Sana Raza,Archana Tewari,Sangam Rajak,Rohit A.Sinha

Department of Endocrinology,Sanjay Gandhi Postgraduate Institute of Medical Sciences,Lucknow,India

Keywords:Anti-oxidant Autophagy Non-alcoholic fatty liver disease (NAFLD)Non-alcoholic steatohepatitis (NASH)Vitamin A Vitamin B Vitamin C Vitamin D Vitamin E

ABSTRACT The incidence of non-alcoholic fatty liver disease (NAFLD) is rising rapidly across the globe.NAFLD pathogenesis is largely driven by an imbalance in hepatic energy metabolism,and at present,there is no approved drug for its treatment.The liver plays a crucial role in micronutrient metabolism,and deregulation of this micronutrient metabolism may contribute to the pathogenesis of NAFLD.Vitamins regulate several enzymatic processes in the liver,and derangement in vitamin metabolism is believed to play a critical role in NAFLD progression.The anti-oxidant activities of vitamins C and E have been attributed to mitigate hepatocyte injury,and alterations in the serum levels of vitamin D,vitamin B12 and folate have shown a strong correlation with NAFLD severity.This review aims to highlight the role of these vitamins,which represent promising therapeutic targets for the management of NAFLD.

1.Introduction

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic hepatopathy worldwide.It is recognized as a hepatic manifestation of the metabolic syndrome,and characterized by lipid infiltration in the hepatocytes.NAFLD comprises a range of diseases from simple steatosis to non-alcoholic steatohepatitis(NASH),fibrosis,cirrhosis,and may progress to hepatocellular carcinoma (HCC).1,2The worldwide prevalence of NAFLD is estimated to be 24%,3while it is reported to have much higher incidence in patients with metabolic syndrome and type 2 diabetes(T2D).4The mortality rate and the number of liver transplantations owing to NAFLD and NASH are increasing,making it the second leading cause of liver transplant in the United States.5,6A steady progress has been seen in the understanding of NAFLD epidemiology and pathogenesis;however,there is slow advancement in the development of therapeutics.Although several potential targets have been identified,currently,there is no the Food and Drug Administration (FDA) approved therapy for NAFLD/NASH.7

NAFLD is a multifactorial disease having various modifiers such as lifestyle,diet,and gut microbiota,which act together in a suitable genetic/epigenetic environment and alter the response to lipid excess.Two significant metabolic abnormalities commonly linked to NAFLD are insulin resistance (IR) and increased supply of fatty acids to the liver,which are largely attributed to obesity induced by high energy food consumption and sedentary lifestyle adopted by the modern society.The consumption of an obesogenic diet,and deranged energy balance,result in lipid accumulation in the hepatocytes,leading to lipotoxicity,oxidative stress,endoplasmic reticulum (ER) stress,and liver inflammation,which are the key drivers of NAFLD.The pathogenesis of NAFLD is substantially affected by individual lifestyle choices including diet and exercise.Lifestyle modifications (diet,physical activity and exercise),which primarily aim at controlling bodyweight and metabolic disorders,remain the mainstay approach of disease management.8However,a reduction in energy alone does not suffice and studies have highlighted that dietary composition also plays a critical role in the manifestation and development of NAFLD.9

The liver is involved in micronutrient metabolism,the impairment of which,may contribute to the pathogenesis of NAFLD.10In addition to hepatocytes,the liver contains monocytes,lymphocytes,Kupffer cells,natural killer (NK) cells,dendritic cells (DCs)and several other immigrant cells,which are closely associated with the immune system.Diet contains macronutrients which provide energy for body functions,and micronutrients that perform regulatory functions.Vitamins are essential nutrients of our diet which are well known to affect the immune system,including both innate and adaptive response.11The immune system is particularly associated with the entero-hepatic axis and involved in the development and progression of NAFLD.Recent studies have highlighted the relation between dietary vitamins and fat accumulation in the liver.12Some researchers have focused on the role of a specific vitamin on hepatic metabolism;however,a broader outlook of the role of vitamins in NAFLD remains unexplored.13In this review,we discuss the hepatic pathophysiology linking vitamins to NAFLD,aiming to summarize the roles of some key vitamins in NAFLD progression and to highlight their potential in NAFLD management.

