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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 35  |  Issue : 1  |  Page : 67-73

The potential effect of vitamin D on rats with fatty liver induced by a choline-deficient diet


Department of Physiology, Faculty of Medicine, Benha University, Benha, Egypt

Date of Submission09-Jan-2017
Date of Acceptance16-Mar-2017
Date of Web Publication28-Feb-2018

Correspondence Address:
Dania A Mohammed
Department of Physiology, Faculty of Medicine, Benha University, Benha, 13511
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bmfj.bmfj_3_17

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  Abstract 


Background Nonalcoholic fatty liver disease (NAFLD) is present worldwide and is considered the most common cause for abnormal liver function tests and chronic liver disease in both developed and developing countries.
Aim This study aimed to investigate the effect of pretreatment with vitamin D on NAFLD and the role of transforming growth factor-β1 (TGF-β1) in vitamin D-mediated hepatoprotective effect.
Materials and methods NAFLD was induced in albino rats by administration of a choline-deficient diet. Serum activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), serum levels of albumin, bilirubin, and 25 hydroxyvitamin D [25(OH)D], body weight, liver weight, and liver index were measured in different experimental groups to assess liver affection. In addition, TGFβ1 was immunohistochemically determined.
Results NAFLD caused significant increases (P<0.005) in serum levels of AST and ALT, liver weight, and liver index, with a significant decrease (P<0.005) in serum levels of 25(OH)D. Administration of vitamin D (1, 5, and 10 μg/kg) caused a significant reduction (P<0.005) in serum levels of AST and ALT, liver weight, and liver index with a significant increase (P<0.005) in serum levels of 25(OH)D. TGFβ1 expression showed improvement in a dose-dependent manner.
Conclusion Vitamin D has a hepatoprotective effect in NAFLD induced by a choline-deficient diet, and TGFβ1 contributes to this protection.

Keywords: choline-deficient diet, nonalcoholic fatty liver disease, transforming growth factor β1, vitamin D


How to cite this article:
El Talees AA, Hussien NI, Allam MM, Mohammed DA. The potential effect of vitamin D on rats with fatty liver induced by a choline-deficient diet. Benha Med J 2018;35:67-73

How to cite this URL:
El Talees AA, Hussien NI, Allam MM, Mohammed DA. The potential effect of vitamin D on rats with fatty liver induced by a choline-deficient diet. Benha Med J [serial online] 2018 [cited 2018 Sep 22];35:67-73. Available from: http://www.bmfj.eg.net/text.asp?2018/35/1/67/226418




  Introduction Top


Nonalcoholic fatty liver disease (NAFLD) has become the most common form of chronic liver disease exceeding viral hepatitis and alcoholic liver disease [1]. NAFLD represents a continuum of hepatic injuries that progress from simple fatty liver to nonalcoholic steatohepatitis (NASH), cirrhosis, or even hepatocellular carcinoma [2].

In laboratory animals, a choline-deficient (CD) diet may induce a histological and metabolic phenotype resembling human fatty liver. This phenotype is characterized by fatty liver, inflammation, fibrosis, cirrhosis, and even hepatocellular carcinoma [3].

Vitamin D has an immunomodulatory, antioxidant, anti-inflammatory, antifibrotic, and antiapoptotic role in rodents [4].

This study was designed to evaluate the effect of vitamin D on fatty liver induced by a CD diet and to determine whether transforming growth factor β1 (TGFβ1) contributes toward vitamin D-mediated protection.


  Materials and methods Top


Animals

This study was conducted on 40 adult male albino rats, aged 6–8 weeks, weighing between 180 and 220 g. They were obtained from the Animal Unit of Moshtohor Faculty of Agriculture.

Animals were acclimatized to the laboratory conditions for 10 days before initiation of the experiment. They had free access to water and diet. The study period lasted for 12 weeks. Experimental rats were housed under completely healthy conditions throughout the experiment and under care of a professional technician and a qualified researcher. None of the rats died during the experiment. At the end of the study, rats were incinerated at the Benha University Hospital incinerator.

