Nonalcoholic Fatty Liver Disease and Reduced Serum Vitamin D 3 Levels

Abstract

Nonalcoholic fatty liver disease (NAFLD) and vitamin D₃ deficiency are two highly prevalent pathologic conditions worldwide that share several cardiometabolic risk factors. In addition to its traditional calcium-related effects on the skeleton, vitamin D₃ deficiency has now been recognized to exert nonskeletal adverse effects on several other organ systems. Accumulating epidemiological evidence suggests that low levels of serum 25-hydroxyvitamin D₃ are associated with the presence and severity of NAFLD, independently of several potential confounders, including features of the metabolic syndrome. The molecular mechanisms of this association remain incompletely understood. A variety of biologically plausible mechanisms may mediate a hepato-protective role for the active metabolite of vitamin D₃. 1α,25-dihydroxyvitamin D₃ modulates the insulin signaling pathway/insulin resistance, suppresses fibroblast proliferation and collagen production, exerts anticoagulant and profibrinolytic effects, and modulates macrophage activity and inflammatory cytokine generation. Overall, the high prevalence of vitamin D₃ deficiency and the plausible biological mechanisms linking this to NAFLD suggest that treatment of vitamin D₃ deficiency to prevent and/or treat NAFLD is a promising field to explore. Large placebo-controlled randomized clinical trials are urgently needed to determine whether vitamin D₃ supplementation could have any potential benefit in reducing the development and progression of NAFLD.

Introduction

N onalcoholic fatty liver disease (NAFLD) is a spectrum of progressive liver disease that encompasses simple steatosis, nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. NAFLD is strongly associated with features of the metabolic syndrome and is now recognized as the hepatic component of the syndrome.¹ With the advent of increasingly sedentary lifestyles and changing dietary patterns, the prevalence of obesity and insulin resistance has increased and, with this, NAFLD has rapidly become the most common cause of chronic liver disease in many developed countries. Approximately 20%–35% of adults in the general population in Western countries have NAFLD, and its prevalence increases to 70%–90% among persons who are obese or have type 2 diabetes mellitus (T2DM).¹ NAFLD is a complex health problem with important implications far beyond the liver. In fact, NAFLD is associated not only with liver-related morbidity and mortality, but also with an increased risk of developing cardiovascular disease (CVD) and other important morbidities such as T2DM, chronic kidney disease, and certain malignancies (e.g., hepatocellular carcinoma).^2
–4 This underscores the urgent need for prevention and treatment strategies against the development and progression of NAFLD.

To date, vitamin D₃ deficiency is a highly prevalent condition worldwide, reportedly present in approximately 30%–60% of the general adult population.^5
–7 This worldwide pandemic remains generally unrecognized and untreated. Evolving human and animal data suggest that vitamin D₃ deficiency is implicated in the pathogenesis of CVD as well as several other chronic diseases, including common cancers, autoimmune diseases, osteoarthritis, mental disorders, infectious diseases, metabolic syndrome, and T2DM.^5
–7

This review focuses on the rapidly expanding body of evidence that supports a link between inadequate vitamin D₃ status and NAFLD and briefly examines the putative underlying mechanisms by which low vitamin D₃ might contribute to the development and progression of NAFLD.

Vitamin D Basics

Vitamin D is a steroid vitamin—a group of fat-soluble prohormones—that is strongly involved in calcium and bone metabolism. Although multiple forms of this vitamin do exist, vitamin D₂ (ergocalciferol) and vitamin D₃ (cholecalciferol) are the two major forms. Vitamin D₂ is produced by some organisms of phytoplankton, invertebrates, and fungi in response to ultraviolet irradiation, but it is not constitutively produced by vertebrates, whereas vitamin D₃ originates by photochemical reaction in the skin of most vertebrates, including humans, from 7-dehydrocholesterol, after irradiation of 7-dehydrocholesterol with ultraviolet light (UVB).^5
–7 The first metabolite to be generated is precholecalciferol (previtamin D₃), which undergoes a spontaneous thermal isomerization to cholecalciferol (vitamin D₃). Regardless of its skin synthesis or dietary intake, vitamin D₃ is then hydroxylated within the liver by the microsomal enzyme vitamin D-25-hydroxylase (i.e., CYP2R1-hydroxylase) to generate 25-hydroxycholecalciferol [25(OH)D].^5
–7 The 25(OH)D is then released into the bloodstream, where it circulates bound to the vitamin D–binding protein, making this compound the most represented and stable vitamin D₃ isoform in the plasma (Fig. 1). Due to its long plasma half-life (∼3 weeks) and because the 25-hydroxylation step is unregulated, the concentration of 25(OH)D is thereby the most suitable indicator of total vitamin D stores in the body, reflecting both vitamin D intake and endogenous production.^5
–7 25(OH)D is finally transported to the proximal tubules of the kidney, where it undergoes another hydroxylation at the 1α position to generate 1α,25-dihydroxycholecalciferol [1,25(OH)₂D] by the enzyme 1α-hydroxylase (i.e., CYP27B1-hydroxylase), which is tightly regulated by serum calcium, phosphorus, parathyroid hormone, fibroblast growth factor-23, and other factors. The concentration of 1,25(OH)₂D is roughly 0.1% of that of 25(OH)D.^5
–7 1,25(OH)₂D is the biologically active form of vitamin D₃; it is a powerful specific ligand of the nuclear vitamin D receptor (VDR), which mediates most of its biological actions. The VDR is a ligand-dependent transcription factor that belongs to the superfamily of nuclear hormone receptors. The VDR binds to its ligand 1,25(OH)₂D, dimerizes with the retinoid X receptor, and attaches to specific genomic sequences (named vitamin D response elements). 1,25(OH)₂D decreases its own synthesis through negative feedback and decreases the synthesis and secretion of parathyroid hormone by the parathyroid glands. 1,25(OH)₂D also increases the expression of 25-hydroxyvitamin D-24-hydroxylase (24-OHase) to catabolize 1,25(OH)₂D to the water-soluble, biologically inactive calcitroic acid, which is excreted in the bile.^5
–7