2.Vitamin E

Vitamin E,a fat-soluble and powerful chain breaking antioxidant in the human body,has the most significant evidence of therapeutic benefit in liver disease.4Vitamin E has the ability to reduce oxidative stress and decelerate the pathogenesis of NASH(Fig.1).14The American Association for the Study of Liver Disease(AASLD) and the European Association for the Study of the Liver(EASL) recommend the use of vitamin E (800 IU/day) in nondiabetic adults with biopsy-proven NASH.4,15Despite the availability of no standard protocol for the treatment of NAFLD/NASH,vitamin E supplements are commonly prescribed to NAFLD patients.16

Fig.1.The roles of vitamin E in regulating hepatic oxidative stress,inflammation and apoptosis.

2.1.Molecular structure and function of Vitamin E

Vitamin E has eight natural forms including four tocopherols(α,β,γ,δ)and four tocotrienols(α,β,γ,δ),with α-tocopherol being the most abundant and efficacious in inhibiting lipid oxidation.Vitamin E reduces peroxidation and inhibits the expression of transforming growth factor-beta (TGF-β) which is involved in hepatic fibrosis and hepatocyte apoptosis through activation of hepatic stellate cells (HSCs).17It improves liver integrity by downregulating the hepatic cluster of differentiation 36 (CD36),a membrane transporter protein responsible for the uptake of fatty acids into the liver.18A rat model based study for NASH showed that vitamin E enrichment caused a lowering in lipid peroxidation.19Elevated hepatic transaminase levels were normalized in chickens fed with a high-oxidant diet along with vitamin E supplementation.20PIVENS (previously reported randomized controlled trial,adult patients) and TONIC(Treatment of NAFLD in Children) trials (pediatric population),showed significant improvement in steatosis and inflammation in patients treated with vitamin E for 96 weeks compared to placebo.21,22However,one study reported no significant improvement in alanine transaminase(ALT)upon daily administration of 800 IU vitamin E for 96 weeks,although NAFLD activity score (NAS) and hepatocellular ballooning score were improved.21Similar results were seen in a pediatric population by Vos et al.,23where they reported that insufficient vitamin E consumption is related to a higher grade of hepatic steatosis.Nobili et al.24showed improvement in transaminases and liver histology in children receiving vitamin E treatment.Similar results were reported by Nobili et al.,25who showed that vitamin E supplementation provided no improvement in NAFLD,in a cohort of 53 children and adolescents.

2.2.Anti-oxidant effect

Patients with NAFLD show increased oxidative stress.This oxidative stress is developed when reactive oxygen species (ROS)generation overcomes the cellular anti-oxidant mechanisms.26,27Anti-oxidant deficit may lead to increased lipid peroxidation and cell death caused due to mitochondrial compromise.Vitamin E has an inherent anti-oxidative activity;it can scavenge lipid peroxyl radicals by donating a hydrogen ion from its chromanol ring.28,29It acts as a scavenger of hydroxyl,peroxyl and superoxide radicals and provides protection against plasma lipid and low-density lipoprotein peroxidation.The ability of vitamin E to scavenge radicals is not limited to ROS,it has also been found to be active against reactive nitrogen species (RNS).30Higher vitamin E intake may,therefore,help to counteract the rise in oxidative stress in NAFLD patients.