Experimental design

Rats were randomly classified into three main groups: group I (control group) (n=8) was fed a balanced animal diet without exposure to drugs for 12 weeks; group II (CD diet group) (n=8) received a CD diet for 12 weeks; and group III (n=24 rats) received a CD diet+vitamin D supplementation and was divided into three subgroups according to the dose of vitamin D as follows − group IIIA (n=8) received vitamin D 1 μg/kg intraperitoneally twice a week for 12 weeks, Group IIIB (n=8) received vitamin D 5 μg/kg intraperitoneally twice a week for 12 weeks, and group IIIC (n=8) received vitamin D 10 μg/kg intraperitoneally twice a week for 12 weeks.

Biochemical analysis

Estimation of aspartate aminotransferase (AST) was performed using BT29 4QY kits (Randox Laboratories Ltd, Ardmore, County Antrim, UK). Estimation of alanine aminotransferase (ALT) was performed using Egyptian Company for Biotechnology kits (Spectrum Diagnostics, Cairo, Egypt). Estimation of albumin was carried out using Biostc Diagnostic Reagents kits (Biostc, Cairo, Egypt). Estimation of bilirubin was carried out using Egyptian Company for Biotechnology kits. Estimation of vitamin D was carried out using Calbiotech kits (CALBIOTECH, Spring Valley, CA, USA). Estimation of TGFβ1 was carried out using US Biological Life Science kits (United States Biological, Salem, Massachusetts, USA). Liver weight and body weight were measured in all rats. The liver index was estimated by dividing liver weight by body weight.

Chemicals used

  1. The CD diet was formulated according to NRC.
  2. Vitamin D was purchased from Memphis Pharmaceutical and Chemical Industry (Cairo, Egypt).


Statistical analysis

Data were analyzed using the computer program statistical Package for Social Science, version 16 (SPSS Inc., Chicago, Illinois, USA). The significance of difference was tested using analysis of variance to compare between more than two groups of numerical (parametric) data. For continuous nonparametric data, the Kruskal–Wallis test was used. For intergroup analyses, the post-hoc test least significant difference (LSD) was used. A P value less than 0.05 was considered statistically significant.


  Results Top


There was a significant increase (P<0.05) in serum AST and ALT activity levels in group II when compared with the control group; on the contrary, there was a nonsignificant (P>0.05) increase in serum AST and ALT activity levels in group III and IV when compared with the control group. There was a nonsignificant (P>0.05) increase in group V when compared with group I.

Serum AST and ALT activity levels in group V showed a significant decrease (P<0.05) when compared with that of group II. On the contrary, there was a nonsignificant (P>0.05) decrease in serum AST and ALT activity levels in group III and IV when compared with group II.

Serum albumin levels and total bilirubin in groups II, III, IV, and V showed a nonsignificant (P>0.05) decrease when compared with the control group ([Table 1] and Chart 1a–c).
Table 1 Effect of vitamin D on aspartate aminotransferase, alanine aminotransferase, albumin, and total bilirubin in different experimental groups

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Serum 25 hydroxyvitamin D [25(OH)D] levels were significantly decreased (P<0.05) in groups II, III, and IV when compared with the control group. On the contrary, there was a nonsignificant (P>0.05) decrease in serum levels of 25(OH)D in group V when compared with the control group.

There was a significant increase (P<0.05) in the serum levels of 25(OH)D in groups III, IV, and V when compared with group II. In contrast, there was a significant increase (P<0.05) in serum levels of 25(OH)D in group IV and V when compared with group III ([Table 2] and Chart 2).
Table 2 Mean serum 25 hydroxyvitamin D in animals of different experimental groups (n=8)

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There was a significant (P<0.05) increase in liver weight in groups II, III, and IV compared with the control group (I). On the contrary, there was a nonsignificant (P>0.05) increase in liver weight in group V when compared with the control group (I).

Liver weight in group V showed a significant decrease (P<0.05) when compared with that of groups II, III, and IV.



Body weight in groups II, III, IV, and V showed nonsignificant changes (P>0.05) when compared with group I.

There was a significant (P<0.05) increase in the liver index in groups II, III, and IV when compared with the control group. On the contrary, there was a nonsignificant (P>0.05) increase in liver index in group V when compared with the control group.

The liver index in group V showed a significant decrease (P<0.05) when compared with groups II, III, and IV ([Table 3] and Chart 3a–c).
Table 3 Effect of vitamin D on liver weight, body weight, and liver index in different experimental groups (n=8)

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Hematoxylin and eosin results

Liver sections of the control group showed normal histological features of hepatic tissue ([Figure 1]). In group II, liver specimens of rats that received CD diet alone exhibited fatty liver in the form of severe steatosis of hepatocytes and inflammatory cells infiltration ([Figure 2]). Groups treated with vitamin D showed improvement, as they exhibited minimal activation of Kupffer cells with very minimal hydrobic degeneration of some hepatocytes ([Figure 2]).
Figure 1 Hematoxylin and eosin-stained samples of the control group (normal liver).