FIG. 1.

A schematic illustration showing the processes and tissues involved in the activation of vitamin D prior to binding to the vitamin D receptor in liver.

The leading and most widely known physiological function of 1,25(OH)₂D is to regulate mineral and skeletal homeostasis. Briefly, vitamin D₃ stimulates calcium absorption in the gut and maintains adequate concentrations of both calcium and phosphate in the serum to enable normal mineralization of bone and prevent osteoporosis and hypocalcemic tetany.^5
–7 The recent discovery that many extrarenal tissues also possess the VDR and the enzyme 1α-hydroxylase to convert circulating 25(OH)D to its active form locally has provided new insights into the important physiologic paracrine and autocrine roles of vitamin D₃ in various tissues and organs. Indeed, the presence of nuclear VDRs into several cell types, including cardiomyocytes, vascular endothelial cells, immune cells, and hepatocytes has stimulated considerable interest in understanding the putative pleiotropic properties of vitamin D₃ that may regulate hundreds of different genes and play a significant role in regulating cell proliferation, differentiation, and apoptosis in many cells and also have immunomodulatory anti-inflammatory effects.^5
–7

Vitamin D₃ Deficiency: Definition and Epidemiology

Decreased sun exposure, skin pigmentation, and aging are among the most important factors in modulating the skin synthesis of vitamin D₃. Other risk factors and important co-morbidities may also reduce skin synthesis or decrease bioavailability of vitamin D₃, such as being institutionalized, malabsorption syndromes, severe obesity, liver failure, chronic kidney disease, and use of medications that may accelerate vitamin D metabolism (e.g., anticonvulsants, ketoconazole, corticosteroids).^5
–7 Regardless of the skin production, vitamin D can also be consumed in the diet, although its content in most foods is low. The Recommended Dietary Allowances for vitamin D to maintain bone and skeletal health recently issued by the Food and Nutrition Board at the Institute of Medicine of the National Academies are as follows: 400 IU/day at 0–12 months of age, 600 IU/day at 1–70 years, and 800 IU/day >70 years.⁸ However, much evidence suggests that these recommended vitamin D intakes are actually inadequate and need to be increased to at least 1000 IU of vitamin D₃ per day in adults.^5
–7

Although a consensus regarding the optimal 25(OH)D level has not yet been established, most experts define vitamin D deficiency as a serum 25(OH)D level of less than 20 ng/mL (<50 nmol/L) and vitamin D insufficiency as 21–29 ng/mL. The optimal 25(OH)D level is at least 30 ng/mL (≥75 nmol/L) and vitamin D toxicity is defined as a 25(OH)D level≥150 ng/mL (≥375 nmol/L).^5
–7

A rapidly evolving knowledge base indicates that vitamin D₃ deficiency is much more prevalent than previously recognized. A number of epidemiologic studies have estimated that vitamin D₃ deficiency or insufficiency is an extremely common condition among adults in Western countries, especially among selected patient populations at particularly high risk for hypovitaminosis D₃, such as residents of nursing homes, older patients, and those with hip fractures.^9
–11 It has been estimated that approximately 50%–100% of elderly men and women in the United States and Europe are vitamin D deficient.^{5
–7,9
–11} Overall, an estimated 30%–50% of young people in Europe have some degree of vitamin D₃ deficiency or insufficiency and, unpredictably, it seems more prevalent in sunny Mediterranean countries than in northern ones, such as Norway.¹² The prevalence of vitamin D₃ deficiency varies widely also in developing countries, with prevalences ranging from 30% to 90%, according to the threshold values.¹³

Vitamin D₃ Deficiency and NAFLD: Data from Epidemiological Studies

A growing body of epidemiological evidence suggests that that low serum 25(OH)D levels are strongly associated with features of the metabolic syndrome and play an important role in modifying risk for cardiometabolic outcomes, including T2DM, hypertension, and CVD.^5
–7,14,15 Low 25(OH)D levels are also associated with insulin resistance and pancreatic β-cell dysfunction among individuals at risk of T2DM.¹⁶ A recent meta-analysis showed that vitamin D₃ supplementation improves insulin resistance compared to placebo.¹⁷