The interaction of vitamin E with cellular components may help to foster the anti-oxidative environment.28Superoxide dismutase(SOD) is a crucial anti-oxidative enzyme,which can partition superoxide radicals into oxygen or hydrogen peroxide.Vitamin E supplementation is known to stimulate SOD levels.27,31-34Vitamin E also stimulates the action of other antioxidative enzymes including catalase and glutathione peroxidase.27,31,33,34Dietary vitamin E lowers c-myc and transforming growth factor-alpha(TGF-α) expression,causing reduced secretion of nitric oxide synthase (iNOS) and reduced form of nicotinamide adenine dinucleotide phosphate(NADPH)oxidase,which significantly contribute to oxidative stress.31,35

2.3.Anti-inflammatory and anti-apoptotic effect

Vitamin E may mitigate hepatic fibrosis and prevent cirrhosis by modulating inflammatory response.14,28Vitamin E supplementation has been linked to increased adiponectin messenger RNA(mRNA) and protein levels.36,37Adiponectin suppresses hepatic fatty acid synthesis and reduces inflammation in patients with NASH.36Vitamin E suppresses the expression of pro-inflammatory cytokines such as TNF-α,interleukin(IL)-1,IL-2,IL-4,IL-6,and IL-8 and lowers inflammatory response in NAFLD.28,31,38

Vitamin E is also reported to play a role in cell death and apoptosis.36,39Studies have suggested that vitamin E increases the levels of anti-apoptotic protein BCL-2,and lowers the levels of proapoptotic proteins BAX and p53.39It decreases the activity of caspase-9 and cytochrome C,which are involved in the mitochondrial apoptosis pathway,and caspase-8 and caspase-3 involved in the Fas/FasL apoptotic pathway.39Some studies have also shown the drawbacks of vitamin E.A study linked the supplementation of high-dose vitamin E with increased mortality.40Further studies are needed to evaluate the safety and effectiveness of using vitamin E as a therapeutic agent in NAFLD.

3.Vitamin D

Vitamin D is an essential nutrient and a steroid hormone well known for its role in calcium homeostasis and mineral metabolism.Vitamin D is derived from 7-dehydrocholesterol present in the skin,which upon ultraviolet light B (UVB) irradiation converts to an inactive precursor (cholecalciferol) that further undergoes a twostep metabolism in the liver and the kidney,and gets converted into its biologically active form,1,25-dihydroxy vitamin D3 (calcitriol).Vitamin D mediates its action by interaction with the nuclear vitamin D receptor (VDR),which belongs to the nuclear receptor superfamily of ligand activated transcription factors.41The genomic actions of vitamin D involve the heterodimerization of the ligand activated VDR with the retinoid-X-receptor (RXR) causing transcriptional activation or repression of a number of target genes through binding to vitamin D response elements (VDREs) in the promoter region of target genes.The non-genomic actions of vitamin D are manifested through the activation of signaling molecules including phosphatidylinositol-3 kinase (PI3K),generation of secondary messengers(cyclic adenosine monophosphate(AMP),Ca2+,etc.) and activation of protein kinases including protein kinase A,mitogen-activated protein (MAP) kinases,src,protein kinase C(PKC)and Ca2+-calmodulin kinase II.42Remarkable scientific interest in the extra-skeletal actions of vitamin D in recent years has expanded its actions to cardiovascular disease,diabetes,immune modulation,cell proliferation and differentiation.Consistent with the multifaceted nature and diverse properties of vitamin D,circulating evidences provide a link between vitamin D and fatty liver disease.While vitamin D deficiency is widely prevalent in the world,concurrently,NAFLD is a rapidly emerging manifestation of the metabolic syndrome worldwide.Several epidemiological studies have reported the coexistence of hypovitaminosis D and NAFLD,and provide evidence that these conditions share multiple risk factors.It is notable that low serum vitamin D levels correlate with the severity of steatosis and necro-inflammatory damage.43,44

