Examination Survey (NHANES, 20032006) reported that serum folate levels were significantly reduced in obese patients (12.4 g/L) compared with normalweight individuals (13.1 g/L) (Bird et al., 2015). Regression analysis revealed that the serum folate level is negatively correlated with body mass index (BMI) regardless of adjustment for vitamin intakes and demographic variables such as gender, age, ethnicity, smoking status, and
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alcohol use. However, the BMI values were positively associated with folate levels in red blood cells (Bird et al., 2015). The mechanism and clinical significance of such a discrepancy in the correlations between BMI and folate levels in serum and RBC remain to be investigated. In another study, gastric bypass surgery significantly improved folate status in morbidly obese patients (Updegraff and Neufeld, 1981). It is plausible that obesity may be one of the underlying causes for the imbalance of endogenous folate levels. Low circulating folate levels were detected in patients with type 2 diabetes (Nilsson et al., 2015). The low blood folate levels were correlated to high levels of fasting blood glucose and increased expression of hepatic genes involved in the development of diabetes. Folate depletion also caused epigenetic and transcriptional alterations in the liver, which might contribute to the pathogenesis of type 2 diabetes (Nilsson et al., 2015). The reduction of serum folate levels found in patients with metabolic disease suggests a potential relationship between the abnormal metabolic processes and perturbed folate status.
17.2.7 Folate status and NAFLD Several reports have indicated the association between the circulating folate level and NAFLD. Obese female patients with severe NAFLD showed significantly reduced serum folate levels compared with individuals with normal liver morphology or minimal liver damage (21 vs 27 nmol/L) (Hirsch et al., 2005). Another cohort study showed a correlation of low serum folic acid levels with the presence and severity of steatosis (Xia et al., 2018). Such a correlation was independent of gender, BMI, insulin resistance, and parameters of metabolic syndrome. Low serum folate and vitamin B12 levels were also correlated with increased NASH severity (Mahamid et al., 2018). While clinical evidence suggests that patients with NAFLD may be at a higher risk for folate deficiency, a causal relationship has yet to be confirmed. Further clinical investigation is necessary to establish proper folate intake guidelines for patients with NAFLD. Animal models that mimic the histology and pathology of NAFLD have been established. High-fat diets can induce histopathological changes that resemble the NAFLD features including hepatic lipid accumulation, oxidative stress, and inflammation in rodents (Sarna et al., 2012; Nakamura and Terauchi, 2013; Sid et al., 2018a,b). In a recent study, a high-fat diet feeding caused a significant decrease in serum and liver folate
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levels in mice (Sid et al., 2018b). This was attributed to impaired expression of hepatic folate transporters (PCFT and RFC). It appears that prolonged consumption of diets that are rich in fat may exert a negative impact on folate homeostasis (Sid et al., 2018b). Another study reported that a depletion of dietary folate in rodents was associated with an increased expression of lipid biosynthetic genes, which contributed to abnormal lipid metabolism in the liver (Champier et al., 2012). Hepatic lipid transport via very low density lipoprotein (VLDL) was impaired in folate-deficient mice leading to hepatic lipid accumulation (Christensen et al., 2010). These results suggest that alterations in folate metabolism may have an important implication in NAFLD.