More recently, as summarized in Table 1, accumulating epidemiological data suggest that low levels of serum 25(OH)D are associated with NAFLD as diagnosed either by biochemistry,^18
–20 imaging,^21

–25 or biopsy.^26
–28 With regard to biochemistry-diagnosed NAFLD, Liangpunsakul et al.¹⁸ reported that in a subset of 1287 adult participants from the National Health and Nutrition Examination Survey (NHANES III 1988–1994), those with unexplained elevation in serum alanine aminotransferase (ALT) levels, a proxy of NAFLD,^1,2,4 had lower 25(OH)D levels than those with normal ALT levels (24.7±10.4 vs. 26.8±10.9 ng/mL, P<0.001). Compared to the lowest quartile, patients with the top two quartiles of serum 25(OH)D levels had a lower prevalence of unexplained elevation in serum ALT, independently of metabolic syndrome features.¹⁸ Similar results were found by Kayaniyil et al.¹⁹ in a multiethnic sample of 654 Canadian nondiabetic adults at high risk for T2DM. Conversely, in a study involving 1630 adolescents from the NHANES III 2001–2004 database, Katz et al.²⁰ reported that those with unexplained elevation in serum ALT had lower 25(OH)D levels than those with normal ALT levels (23.0±1.4 vs. 25.6±0.8 ng/mL, P=0.026). These investigators also reported that elevated serum ALT was associated with lower 25(OH)D levels, independently of age, sex, race, and poverty status, but that this association was lost after further adjustment for obesity.²⁰

Table 1.

Principal Observational Studies of the Association Between Low Serum 25-Hydroxyvitamin D₃ Levels and NAFLD (As Detected by Means of Biochemistry, Imaging, or Histology), Ordered by Year

Investigators, year (ref.)	Study population	Total no./no. of NAFLD cases	Method of NAFLD ascertainment	25(OH)D level cut-off for vitamin D deficiency	Main findings
Targher et al. (2007)²⁶	Sample of 60 consecutive Italian adult outpatients with NAFLD and 60 control subjects matched for age, sex, BMI, and season measurement (winter)	120/60	Biopsy	<15 ng/mL (38.3% had vitamin D deficiency)	NAFLD is associated with low 25(OH)D levels independently of age, sex, BMI, serum calcium, creatinine, HOMA-IR score, and presence of metabolic syndrome. Low 25(OH)D levels are also associated with the severity of NAFLD histology (steatosis, necroinflammation, and fibrosis), independently of the above covariates.
Manco et al. (2010)²⁷	Selected sample of 64 Italian obese or overweight children with NAFLD	64/64	Biopsy	<20 ng/mL (54.7% had vitamin D deficiency)	Severity of NAFLD histology (necroinflammation and fibrosis) is associated with low 25(OH)D, independently of metabolic characteristics.
Katz et al. (2010)²⁰	Population-based cohort of 1630 United States adolescents (aged 12–19 years)	1630/143	Elevated serum ALT (>30 IU/L)	Quartiles (I: <20; II: 20 to 25; III: 26 to 31; IV: >31 ng/mL)	Suspected NAFLD is associated with lower 25(OH)D levels independently of age, sex, race, and poverty status but this association is lost after further adjustment for BMI.
Barchetta et al. (2011)²¹	Sample of 262 consecutive Italian adult outpatients with normal serum liver enzymes referred to the diabetes and metabolic diseases clinic for metabolic evaluation	262/162	Ultrasonography	<20 ng/mL (∼64% had vitamin D deficiency)	NAFLD is associated with low 25(OH)D levels independently of age, sex, BMI, diabetes status, triglycerides, HDL-C, and fasting glucose level.
Nseir et al. (2011)²³	Sample of 247 Israel adult hospitalized patients with NAFLD and 100 age- and sex-matched control subjects	347/247	Ultrasonography	<20 ng/mL	NAFLD is associated with low 25(OH)D levels (no adjustment was made for potential confounders other than age and sex).
Foster et al. (2011)²⁵	Community-based cohort of 1394 United States adults with available serum 25(OH)D measurements	1394/223	Computed tomography (liver/spleen ratio<1)	<30 ng/mL (81% had vitamin D deficiency)	NAFLD is associated with low 25(OH)D levels, but this association was not independent of age, sex, race, and features of the metabolic syndrome.
Kayaniyil et al. (2011)¹⁹	Community-based cohort of 654 Canadian nondiabetic adults	Not reported	Elevated serum ALT (>37 IU/L)	Not reported	Suspected NAFLD is associated with lower 25(OH)D levels independently of age, sex, race, season measurement, supplement use, physical activity, waist circumference, and parathyroid hormone level.
Liangpunsakul et al. (2011)¹⁸	Population-based cohort of 1287 United States adults with or without unexplained elevation of serum ALT levels matched for age, sex and BMI (case–control study design)	1287/308	Elevated serum ALT (>40 IU/L)	Quartiles (I: <18; II: 18 to 25; III: 25 to 33; IV: >33 ng/mL)	Suspected NAFLD is associated with lower 25(OH)D levels independently of the presence of metabolic syndrome and insulin resistance (i.e., HOMA-IR score>3.5) and plasma triglycerides.
Dasarathyet al. (2012)²⁸	Sample of 87 United States adult outpatients with NAFLD and 32 healthy controls without steatosis	119/87	Biopsy	Not reported	NAFLD is associated with lower 25(OH)D levels. Severity of NAFLD histology (steatosis, necroinflammation and fibrosis) is associated with low 25(OH)D, independently of body fat mass.
Rhee et al. (2013)²⁴	Occupational cohort of 6567 Korean adult men	6567/2863	Ultrasonography	Tertiles (I: <13.5; II: 13.5 to 16.9; III: >16.9 ng/mL)	NAFLD is associated with low 25(OH)D levels independently of age, season measurement, smoking, physical exercise, BMI, systolic blood pressure, calcium, total cholesterol, HDL-C, ALT, and HOMA-IR score.
Jablonski et al. (2013)²²	Retrospective cohort of 1214 United States adult outpatients with and without NAFLD matched for age, sex, race, and season of measurement (case–control study design)	1214/607	Ultrasonography	<30 ng/mL (57.2% had vitamin D deficiency)	NAFLD is associated with low 25(OH)D levels independently of BMI, hypertension, history of diabetes, renal disease, peripheral vascular disease, and liver diseases.