3.1.Role of vitamin D in insulin resistance

Vitamin D deficiency occurs in 55%of the patients with NAFLD.45Several experimental models have proposed vitamin D as a modulator of insulin sensitivity.46Epidemiological data reports an association between vitamin D levels and the presence of obesity,T2D and IR.47IR is a well-known precursor and accelerant of NAFLD,and is considered as a hallmark of the disease linked to the development of oxidative stress and lipotoxicity.48Vitamin D deficiency is reported to affect insulin secretion in both animal and human models.Vitamin D has a positive role in the modification of pancreatic β-cell function and may directly stimulate pancreatic insulin secretion owing to the presence of VDRs in the β-cells of pancreas,and by the expression of 1-α-hydroxylase enzyme.49Insulin secretion is a calcium-dependent process,and changes in calcium flux may have an adverse effect on pancreatic β-cell secretion (Fig.2).Vitamin D deficient-hypocalcemic rats show lower glucose-stimulated insulin secretion without correction of hypocalcemia.50Vitamin D deficiency may,therefore,contribute to IR and NAFLD.

Fig.2.Vitamin D stimulates insulin secretion in pancreatic β-cells.

3.2.Vitamin D as an immunomodulator

Various studies have shown the role of vitamin D in the development and function of the immune system.The detection of functional VDR in almost all immune cells including antigenpresenting cells (APCs),i.e.the macrophages and DCs,and activated T lymphocytes,provides an indirect evidence of the potential role of vitamin D as an immunomodulator.Vitamin D regulates both innate and adaptive immune system through VDR.The activated nuclear VDR is involved in the transcriptional regulation of the proliferation and differentiation of immune cells.51The immunomodulatory effects of vitamin D may be attributed to the ability of its biologically active form,calcitriol,to regulate the expression of various genes.The innate immune response involves activation of Toll-like receptors(TLRs)on polymorphonuclear cells,monocytes,macrophages,and epithelial cells.The TLRs are pattern recognition receptors,which play a key role in host cell recognition and response to pathogens.Among the 13 TLRs identified in mammals,TLR2,TLR4 and TLR9 are involved in the pathogenesis of NAFLD.52Vitamin D deficiency exacerbates the development and progression of NAFLD by the activation of TLR2 and TLR4,and stimulates downstream inflammatory signaling molecules causing steatosis and inflammation.52

Adiponectin,the prototypic adipokine which is described as an anti-inflammatory agent,has been shown to decrease necroinflammation and steatosis in NAFLD and improve IR.53In a large cohort of patients,a positive association was observed between 25-hydroxy vitamin D concentrations and adiponectin levels,independent of body mass index (BMI).54In another study,55mice fed with a high-fat Western diet(WD)along with vitamin D depletion(VDD) were compared to the WD alone group.The WD/VDD mice had increased hepatic mRNA levels of resistin,IL-4,IL-6 and TNF-α markers known to be implicated in oxidative stress and hepatic inflammation.The vitamin D deficient mice had increased levels of mRNA of TLR-2,TLR-4,and TLR-9 following WD.Similarly,in a study based on a rat NASH model,25 hydroxy vitamin D and 1,25-dihydroxy vitamin D levels were increased by phototherapy while lowering hepatocyte inflammation,fibrosis and apoptosis as compared to controls.54In association with the reduced hepatic expression of inflammatory genes TNF-α and TGF-β,phototherapy improved IR and increased serum adiponectin.

3.3.Vitamin D attenuates hepatic steatosis by inducing autophagy

Autophagy is a major cellular recycling process leading to the degradation of cytoplasmic contents,which are delivered to and degraded in the lysosomes,to supply energy and regenerate organelles.It helps to remove the misfolded cytosolic proteins,glycogen,lipids,nucleic acids,and clears damaged organelles including mitochondria,endoplasmic reticulum and peroxisomes.Following degradation,the breakdown products are released and recycled into macromolecular components;therefore,autophagy is believed to be a cell-survival mechanism to maintain cell viability under unfavourable conditions and balances energy sources under nutrient stress.The deregulation of autophagy may contribute to the pathogenesis of human diseases including liver associated diseases,neurodegenerative diseases and cancer.56Autophagy has been implicated as a disease associated factor in people with liverrelated disorders,and is reported to contribute to the development of hepatic steatosis,fibrosis,cirrhosis,and HCC.57Hormone induced autophagy helps to maintain intracellular homeostasis in response to nutrient and energy deficit.58Hepatic autophagy serves as a means to regulate hepatic lipid content through delivery of lipid droplets to lysosomes in a process known as “Lipophagy”.58Lipophagy has been implicated in a number of anti-NAFLD action of drugs and hormones.59,60Murine models of NAFLD showed that pharmacological or genetic inhibition of autophagy increased hepatic fat content.61Furthermore,the down-regulation of hepatic autophagy is a consistent finding in human studies on NAFLD.62