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Figure 2 Hematoxylin and eosin-stained samples from group IIIC (minimal hydrobic degeneration of liver).

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Immunohistochemical examination of transforming growth factor β1

In the control group (group I), liver sections showed negative immunochemical reaction ([Figure 3]). In group II, liver specimens of rats that received the CD diet alone exhibited strong, positive expression of TGFβ1 (immunopositivity indicated by brown color) ([Figure 4]). Groups treated with vitamin D showed improvement as they exhibited very weak positive expression of TGFβ1 ([Figure 5]).
Figure 3 Group I (negative immunochemical reaction).

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Figure 4 Group II (strong positive expression of transforming growth factor β1).

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Figure 5 Group IIIc (very weak positive expression of transforming growth factor β1).

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  Discussion Top


NAFLD is defined as the abnormal accumulation of lipids, primarily in the form of triglycerides, in individuals who do not consume significant amounts of alcohol (≤20 g ethanol/day). NAFLD is a spectrum of disorders ranging from simple steatosis to NASH. NAFLD is a very common metabolic liver disorder, which is linked with obesity, type-2 diabetes, and metabolic syndrome. At present, it is considered the most prevalent liver disorder [5].

Vitamin D is an important steroid hormone with various effects that extend well beyond its established regulatory role in calcium and bone homeostasis [6]. Vitamin D has been shown to act as an immunomodulatory, antioxidant, anti-inflammatory, and antiapoptotic compound in rodents [4].

In our study, administration of a CD diet for 12 weeks resulted in development of NAFLD. The CD diet rats (group II) showed significant increases (P<0.05) in serum liver biomarkers such as ALT and AST, liver weight, and liver index when compared with control rats (group I). On the contrary, there were nonsignificant changes in serum albumin, total bilirubin, and body weight. There was a highly significant decrease in serum 25(OH)D in rats that were fed the CD diet (group II) when compared with control rats (group I).

Significant increases (P<0.05) in serum liver biomarkers such as ALT and AST in group II can be explained by the fact that choline is an important element for the formation of phosphatidylcholine, a critical component of very-low-density lipoproteins, which are responsible for transporting triglycerides out of the liver [7].

Moreover, a CD diet was reported to induce NASH by causing mitochondrial dysfunction, which is a central mechanism in the pathogenesis of NAFLD [8].

Significant increases (P<0.05) in liver weight and liver index were observed, as low choline leads to reduced secretion of liver triglycerides such as very-low-density lipoproteins, resulting in accumulation of liver triglycerides without affecting the enzymes involved in de-novo lipogenesis. Therefore, the liver weight increase without change in body weight is observed [9].

The results of our study also indicate that there were nonsignificant (P>0.05) changes in serum albumin and total bilirubin in rats fed the CD diet (group II) in comparison with control rats (group I), as total bilirubin and albumin are usually normal in NASH and NAFLD. Hyperbilirubinemia and hypoalbuminemia are seen only in cases associated with cirrhosis that develops in end-stage disease [10].

Our results show that there is a highly significant decrease in serum 25(OH)D in rats fed the CD diet (group II) when compared with control rats (group I), as a lower level of serum 25(OH)D is associated with the presence and severity of NASH [11]. Numerous studies have found that liver tissues of individuals with low levels of vitamin D have increased hepatic mRNA levels of resistin, interleukin-4, interleukin-6, and tumor necrosis factor α − markers known to be implicated in oxidative stress and hepatic inflammation [12].

Our results are supported by the histopathological study of the liver of rats. We found that there were fatty liver changes in the form of severe steatosis of hepatocytes and inflammatory cells infiltration, indicating occurrence of fatty liver under the effect of diet, as seen in [Figure 6]; these results are in agreement with Han et al. [13].
Figure 6 Hematoxylin and eosin-stained samples from group II (severe fatty liver).