NAFLD, nonalcoholic fatty liver disease; BMI, body mass index; 25(OH)D, 25-hydroxyvitamin D₃; HOMA-IR, homeostasis model assessment of insulin resistance; HDL-C, high-density lipoprotein cholesterol; ALT, alanine aminotransferase.

With regard to imaging-diagnosed NAFLD, Barchetta et al. ²¹ studied 262 consecutive patients referred to the diabetes and metabolic diseases clinics for metabolic evaluation. They found that NAFLD patients had lower serum 25(OH)D levels than those without NAFLD (14.8±9.2 vs. 20.5±9.7 ng/mL, P<0.001), and that the significant, inverse relationship between 25(OH)D levels and NAFLD was independent of age, sex, and metabolic syndrome features.²¹ In a large case–control study involving 607 US adult cases with NAFLD and 607 controls matched for age, sex, race, and measurement season, Jablonski et al.²² reported serum 25(OH)D levels were lower in the group with NAFLD as compared to that in the matched control group (30±7 vs. 34±8 ng/mL, P<0.001). The relationship between NAFLD and inadequate vitamin D₃ status remained statistically significant after adjustment for BMI, hypertension, history of diabetes, renal disease, peripheral vascular disease, and liver diseases.²² In a smaller case–control study, Nseir et al.²³ reported that hospitalized patients with NAFLD had lower 25(OH)D levels than their age- and sex-matched counterparts without NAFLD (22.9±9.8 vs. 31±6 ng/mL, P<0.001). In a large occupational cohort of Korean adult men, Rhee et al.²⁴ reported that NAFLD patients had lower 25(OH)D levels than those without steatosis (15.5±3.6 vs. 16.0±3.9 ng/mL, P<0.001), and that the inverse relationship between NAFLD and 25(OH)D tertiles was statistically significant even after controlling for a wide range of potential confounders. Finally, in a recent abstract, Foster et al.²⁵ reported that NAFLD (as detected by computed tomography) was significantly associated with lower 25(OH)D levels among 1394 adult participants of the Multi-Ethnic Study of Atherosclerosis. However, this association was lost after adjusting for metabolic syndrome features.²⁵

With regard to biopsy-diagnosed NAFLD, Targher et al.²⁶ first studied circulating 25(OH)D levels in 60 consecutive Italian outpatients with NAFLD and 60 healthy nonsteatotic controls of comparable age, sex, BMI, and season measurement and found reduced levels in those with NAFLD (20.4±8.8 vs. 29.8±6 ng/mL, P<0.001). The prevalence of vitamin D₃ deficiency was also remarkably greater in NAFLD patients than in healthy controls (48.3% vs. 28.3%, P<0.001). The differences in 25(OH)D levels observed between the groups were little affected by adjustment for multiple confounding variables, including serum creatinine level, insulin resistance, and metabolic syndrome features.²⁶ Notably, as shown in Fig. 2, serum 25(OH) levels were lowest in patients with NASH, intermediate in those with simple steatosis, and greatest in matched healthy controls. Furthermore, among NAFLD patients, decreased 25(OH)D levels were independently associated with the histological severity of hepatic steatosis, necroinflammation, and fibrosis.²⁶ These findings have been confirmed by Manco et al.²⁷ in overweight/obese children with NAFLD and by Dasarathy et al.²⁸ in a sample of 119 adults with and without NAFLD.

FIG. 2.

(A) Means (±standard deviations) of serum 25-hydroxyvitamin D₃ levels in nonsteatotic healthy controls, patients with simple steatosis and those with nonalcoholic steatohepatitis (NASH). (B, C, and D) Means (±standard deviations) of serum 25-hydroxyvitamin D₃ levels in relation to the histological severity of hepatic steatosis (including patients with simple steatosis and those with NASH), necroinflammation, and fibrosis. P values for trends are adjusted for age, sex, body mass index, serum calcium, serum creatinine, insulin resistance as estimated by homeostasis model assessment, and presence of the metabolic syndrome. (Adapted with permission from Targher et al.²⁶).