1,25-dihydroxy vitamin D has been shown to protect against high-fat diet(HFD)or free fatty acid(FFA)induced hepatic steatosis by inducing autophagy in FFA-treated HepG2 cells (Fig.3).63Furthermore,HFD-fed mice treated with 1,25-dihydroxy vitamin D showed increase in autophagic flux and increased expression of the markers of autophagy.63The protective effects of 1,25-dihydroxy vitamin D were abrogated on the inhibition of autophagy by 3-methyladenine.63These findings suggest that 1,25-dihydroxy vitamin D may modulate hepatic steatosis by inducing autophagy,however,the mechanism underlying this effect is not well understood.Some studies indicate a direct role of vitamin D in modulating liver inflammation and fibrogenesis,and improving hepatic response to insulin by its binding to specific VDR expressed on different cell types in the liver.64While the VDR is expressed at low levels in hepatocytes,their levels can be induced by lipotoxic insults.65,66Additionally,high expression of both nuclear and membrane VDR is seen in the non-parenchymal liver cells including HSCs,Kupffer cells,natural killer T (NKT) cells and sinusoidal endothelial cells.67HSCs are major effectors of hepatic fibrosis,and the expression of VDR in the HSCs has been reported to antagonise TGF-β signaling,which is the major pro-fibrogenic pathway in the liver.68

Fig.3.Modulation of hepatic pathophysiology by vitamin D.

Although epidemiological studies correlating hypovitaminosis D to the presence of NAFLD reinforce the belief that restoration of optimal vitamin D levels may be a therapeutic approach to the management of NAFLD,the lack of consistent data from vitamin D supplementation trials warrants further studies to explore its therapeutic potential.47,69

4.Vitamin B

The vitamin B group includes eight types of compounds including thiamine (B1),riboflavin (B2),niacin (B3),pantothenic acid (B5),pyridoxine (B6),biotin (B7),folic acid (B9),and cyanocobalamin (B12).Vitamin B3,also known as niacin,acts as a precursor of the coenzyme nicotinamide adenine dinucleotide (NAD)and nicotinamide adenine dinucleotide phosphate(NADPH),which have a crucial role in lipid metabolism.70It has been used for the treatment of dyslipidemia and cardiovascular diseases.71Vitamin B3 supplementation increased redox potential,lowered hepatic cholesterol content,and blocked the gain in liver weight in obesogenic diet-induced rat model of NAFLD.A protective effect was also seen on the pre-existing hepatic steatosis,displaying the therapeutic potential of vitamin B3 in NAFLD.72Niacin treated human hepatoblastoma cells showed a reduction in hepatic lipid accumulation by 40-60%,likely through the inhibition of diglyceride acyltransferase 2 and NADPH oxidase.73Plasma triglyceride concentration,hepatic fat content,and improved liver enzymes were lowered in dyslipidemic patients treated with niacin.74However,niacin treatment in a random controlled trial based on 27 obese individuals with NAFLD,failed to demonstrate a decrease in hepatic fat deposition although serum triglyceride and very lowdensity lipoprotein were reduced and insulin sensitivity was improved.75Some studies have demonstrated that long-term niacin supplementation may lead to IR.76As the pathogenesis of NAFLD involves IR and patients with NAFLD already have reduced insulin sensitivity,niacin may have some adverse side effects despite reducing the hepatic fat content.