17.3 Nonalcoholic fatty liver disease 17.3.1 Prevalence and pathogenesis of NAFLD The global prevalence of NAFLD is approximately 20%30% and increases to 70%90% in patients with obesity and type 2 diabetes (Younossi et al., 2016, 2018a). NAFLD is considered as the hepatic manifestation of metabolic syndrome. Patients with NAFLD tend to be obese (BMI . 30 kg/m2) and often exhibit comorbidities associated with insulin resistance, dyslipidemia, hypertriglyceridemia, and hypertension (Farrell and Larter, 2006). Although insulin resistance is an independent risk factor for NAFLD, some patients may have normal insulin function (Cohen et al., 2011). Aside from metabolic risk factors, other nonmodifiable risk factors such as age, gender, ethnicity, and certain gene variants may also influence the susceptibility to NAFLD. Men are more likely to accumulate abdominal fat, which increases their risk towards NAFLD development. Moreover, Hispanics and Asians are more likely to develop fatty liver compared to individuals of African descent (Browning et al., 2004). Hispanic individuals often carry a gene variant of patatin-like phospholipase domain-containing protein 3, which results in a twofold increase in hepatic triglyceride content and confers susceptibility towards NAFLD. In addition, mutations in other genes involved in lipid metabolism can also increase the risk of NAFLD (Birkenfeld and Shulman, 2014). Steatosis is the main histological feature of NAFLD which is defined as lipid accumulation in more than 5% of hepatocytes (Tiniakos et al., 2010). Steatosis is often a self-limiting condition; however, it can advance to NASH that is characterized by steatosis, inflammation, and hepatocyte injury (Cohen et al., 2011). Hepatocyte ballooning is a form of
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hepatocellular injury that is characterized by cell swelling, which is an essential feature that distinguishes NASH from steatosis. Liver fibrosis is frequently detected in NASH patients (Yeh and Brunt, 2014). The presence of advanced hepatic fibrosis increases the risk for cirrhosis and HCC. Although the progression of steatosis to NASH is reversible, NASH may irreversibly advance to cirrhosis that is increasingly susceptible to the development of portal hypertension and HCC (Kessoku et al., 2014). The pathogenesis of NAFLD is complex and incompletely understood. The two-hit hypothesis was initially proposed to describe NAFLD pathogenesis. This hypothesis suggests that perturbations in lipid metabolism lead to steatosis (first hit), which sensitizes the liver to the secondary hit, such as inflammation, oxidative stress, and cell injury (Day, 2005). However, steatosis may not always precede inflammation. It is plausible that inflammation could precede lipid accumulation in NASH patients (Tiniakos et al., 2010). Furthermore, the two-hit theory does not account for many other metabolic changes that occur in NAFLD. This has led to the development of the multiple-parallel-hit hypothesis, which suggests that multiple factors such as insulin resistance, lipotoxicity, oxidative stress, inflammation, gut-derived endotoxins, adipokines, or genetic factors may simultaneously induce NAFLD (Tilg and Moschen, 2010). As NAFLD is a multifaceted disease associated with several metabolic abnormalities, there are currently no therapeutic drugs approved for its treatment. Novel therapeutic strategies are urgently required for the management of NAFLD (Younossi et al., 2018b).
17.3.2 Current treatment for nonalcoholic fatty liver disease Lifestyle modifications such as increasing physical activity and healthy diet are considered to be the most safe and effective strategies for NAFLD management. Such modifications can promote weight loss, alleviate steatosis, and improve liver function (Chalasani et al., 2012). Minimal weight loss (3%5%) is sufficient to reduce hepatic lipid accumulation in NAFLD patients. However, greater than 7% weight reduction is required to improve histological features associated with NASH (Glass et al., 2015). Patients who receive bariatric surgery have profound weight loss, which is associated with improved liver histology and insulin sensitivity (Mechanick et al., 2009). However, bariatric surgery is not recommended for cirrhotic patients due to concerns of liver failure after a rapid weight loss. Although gradual weight loss appears to be beneficial for NAFLD
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patients, lifestyle changes and adherence to healthy diets can be a challenge. Several pharmacological agents that target certain metabolic risk factors have been proposed for the treatment of NAFLD (Chalasani et al., 2012). Metformin, a first line antidiabetic agent, reduces insulin resistance and alanine transaminase levels but has limited effects on inflammation and steatosis in NAFLD patients (Bugianesi et al., 2005). Thiazolidinediones are insulin sensitizers and have been shown to improve liver function and histology in patients with NASH (Neuschwander-Tetri et al., 2003). However, their long-term safety and efficacy for NAFLD treatment have not been established (Chalasani et al., 2012). Oxidative stress is a key mediator in liver injury in NAFLD. While treatment with antioxidants such as vitamin E has been shown to ameliorate steatosis, inflammation, and hepatocyte ballooning, further investigation is required to determine the effectiveness of antioxidants in NAFLD treatment (Sanyal et al., 2004). NAFLD patients have a high cardiovascular risk and therefore statins have been used to treat dyslipidemia and improve cardiovascular outcomes in these patients. The use of statins appears to be safe and effective for correcting lipid abnormalities in NAFLD patients (Chalasani et al., 2012). There is currently no single agent that can target all metabolic risk factors associated with NAFLD. Researchers are still searching for therapeutic agents that can target multiple pathways associated with NAFLD pathogenesis (Younossi et al., 2018b).