Collectively, therefore, the current evidence from the published studies indicates that serum 25(OH) levels are remarkably lower in patients with NAFLD than in those without steatosis, and that NAFLD cases are more likely to be vitamin D₃ deficient compared to control subjects. However, some important limitations of these studies merit comment. First, although the inverse, graded relationship between serum 25(OH)D levels and the severity of NAFLD histology suggests that vitamin D₃ might exert a beneficial, dose-dependent effect on hepatic fat content, the cross-sectional design of these studies does not allow establishing a causative nature of the association between hypovitaminosis D₃ and NAFLD. Second, the method of NAFLD diagnosis varied across studies. Only a few studies used liver biopsy, which is considered the gold standard for the diagnosis,¹ whereas some studies used imaging techniques or elevated serum transaminase levels as to identify NAFLD cases. The use of imaging and particularly of biochemical methods to assess NAFLD may be associated with considerable measurement error and may substantially underestimate the association between vitamin D₃ and NAFLD. Third, the cutoff level for defining vitamin D₃ deficiency also varied across studies. The normal range of serum 25(OH)D levels remains a controversial area of research and has been the subject of debate over the past several years.^5
–7 Fourth, many of these studies did not adjust for potentially important confounders, such as kidney function parameters, insulin resistance, or season of measurement.

Currently, the association between vitamin D₃ status and NAFLD and its potential therapeutic role warrants further research. In a pilot intervention trial, Orsi et al.²⁹ studied 9 obese adolescents with a diagnosis of NAFLD measured by proton magnetic resonance spectroscopy and vitamin D₃ insufficiency. All subjects were given a minimum of 2000 IU/day of vitamin D₃ for a 6-month period, and doses were increased as needed based on serum 25(OH) levels at monthly visits. Interestingly, the investigators found that normalization of serum 25(OH)D levels led to decreased hepatic fat content (from 31.2% to 23.1%, P=0.07); no significant effects were observed in BMI, waist circumference, plasma lipids, and fasting insulin level.²⁹

Putative Underlying Mechanisms Linking Vitamin D₃ Deficiency to NAFLD

Vitamin D₃ signaling

Vitamin D₃ signals within cells expressing the VDR, and it has been estimated that VDR regulates over 200 genes involved in glucose and lipid metabolism, inflammation, angiogenesis, apoptosis, cell growth, proliferation, and differentiation.^5
–7 A schematic illustration of the processes leading to vitamin D₃/VDR regulation of gene expression in the liver is shown in Fig. 1. Normal functioning of the VDR is crucial for liver fat accumulation, and knocking out the VDR in mice protects the liver from hepatic steatosis, despite high-fat feeding.³⁰ Extensive evidence is available to show that the VDR function in the liver regulates not only hepatic lipid metabolism but also hepatic necroinflammation and fibrosis.³¹ Recent evidence shows that the VDR is widely expressed throughout the liver (in hepatocytes, cholangiocytes, and lymphocytes); in NAFLD, VDR expression is negatively associated with the severity of liver disease.³² Furthermore, in this study, serum 25(OH)D₃ levels correlated inversely with hepatocyte damage, as expressed by cellular ballooning, in NASH patients. VDR expression on cholangiocytes and hepatocytes was also significantly lower than that observed in the comparator group without liver disease and was negatively associated with the severity of NAFLD histology. In multivariate regression analysis, there was a strong association between decreased liver VDR expression and a diagnosis of NASH, independent from other metabolic determinants, such as BMI, insulin resistance, and adiponectin.³²

Immunomodulatory effects of vitamin D₃

The immunomodulatory effects of 1,25(OH)₂D are well known (e.g., vitamin D₃ activates monocytic cells exposed to antigen and enhances the innate immune response). However, recent evidence suggests that low vitamin D₃ levels may also contribute to NAFLD disease progression by involvement of the immune system. For example, in a rodent model of NAFLD, animals were fed a vitamin D–deficient diet and deprived of UV light, to investigate mechanisms of immune modulation and hepatic necroinflammation.³³ In this model, serum 25(OH)D levels were decreased by almost 70% in affected animals. From the results of this study, the authors suggested that the mechanism of necroinflammation in NAFLD progression involves the activation of Toll-like receptors (TLR) 2 and 4.³³ TLR2 and TLR4 are involved in lipopolysaccaride signaling and bacterial recognition, by way of CD14 (endotoxin receptor) and the lipopolysaccaride-binding protein, suggesting that the gut–liver axis may be involved in NAFLD pathogenesis. The intestine is rich in bacteria and bacterial products such as lipopolysaccarde, and these products are delivered to the liver via the portal vein. Recent data have demonstrated that the liver is exposed to a high load of TLR ligands due to bacterial overgrowth and increased intestinal permeability in NAFLD,³⁴ emphasising the potential importance of the gut–liver interaction in immune modulation affecting development of NAFLD.

