Nonalcoholic fatty liver disease (NAFLD) is a wide spectrum of fatty liver changes ranging from hepatic steatosis (i.e. abnormal retention of lipids within a cell) to steatohepatitis (nonalcoholic steatohepatitis), which can progress to liver fibrosis and cirrhosis in the absence of alcohol consumption. NAFLD is rapidly becoming a worldwide public health problem. It is present in 10-24% of the world's population and its prevalence is even greater in obese individuals, ranging from 57.5 to 74.0% [1]. In Egypt, its prevalence in the morbidly obese population has been estimated as 22% for male and 48% for female [2]. Evidence from human studies and animal models have suggested that lipid accumulation in the liver (fatty liver) plays an important role in the pathogenesis of heart failure, obesity, diabetes and metabolic syndrome [3]. Treatment strategies for NAFLD aim to improve insulin sensitivity and modify metabolic risk factors, or protect the liver from oxidative stress. Therefore, the discovery of nutrients that ameliorate fatty liver is of interest.

Whey proteins (WP) represent a heterogeneous group of proteins (β-lactoglobulin, α-lactalbumin, serum albumin, immunoglobulins ...etc.). It has been suggested that WP has an antioxidant activity probably depending on the abundance of cysteine in WP or on the presence of glutamylcysteine groups which are in other food proteins. Therefore, WP may be considered as a possible therapeutical tool in oxidative stress correlated diseases [4]. An undenatured cysteine-rich whey protein isolate (WPI) has been proven to raise glutathione levels by supplying the precursors required for intracellular glutathione synthesis. This has been demonstrated in several glutathione-deficient patient groups including those with advanced human immunodeficiency virus (HIV) infection [5]. Whey protein concentrate (WPC), beta-lactoglobulin and alpha-lactalbumin improve the liver damage induced by CCl₄[4, 6]. Oral administration of colostrum, which contains high amount of whey proteins, significantly decreased total cholesterol and triglycerides (TG) levels in both the men and women after 4 weeks [7]. Few studies used total whey proteins or WPI to study their effect on fatty liver [8, 9]. Hence, this study aimed to investigate the protective effect of oral administration of different WP products against fatty liver in an experimental rat model.

Nonalcoholic fatty liver disease (NAFLD) occurs across all age groups and occurs in 14%-30% of the general population [10]. In this study, whey proteins products were assessed for their ability to improve the biochemical and histological features of disease activity in NAFLD induced by a high carbohydrate fat free diet in Wistar rats. Recently, few studies were concerned with the effect of whey proteins on the parameters of fatty liver [8, 9]. Most of these studies focused on the use of total whey proteins instead of using their fractions individually. After 4 weeks of feeding high carbohydrate fat free diet, the histological appearance (Figure 2) of the positive control group (HCFFD) showed a typical appearance of fatty liver as reported by [11]. All tested whey proteins products led to an improvement in the results of histological examination of their livers (Figure 2). The hepatic triglycerides and the serum AST and ALT levels were improved in all whey protein groups. Further more, oral administration of all whey proteins products improved malondialdehyde levels.

In the current study, the growth parameters observation showed a reduction in the final body weight with oral administration of some whey proteins. These results are consistent with a variety of epidemiological trials whereas whey proteins or dairy products enhanced weight and fat mass loss in humans and animals [12, 13]. However, not all weight loss studies were in agreement with this conclusion [14]. Therefore, the results of the current study confirm that oral administration of whey proteins may reduce weight gain in rats.

The key factor in nonalcoholic fatty liver disease is insulin resistance. Recent data clearly implicate hepatic insulin resistance as a responsible factor for the accumulation of free fatty acids as triglycerides in hepatocytes. Therefore, treatment strategies for fatty liver disease, at present, are primarily focused on weight loss and use of insulin sensitizing agents [15]. In this study, oral administration of all the tested whey proteins reduced serum glucose levels. At the same time, this beneficial effect was associated with a reduction in the hepatic triglycerides. Consequently, the histology of the livers of all whey protein groups appeared with less fatty infiltration in hepatocytes (Figure 2). This effect is similar to that observed by [8] who found that whey + calcium containing diet significantly decreased blood glucose, serum insulin, and hepatic triglycerides in lean and obese C57Bl/6J mice.

In addition, alanine transaminase (ALT) is an important enzyme found predominately in the liver. Significantly elevated activity of ALT in serum often suggests its leakage from damaged hepatic cells and reflects hepatocyte damage. Aspartate aminotransferase (AST) is similar to ALT in that it is another enzyme associated with liver. The ALT is a more specific indicator of liver inflammation than the AST, as the AST may also be elevated in diseases affecting other organs. For this reason, ALT is commonly used as a way of screening for liver problems. Elevated ALT levels correlated strongly with NAFLD [16, 17]. In the present study, oral feeding of whey proteins lowered levels of ALT and AST in rats' model of fatty liver (Figure 1). This positive effect on ALT and AST might account for the improvement of the liver histology and less fatty infiltration in hepatocytes. These findings are in agreement with [9], who reported that a daily dose of 20 g whey protein isolate for 12 weeks given to fatty liver patients led to a significant reduction in ALT and AST.

Mitochondrial dysfunction is thought to play a central role in NLFLD as production of reactive oxygen species is increased and lead to inflammation, hepatocellular apoptosis, or in other words increased oxidative stress [9]. Hepatocyte and plasma glutathione (reduced form, GSH) decreased and glutathione disulfide (oxidized form, GSSG) increased in NAFLD patients [18]. As a consequence, strategies to increase glutathione concentrations to reduce oxidative stress might play an important role in pathogenesis of NAFLD. In the results of our study, a significant elevation in the GSH content was observed only when rats were fed on WPI, WPH or βLG (P < 0.05, compared with HCFFD). An undenatured cysteine-rich whey protein isolate has been proven to raise glutathione levels by supplying the precursors required for intracellular glutathione in several glutathione-deficient patient groups including those with advanced human immunodeficiency virus (HIV)-infection [19]. Also, total milk proteins did not decrease total liver GSH compared with the high sucrose diet group [20].

In addition, lipid peroxidation leads to the generation of by-products, such as malondialdehyde (MDA), which are involved in activation of the inflammatory response and consequently, cellular damage [19]. The results of high hepatic MDA levels in the steatosis group are in agreement with the results of [17]. Oral administration of whey proteins products was associated with lowering content of MDA in liver (Table 3). It is expected that the potential effect of whey proteins in preventing further accumulation of free radicals as well as oxidative stress could be due to their anti oxidant capacity and/or their anti-inflammatory effect. Previously, it was observed that whey protein led to an increase in the antioxidant enzymes and a decrease in the oxidative stress. Whey proteins also showed an anti-inflammatory effect in humans [21, 22].

Oral administration of some whey proteins products (specially WPH and WPI) improved the fatty liver state in a rat model of NAFLD, which is in correlation with the results of histological examination of rats' liver and a reduction in hepatic triglycerides, liver enzymes levels (ALT and AST), serum glucose and lipid peroxidation (MDA). Further studies are needed to give further insight into the underlying mechanism.

This study was approved by the Committee of Scientific Ethics at Cairo University and according to its guidelines.