17.3.3 Role of folic acid supplementation in nonalcoholic fatty liver disease It was reported that folic acid supplementation during high-fat diet feeding in mice could effectively improve lipid and glucose metabolism in the liver (Sid et al., 2015). Such effects were mediated through the regulation of AMP-activated protein kinase, a key regulator of whole body energy balance and metabolic homeostasis (Sid et al., 2015). Folate and folic acid exhibit antioxidant functions due to their ability to directly scavenge reactive oxygen species (ROS) (Gliszczynska-Swiglo and Muzolf, 2007) and regulate ROS-generating or metabolizing enzymes (Woo et al., 2006; Hwang et al., 2011; Sarna et al., 2012). It was shown in rodents that folic acid supplementation could confer protective effects against oxidative stress in the liver (Woo et al., 2006; Sarna et al., 2012) and kidney (Hwang et al., 2011). Hepatic oxidative stress is associated with increased expression of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in animal models of obesity, metabolic
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syndrome, and NAFLD. Folic acid supplementation effectively inhibited NADPH oxidase-mediated superoxide production, restoring the antioxidant response in hyperhomocysteinemic rats and in high-fat diet-induced obese mice (Woo et al., 2006; Hwang et al., 2011; Sarna et al., 2012). Patients with NAFLD also have elevated levels of proinflammatory cytokines in the circulation, immune cells, liver, and adipose tissue (Day, 2006; Braunersreuther et al., 2012). Macrophages grown in a folatedepleted medium had high expression of proinflammatory cytokines including interleukin-1β, monocyte chemoattractant protein-1, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) (Kolb and Petrie, 2013). Incubation of macrophages with folic acid attenuated homocysteine-induced proinflammatory cytokine expression (Au-Yeung et al., 2006). Folic acid supplementation in high-fat diet-fed mice significantly decreased the expression of hepatic proinflammatory cytokines (IL-6, TNF-α) through inhibition of nuclear factor kappa B, a key transcriptional factor of proinflammatory cytokine genes (Sid et al., 2018a). Folic acid supplementation in overweight individuals or hyperhomocysteinemic patients was associated with a reduction of proinflammatory cytokine levels (Wang et al., 2005; Solini et al., 2006). Those results indicate that folic acid possesses lipid and glucose lowering effects, as well as antioxidant and antiinflammatory functions, which makes it a promising candidate for managing NAFLD. Although folic acid supplementation appears to be beneficial for alleviating metabolic abnormalities, clinical studies are required to further validate the effect of folic acid supplementation in patients with NAFLD.
17.4 Conclusions Vitamins are micronutrients that support physiological functions in the body. Folate (vitamin B9) plays a fundamental role in one-carbon metabolism that is essential for gene expression and sulfur-containing amino acid homeostasis. Although low folate status is observed in patients with malabsorption, kidney dysfunction, or liver disease, dietary folate deficiency is uncommon in generally healthy populations in countries that have implemented mandatory folate fortification policies. However, obese individuals with NAFLD are reported to have low folate levels in the serum despite the adequate intake of folate from diets and/or supplements (Hirsch et al., 2005; Xia et al., 2018; Mahamid et al., 2018). A recent study has revealed that fatty liver is associated with impaired folate
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transporter expression and low folate levels in diet-induced obese mice (Sid et al., 2018b). In animal studies, folic acid supplementation can restore hepatic redox balance, improve lipid and glucose metabolism, and attenuate inflammatory cytokine production in rodents with diet-induced metabolic abnormalities (i.e., overweight, fatty liver, hyperglycemia) (Woo et al., 2006; Sarna et al., 2012; Sid et al., 2015, 2018a). Nutritional therapy including folic acid supplementation appears to be an alternative approach for the management of metabolic diseases, such as NAFLD and diabetes. Future research is warranted to address whether the regulation of folate homeostasis can improve the clinical outcomes in patients with NAFLD or other metabolic diseases. If proven effective, folic acid supplementation may be an economical and effective approach to control this emerging public health threat (NAFLD) globally.
Acknowledgements This work was supported, in part, by grants from the Natural Sciences and Engineering Research Council of Canada and St. Boniface Hospital Foundation.
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