Vitamin D₃: Adipose tissue, bile, and the intestine

There is now extensive evidence that functioning of visceral adipose tissue contributes to NAFLD,^35,36 and evidence is accumulating to show that vitamin D₃ treatment may favorably affect insulin sensitivity and inflammation in adipose tissue. For example, in a study of 35 obese adolescents treated orally with either 4000 IU of vitamin D₃/day or placebo for 6 months, correction of vitamin D deficiency improved markers of insulin sensitivity.³⁷ Within a substudy embedded in a randomized trial, in vitro concomitant incubation of adipocytes with 1,25(OH)₂ was shown to decrease mRNA levels of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and IL-8³⁸; and in support of a favorable effect of vitamin D treatment on adipose tissue function, it has been suggested that vitamin D₃ may protect against adipose tissue inflammation by disrupting the deleterious cycle of macrophage recruitment.³⁹

Recent data, obtained in 3T3L1 adipocytes, has suggested an interesting and plausible mechanism underlying the association between hyperglycemia/insulin resistance and low vitamin D₃ levels.⁴⁰ In vitro, 1,25(OH)₂D treatment of adipocytes caused significant upregulation of GLUT4 total protein expression and its translocation to the cell surface, and an increase in glucose uptake as well as glucose utilization in high glucose–treated cells. Combined treatment with insulin and vitamin D₃ had a greater effect that either treatment used alone. Supplementation also inhibited MCP-1 and stimulated adiponectin secretion in high glucose–treated adipocytes, lending further weight to there being a beneficial effect of vitamin D₃ in adipocyte to decrease adipose tissue inflammation.⁴⁰

Small intestinal function and hepatic synthesis of bile is crucial for affecting dietary vitamin D uptake and, consequently, for regulating vitamin D₃ function. Vitamin D uptake in the small intestine is bile acid (BA)-dependent and linked to hepatic metabolism of BAs. In support of the importance of BA metabolism in this process, it has been noted that patients with cirrhosis (who have altered BA secretion) have markedly lower vitamin D₃ levels than patients with chronic hepatitis. Furthermore, the more severe forms of cirrhosis are associated with lower concentrations of vitamin D₃ and its binding protein. ⁴¹

BA synthesis begins in the hepatocyte with the condensation of fatty acids to form squalene. Squalene is converted to cholesterol, and the key step in the production of bile acids is the hydroxylation of the seventh carbon in the cholesterol sterol ring structure that is regulated by cholesterol 7α-hydroxylase (CYP7A1). Vitamin D₃ is known to regulate CYP7A1 and also fatty acid synthase (FAS) via increasing farnesoid X receptor (FXR) activity. FAS activity is pivotal in contributing to triacylglycerol and diacylglycerol synthesis that is important in increasing hepatic insulin resistance.⁴

BAs can alter gene expression both in the liver and in the small intestine via activation of the FXR, the VDR, and other nuclear receptors, e.g., pregnane X receptor, and also by affecting signalling molecules and pathways, e.g., G protein–coupled receptor for bile acids, and c-Jun aminoterminal kinases (a subfamily of mitogen-activated protein kinases), AKT (a serine/threonine-specific protein kinase regulating insulin action), and extracellular signal-regulated kinases that are involved in growth factor signaling. Among these signaling molecules/nuclear receptors, FXR is an important BA-responsive ligand-activated transcription factor that is a crucial control element for maintaining BA homeostasis. FXR has a high affinity for several major endogenous BAs, notably cholic acid, deoxycholic acid, and chenodeoxycholic acid. By responding to excess BAs, FXR connects liver and small intestine to control BA concentrations to regulate BA synthesis and enterohepatic flow.⁴²

Activation of FXR by vitamin D₃ is a major mechanism in suppressing BA synthesis by reducing the expression of key genes such as CYP7A1.⁴³ In the small intestine, FXR induces an intestinal hormone, fibroblast growth factor (FGF-19), which activates hepatic FGF-receptor 4 (FGFR4) signaling to inhibit BA synthesis. Vitamin D₃ also upregulates FGF-15 (the rodent homolog of FGF-19 in humans).⁴⁴ In the liver, BAs activate FXR, which induces an atypical nuclear receptor small heterodimer partner (SHP) and subsequently inhibits nuclear receptors, liver-related homolog-1, and hepatocyte nuclear factor 4α and results in a feedback inhibition of BA synthesis by inhibiting transcription of the rate-limiting step CYP7A1.⁴⁵ BAs are able to induce FGF-19 in hepatocytes, and CYP7A1 is downregulated by vitamin D₃ and by FGF-19.^45
–47