Vitamin B12 exists in two forms in humans:methyl cobalamin and 5′-deoxyadenosylcobalamine and has been linked to hepatitis,cirrhosis and HCC.It acts as a cofactor for the mitochondrial enzyme,methyl malonyl CoA mutase,which regulates the rate of long-chain fatty acyl-CoA transfer into mitochondria and affects lipid metabolic pathways.77The liver acts as a storage site for vitamin B12.Some studies,in animal models,demonstrated the effect of vitamin B12 deficiency in lipid metabolism.78Low vitamin B12 intake in maternal diet led to high rates of adiposity and T2DM in offspring,along with a change in the expression of genes involved in hepatic lipid metabolism.These alterations were normalized upon reconstitution of vitamin B12.78,79Low serum vitamin B12 was associated with elevated serum ALT levels in NAFLD patients.80Increased body fat mass percentage,decreased fat-free mass,and impaired capacity to secrete insulin was seen in offspring of female weaning rats with restricted vitamin B12.Offspring born to mothers with B12 deficiency displayed dysregulation of fatty acid metabolism,amino acid metabolism,and glycolysis possibly due to alteration of peroxisome proliferatoractivated receptor (PPAR) γ and PPARα hepatic expression.81In some other studies,offspring subjected to maternal vitamin B12 deficiency displayed enhanced plasma total cholesterol.79The protective role of vitamin B12 in NAFLD remains controversial as some studies also reported no protective effect of vitamin B12 in NAFLD.82

Folate or vitamin B9,a water-soluble vitamin,is involved in onecarbon transfer reactions which are essential for cell metabolism.83,84Dysregulated folate-dependent one-carbon metabolism has been implicated in NAFLD-related comorbidities including obesity,T2D,and metabolic syndrome.85-88Genetic mutations in folate pathway have been shown to dysregulate homocysteine metabolism.89,90Homocysteine,a sulphur-containing amino acid,is formed as an intermediate in the biosynthesis of methionine and cysteine.91Hyperhomocysteinemia is known to affect intracellular lipid metabolism and increased homocysteine levels may promote hepatic fat accumulation.92,93A recent study reported that hyperhomocysteinemia promotes hepatic steatosis in mice via activation of the aryl hydrocarbon receptor/CD36 pathway.94Hepatic lipid accumulation was reported in various models of hyperhomocysteinemia.95,96

In mammals,dietary intake of folate is essential to meet physiological requirements as they lack the ability to synthesize folate.97,98Studies have reported an inverse association of serum folate levels with BMI in patients with obesity,NAFLD and T2D.86,88Low serum folate levels were found in obese and overweight patients as compared to individuals with normal weight.87It is suggested that folate deficiency may contribute to the development of steatosis.99,100Depletion of dietary folate was linked to high expression of lipid biosynthetic genes,leading to dysregulation of lipid metabolism in the liver.101Folate deficient mice showed impaired hepatic lipid transport by very low-density lipoprotein(VLDL).85,99

A recent study demonstrated that AMP activated protein kinase(AMPK) activation in HFD fed mice was restored upon folic acid supplementation and improved hyperinsulinemia,lipid and glucose metabolism.102Hepatic IR disturbs lipid metabolism leading to disrupted glucose and lipid production in the liver,a feature commonly observed in NAFLD.103This dysregulated lipid and carbohydrate metabolism is often associated with disturbed AMPK,a key regulator of energy metabolism.104The inactivation of AMPK is linked to hepatic lipid accumulation,hyperinsulinemia and hyperglycemia in animal models with HFD induced NAFLD.102,105Further studies are warranted to elaborate upon the role of folate in metabolic disease and explore its potential as a therapeutic for NAFLD.