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Immunohistochemical examination of TGFβ1 showed a strong positive expression of TGFβ1 in CD diet-fed rats (group II) when compared with the control group (group I), as shown in [Figure 4]

On studying the prophylactic effect of vitamin D on fatty liver, as it was administrated twice weekly (1, 5, and 10 μg/kg) for 12 weeks, there were significant decreases (P<0.05) in liver biomarkers such as AST and ALT (shown in [Table 1] and Chart 1), liver weight, and liver index (shown in [Table 3] and Chart 3a–c).

These results are in agreement with Han et al. [13] who showed that AST and ALT levels significantly decreased in groups treated with vitamin D in a dose-dependent manner, as vitamin D inhibits hepatic fat accumulation, suggesting its involvement in the modulation of lipid metabolism in the liver.

Our results also showed that vitamin D supplementation decreased liver weight and liver index in a dose-dependent manner. These results are in agreement with Han et al. [13], who explained that vitamin D reduces hepatic fat accumulation.

On the other hand, there were significant decreases (P<0.05) in serum levels of 25(OH)D, liver weight, and liver index, as shown in [Table 2] and Chart 2.

Histopathological examination of the liver of rats that received the CD diet with vitamin D supplementation (1, 5, and 10 μg/kg) for 12 weeks confirmed our biochemical results; we found moderate hydrobic degeneration of hepatocytes, portal inflammatory cells infiltration, slight activation of Kupffer cells, and slight cytoplasmic vacuolization of focal hepatocytes, as seen in [Figure 2]. In addition, immunohistochemical examination of TGFβ1 showed that there was a decrease in its expression, indicating regression of the disease as shown in [Figure 5], confirming the protective role of vitamin D mediated through TGFβ1. These results are in agreement with Han et al. [13] and Eliades and Spyrou [14].


  Conclusion Top


We conclude that the CD diet plays a role in the development of NAFLD in experimental rats. Vitamin D administration had a hepatoprotective effect that could be mediated through TGFβ1.

Acknowledgements

The authors offer their gratitude and heartfelt thanks to all who helped them complete this study. They specially thank their technicians for their technical support. They also express my unlimited appreciation for the material support and equipment provided by our laboratory

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Kawaratani H, Tsujimoto T, Kitazawa T, Kitade M, Yoshiji H, Uemura M et al. Innate immune reactivity of the liver in rats fed a choline-deficient l-amino-acid-defined diet. World J Gastroenterol 2008; 14:6655–6661.  Back to cited text no. 3
    
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Kitson MT, Roberts SK. Delivering the message: the importance of vitamin D status in chronic liver disease. J Hepatol 2012; 57:897–909.  Back to cited text no. 6
    
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Noga AA, Vance DE. A gender-specific role for phosphatidylethanolamine N-methyltransferase-derived phosphatidylcholine in the regulation of plasma high density and very low density lipoproteins in mice. J Biol Chem 2003; 278:21851–21859.  Back to cited text no. 7
    
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Cheung O, Sanyal AJ. Recent advances in nonalcoholic fatty liver disease. Curr Opin Gastroenterol 2010; 26:202–208.  Back to cited text no. 8
    
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Smith BW, Adams LA. Nonalcoholic fatty liver disease and diabetes mellitus: pathogenesis and treatment. Nat Rev Endocrinol 2011; 7:456–465.  Back to cited text no. 10
    
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Eraslan S, Kizilgul M, Uzunlulu M, Colak Y, Ozturk O, Tuncer I. Frequency of metabolic syndrome and 25-hydroxyvitamin D3 levels in patients with non-alcoholic fatty liver disease. Minerva Med 2013; 104:447e53.  Back to cited text no. 11
    
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Roth CL, Elfers CT, Figlewicz DP, Melhorn SJ, Morton GJ, Hoofnagle A et al. Vitamin D deficiency in obese rats exacerbates nonalcoholic fatty liver disease and increases hepatic resistin and toll-like receptor activation. Hepatology 2012; 55:1103–1111.  Back to cited text no. 12
    
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Han H, Cui M, You X, Chen M, Piao X et al. A role of 1,25(OH)2D3 supplementation in rats with nonalcoholic steatohepatitis induced by choline-deficient diet. Nutr Metab Cardiovasc Dis 2015; 25:556–561.  Back to cited text no. 13
    
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Eliades M, Spyrou E. Vitamin D: a new player in non-alcoholic fatty liver disease. World J Gastroenterol 2015; 6:1718–1727.  Back to cited text no. 14
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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