Bile acid synthesis and insulin sensitivity

CYP7A1 activity may be more important in the regulation of liver fat content and insulin sensitivity than has previously been appreciated, because modifying expression of this enzyme in mice has been shown to affect obesity and insulin resistance.⁴⁸ It is, therefore, plausible that strategies to modify expression of CYP7A1 may be effective in decreasing liver fat content.⁴⁹ Recent work testing associations between single-nucleotide polymorphisms (SNPs) of the VDR gene and insulin resistance and the effects of these SNPs on changes in insulin sensitivity in response to vitamin D₃ supplementation suggest an association between vitamin D₃ responsiveness and insulin resistance with VDR gene polymorphisms.⁵⁰ Vitamin D₃ has been shown to downregulate forkhead Box O (FoxO1),⁵¹ which is an important regulator of insulin action in the liver, regulating hepatic gluconeogenesis and hepatic glucose output, and hepatic insulin resistance is a feature of NAFLD.⁴ FXR is also expressed in pancreatic β-cells and regulates insulin signaling.⁴² Furthermore, postprandial increases in intestinal FGF-19 expression affect hepatic glucose metabolism independently of FXR, by inhibiting expression of genes involved in gluconeogenesis through a mechanism involving the dephosphorylation and inactivation of the transcription factor cAMP regulatory element-binding protein (CREB).⁵² This effect, in turn, decreases expression of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and other genes that are involved in the regulation of hepatic oxidative metabolism.

FXR agonists have been shown to decrease insulin resistance and decrease hepatic steatosis, and BAs may produce a beneficial effect in NAFLD by promoting intestinal glucagon-like peptide-1 (GLP-1) secretion.⁵³ However, the precise effects of modifying FXR function in the various stages of NAFLD need to be tested. BAs are known to be increased in NASH patients.⁵⁴ Recently, in an attempt to identify potential interactions between BAs and free fatty acid metabolism in NAFLD, liver biopsies and serum samples were taken from 113 morbidly obese patients receiving bariatric surgery. These individuals also included NAFLD patients.⁵⁵ The investigators concluded that BA synthesis and serum BA levels correlated positively with disease severity in NAFLD, whereas serum adiponectin was inversely correlated. Free fatty acid exposure prevented SHP-mediated repression of CYP7A1 expression, which leads to increased BA synthesis. In NASH, BA accumulation induced hepatocyte cell death and late FXR activation failed to prevent hepatocyte injury due to decreased adiponectin levels. It is plausible from these results that early treatment with FXR ligands and/or adiponectin-receptor agonists might act early in NAFLD, but further work in this area is needed. Additionally, a small study investigating the hepatic response to FGF-19 showed that this response was impaired in NAFLD patients compared to healthy controls (although plasma FGF-19 levels were not significantly different between these groups).⁵⁶ Therefore, given the effects of vitamin D₃ to increase FGF-19 levels discussed above, vitamin D₃ treatment may be beneficial.

Vitamin D₃ and NAFLD disease progression

As NAFLD progresses towards end-stage liver disease, activation of hepatic stellate cells plays a key role in the development of fibrosis. Although it has been known for some time that vitamin D₃ has antiproliferative and antifibrotic properties and plays an important role in regulation of the extracellular matrix, little has been known until recently about the effects of vitamin D₃ on hepatic stellate cells. Increasing evidence now suggests that vitamin D₃ signals via the VDR in hepatic stellate cells to inhibit collagen synthesis and secretion. For example, it has been shown that 1,25(OH)₂D decreased human α(1) (I) collagen mRNA and protein and the secretion of type I collagen by hepatic stellate cells after exposure to transforming growth factor-β1 (TGF-β1).⁵⁷ Activation of hepatic stellate cells is associated with a decrease in expression of stellate cell VDR, and incubation of stellate cells with vitamin D₃ has been shown to decrease proliferation and matrix formation and increase VDR and tissue inhibitor of metalloproteinase 1 (TIMP-1) expression.⁵⁸ UV light exposure in a mouse model of NASH has been shown to increase expression of adiponectin in white adipose tissue. It is possible that induction of adiponectin may be a natural defense response for the prevention of liver fibrosis, but more work investigating the role of adiponectin signaling in NAFLD progression is needed.⁵⁹ Low vitamin D₃ levels are also associated with markers of T cell and macrophage activation,⁶⁰ suggesting that correction of low vitamin D₃ levels is beneficial in decreasing levels of inflammation in NAFLD.

As reported previously, lower 25(OH)D levels have been shown in NAFLD patients, both adults and adolescents, compared to controls with a close association to the histological severity of NAFLD that was independent of the metabolic syndrome and insulin resistance.^26,27

Summary

Vitamin D₃ treatment would also be an ideal treatment for NAFLD because it acts not only on the liver, but also on adipose tissue and small intestinal function. Despite a strong association between low levels of vitamin D₃ and glucose intolerance and insulin resistance, an underlying mechanism explaining these associations has proved elusive. Vitamin D₃ could produce a favorable benefit at various stages within the spectrum of NAFLD. These data from animal studies, in vitro experiments, and human studies are summarized in Table 2. The evidence of benefit suggests that vitamin D₃ treatment would be favorable in adipose tissue to facilitate glucose uptake and decrease inflammation. In the liver, vitamin D₃ acts to decrease BA production, favorably influences liver fat content, decreases gluconeogenesis, and decreases inflammation and matrix production. A schematic representation of potential vitamin D₃–mediated mechanisms operating in the small intestine, adipose tissue, and liver that would produce a favorable effect in NAFLD is shown in Fig. 3.

FIG. 3.