5.Vitamin A

Vitamin A or retinoic acid is an essential fat-soluble vitamin obtained from carotenoids such as β-carotene(found in carrots and green leafy vegetables such as spinach) or retinyl esters from rich animal sources like eggs,fish and the liver.They are absorbed and transported as retinyl esters in chylomicrons,to the liver where they are hydrolyzed to form retinol.Retinol is mainly stored in the HSCs,which are responsible for fibrosis,and plays a central role in vitamin A metabolism.106Retinol mainly exerts its action via activation of the retinoic acid receptor (RAR) or RXR.Hepatocytes are known to actively metabolize vitamin A and alter glucose and lipid metabolism in response to the vitamin A metabolites.107,108Vitamin A is crucial for several physiological processes such as vision,cell proliferation and differentiation,immune regulation,embryogenesis,glucose and lipid metabolism.106

Retinoic acid via RARs increases hepatic expression of genes promoting fatty acid oxidation (PPARα,fibroblast growth factor 21(FGF21),carnitine palmitoyltransferase I (CPT1),and uncoupling protein 2 (UCP2)).106In line with these findings retinoic acid treatment causes reduction in liver triacylglycerol content and circulating VLDL fraction,and moderately increase ketogenesis.109

Hepatic lipid accumulation results primarily due to influx of dietary lipids,non-esterified fatty acids(NEFAs)from adipose tissue and hepatic de novo lipogenesis (DNL).Vitamin A is directly or indirectly involved in the regulation of these processes.Retinoic acids,as well as synthetic ligands of RXRα (e.g.,bexarotene),enhance hepatic DNL and plasma TG levels by activating liver X receptor (LXR)/RXR and PPARγ/RXR,which,in turn,enhance the expression of sterol regulatory element-binding protein(SREBP)-1c and carbohydrate-responsive element binding protein(ChREBP).110Both SREBP-1c and ChREBP increase hepatic lipogenic genes such as acetyl-CoA synthase and lipogenic enzymes including acetyl-CoA carboxylase 1 (ACC1),fatty acid synthase,stearyl-CoA desaturase and glycerol-3-phosphate acyltransferase,mitochondrial(GPAT) in the liver.The inhibition of SREBP-1c or ChREBP leads to impaired lipid synthesis and decreases hepatic steatosis.111,112

These apparent opposing effects of retinoic acid signalling may be due to differential action of RARs(lipolytic)and RXRs(lipogenic)transcriptional activities.Furthermore,model systems,ligand and doses of retinoic acid used in different studies may also lead to these contrasting effects.

Along with their anti-steatotic effects in hepatocytes,a highly selective RARβ2 agonist AC261066,was shown to reduce the activation of HSCs,marked by decreased HSC expression of alphasmooth muscle actin(α-SMA),in mice with HFD-induced NAFLD.113

Several genomic loci are known to increase the predisposition to NAFLD,the most prominent being a genetic variant of patatin-like phospholipase domain-containing 3 (PNPLA3-I148 M),a distinguished heritable factor linked to NAFLD.It is known to predispose for disease progression and NAFLD-associated HCC.114PNPLA3 is a multifunctional enzyme which acts as a triglyceride hydrolase,retinyl esterase and acetyl-CoA-independent transacylase.115Its genetic variant,PNPLA3-I148 M has lowered hydrolase activity and promotes triglyceride accumulation in the liver.Overexpression of PNPLA3-I148 M is reported to cause hepatic steatosis in mice and alters circulating retinol levels in humans.116

In human studies involving NAFLD/NASH patients,serum retinoic acid concentrations were lowered and the extent of hepatic steatosis was inversely correlated with RXR mRNA expression.117NAS and hepatic ballooning were also associated with alteration in serum retinoic acid concentration.117,118

6.Vitamin C

The impact of oxidative stress and inflammation is well demonstrated in the development of NAFLD,metabolic syndrome,cardiovascular disease,and diabetes mellitus.119The production of ROS leads to peroxidation,resulting in inflammation and the activation of HSCs,thereby causing fibrosis.Oxidative stress accelerates IR and inflammation in hepatic cells,which are critical mechanisms leading to dislipidemia.120Vitamin C (ascorbic acid) is a powerful anti-oxidant in human health,capable of scavenging free radicals.121In isolated rat liver mitochondria,vitamin C decreases mitochondrial ROS generation and enhances manganese SOD and glutathione peroxidase(GPx)activity.122Vitamin C treatment may,therefore,relieve hepatic oxidative stress.It has also been inversely linked to the inflammatory markers,C-reactive protein (CRP) and myeloperoxidase.123,124Ascorbic acid is also suggested to regulate adiponectin,which lowers lipid accumulation in the liver,systemic IR and inflammation,and protects against NAFLD.125,126Additionally,ascorbic acid supplementation inhibits hepatic steatosis and stress via increase in the mRNA levels of PPARα-dependent fatty acid β-oxidation genes in livers activation of the FGF21/FGFR2/adiponectin pathway.127,128

Vitamin C is believed to play a role in circulating and hepatic lipid homeostasis.129Vitamin C supplementation in rats with glucose intolerance induced by dexamethasone led to improved insulin sensitivity.130A decrease in serum vitamin C levels was related to increase in hepatic ballooning in pediatric patients with NAFLD.23A cross-sectional study by Han et al.131in Korean males showed a positive association between low vitamin C intake and NAFLD.Similarly,an inverse association between dietary vitamin C intake and NAFLD in middle-aged and older adults,especially for the male population and non-obesity population.132In contrast to this,another cross-sectional study showed similar level of vitamin C concentrations in NAFLD patients and healthy controls.100Another study by Madan et al.133reported no difference in plasma vitamin C levels in NAFLD patients and healthy control population.

Vitamin C deficiency is reported to accelerate dyslipidemia and hepatic consequences,while increased intake of vitamin C reduced the severity of dyslipidemia and lipid accumulation in the hepatocytes of guinea pigs.134Another study showed that vitamin C supplementation improved oxidative stress,hepatocellular ballooning and inflammation in a rat model.135Harrison et al.136reported that a combination treatment of vitamin C and E led to improved hepatic fibrosis in NASH patients.A study by Foster et al.137suggested that vitamin C and E,along with atorvastatin(20 mg),effectively reduced hepatic steatosis by 71%in individuals with NAFLD.Some other trials suggested that a combination supplementation of vitamin C and vitamin E did not produce better effect than lifestyle intervention.24,25These studies did not,however,assess the independent effect of vitamin C supplementation.There is a deficit of literature on the role of vitamin C in NAFLD and the association between vitamin C and NAFLD remains controversial.

7.Conclusions

NAFLD is a multifaceted and complex liver disease,having strong association with oxidative stress,IR,lipid profile,metabolic deregulation,obesity,immune regulation and gut microbiota,among others.The deficiency of several micronutrients,particularly several vitamins,is commonly associated with NAFLD and has been correlated with disease severity.Several pieces of experimental data have shown that deficiency of different vitamins may cause metabolic deregulation.Some studies have demonstrated the antiinflammatory and insulin-sensitizing properties exerted by these vitamins in the hepatic cells.Based on these observations,several vitamins such as vitamins E,D,B9,B12,A and C represent potential therapeutic options for liver damage in NAFLD and NASH.More clinical trials are warranted to directly evaluate the efficacy of supplementation of these vitamins on disease progression in NAFLD,as some of them may also have adverse outcomes.It is also crucial to study the interactions between these vitamins and how their intake affects lipid metabolism.Well-defined studies are required to establish the roles of these vitamins as therapeutic agents in NAFLD/NASH.

Authors’ contributions

Study concept and design,drafting of the manuscript,and critical revision of the manuscript for important intellectual content:S.Raza,R.A.Sinha.Literature search:all authors.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by the Indian Council of Medical Research(ICMR)(59/05/2019/ONLINE/BMS/TRM),SERB(SRG/2019/000398) and Wellcome Trust/DBT India Alliance Fellowship (IA/I/16/2/502691) awarded to R.A.Sinha.