A schematic representation of potential vitamin D–mediated mechanisms operating in small intestine, adipose tissue, and liver, potentially producing a favorable effect in NAFLD. Dietary vitamin D is ingested as a fat-soluble vitamin within the diet. Small intestinal function is crucial for affecting dietary vitamin D uptake and for regulating vitamin D₃ function. Vitamin D uptake in the small intestine is bile acid (BA)-dependent and linked to hepatic metabolism of BAs that is regulated by cholesterol 7α-hydroxylase (CYP7A1). BAs and vitamin D can alter gene expression in the liver and small intestine via activating of the nuclear bile acid receptor (also known as the farnesoid X receptor or FXR) and fibroblast growth factor-19 (FGF-19). Vitamin D₃ also signals via the vitamin D receptor, which forms a heterodimer with the retinoid X receptor (RXR), in adipocytes (to affect glucose uptake), and in the liver to affect lipid metabolism and macrophage and stellate cell function. TNF-α, tumor necrosis factor-α; PPARγ, peroxisome proliferators-activated receptor-γ; LCFA, long-chain fatty acids; TAG, triacylglycerols; cAMP, cyclic adenosine monophosphate; HSL, hormone-sensitive lipase; GLUT-4, glucose transporter type 4.

Table 2.

Summary of the Evidence of Potential Benefit of Vitamin D₃ Treatment in NAFLD

	Relationship/effect
Animal and in vitro data	Knocking out the vitamin D receptor in mice causes liver fat accumulation.
	Vitamin D₃ treatment protects mice from diet-induced liver fat development.
	Treating mice with artificial sunlight (to increase vitamin D₃ levels) prevents NAFLD from progressing to more severe fibrotic liver disease.
	Treating adipocytes with 1,25-dihydroxyvitamin D₃ increases GLUT-4–mediated uptake, decreases inflammation, and increases adiponectin production.
Human data	Low serum 25-hydroxyvitamin D₃ levels occur in patients with NAFLD and in people with hyperglycemia, type 2 diabetes mellitus, and insulin resistance.
	There is a graded, inverse relationship between serum 25-hydroxyvitamin D₃ levels and the severity of NAFLD histology.
	Low levels of vitamin D receptor have been detected in the livers of people with NAFLD.
	Vitamin D₃ treatment acting on the vitamin D receptor decreases inflammation in adipocytes and improves insulin sensitivity.
	Vitamin D₃, through interaction with its nuclear receptor, attenuates the inflammatory response and development of fibrosis.
	Vitamin D₃ upregulates fibroblast growth factor-19 (FGF-19) and farnesoid X receptor (FXR) to decrease cholesterol 7α-hydroxylase (CYP7A1), which is pivotal in the regulation of bile acid synthesis.
	Bile acid synthesis is increased in NASH and vitamin D₃ treatment decreases CYP7A1 expression (in vitro).

NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis.

Conclusions

It has been known for a long time that serum 25(OH)D levels are decreased in people with insulin resistance, glucose intolerance, or T2DM. Recent evidence shows that low levels of serum 25(OH)D also occur in NAFLD. Proof of concept that vitamin D₃ treatment can work in NAFLD is established from mechanistic animal data, in vitro studies, and observational studies and small clinical trials in humans. To date, there are no published data testing the effects of vitamin D₃ in NAFLD in rigorous, randomized, placebo-controlled trials. There are currently three clinical trials registered with www.clinicaltrials.gov focused on testing the effects of vitamin D₃ treatment in NASH. None of these three trials (NCT01623024, NCT01083992, NCT01571063) is focused on the early stages of NAFLD, and because hepatic steatosis occurs at an early stage in NAFLD, it is obviously desirable also to test the effect of vitamin D₃ treatment on liver fat content. We suggest that the next step is to build on the existing data to perform phase III clinical trials to evaluate clinical efficacy and obtain estimates of size of effect of vitamin D₃ treatment to decrease hepatic fat content and improve parameters of liver inflammation in NASH. Such trials also need to investigate safety of vitamin D₃ treatment in terms of calcium metabolism in NAFLD patients.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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Nonalcoholic Fatty Liver Disease and Reduced Serum Vitamin D 3 Levels

Abstract

Introduction

Vitamin D Basics

Vitamin D3 Deficiency: Definition and Epidemiology

Vitamin D3 Deficiency and NAFLD: Data from Epidemiological Studies

Putative Underlying Mechanisms Linking Vitamin D3 Deficiency to NAFLD

Vitamin D3 signaling

Immunomodulatory effects of vitamin D3

Vitamin D3: Adipose tissue, bile, and the intestine

Bile acid synthesis and insulin sensitivity

Vitamin D3 and NAFLD disease progression

Summary

Conclusions

Footnotes

Author Disclosure Statement

References

Vitamin D₃ Deficiency: Definition and Epidemiology

Vitamin D₃ Deficiency and NAFLD: Data from Epidemiological Studies

Putative Underlying Mechanisms Linking Vitamin D₃ Deficiency to NAFLD

Vitamin D₃ signaling

Immunomodulatory effects of vitamin D₃

Vitamin D₃: Adipose tissue, bile, and the intestine

Vitamin D₃ and NAFLD disease progression