Research Article
Gene pathways associated with mitochondrial function, oxidative stress and telomere length are differentially expressed in the liver of rats fed lifelong on virgin olive, sunflower or fish oils

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Abstract

This study investigates the effect of lifelong intake of different fat sources rich in monounsaturated (virgin olive oil), n6 polyunsaturated (sunflower oil) or n3 polyunsaturated (fish oil) fatty acids in the aged liver. Male Wistar rats fed lifelong on diets differing in the fat source were killed at 6 and at 24 months of age. Liver histopathology, mitochondrial ultrastructure, biogenesis, oxidative stress, mitochondrial electron transport chain, relative telomere length and gene expression profiles were studied. Aging led to lipid accumulation in the liver. Virgin olive oil led to the lowest oxidation and ultrastructural alterations. Sunflower oil induced fibrosis, ultrastructural alterations and high oxidation. Fish oil intensified oxidation associated with age, lowered electron transport chain activity and enhanced the relative telomere length. Gene expression changes associated with age in animals fed virgin olive oil and fish oil were related mostly to mitochondrial function and oxidative stress pathways, followed by cell cycle and telomere length control. Sunflower oil avoided gene expression changes related to age. According to the results, virgin olive oil might be considered the dietary fat source that best preserves the liver during the aging process.

Introduction

Senescence involves the progressive decline in the cell capacity to respond against oxidative damage, this decline ending in cell death [1], [2]. Although liver has usually been described as a well-preserved organ during the aging process, rats reportedly develop few hepatocytes of greater volume. Likewise, at the cell level, a loss of smooth endoplasmic reticulum has also been noted, together with an increase in dense body-compartment volume (including secondary lysosomes, residual bodies and lipofuscin), and greater polyploidy [3]. In 1980, Ludwig et al. introduced the concept of nonalcoholic steatohepatitis (NASH) to describe liver histologic changes resembling alcoholic hepatitis in individuals without significant alcohol intake [4]. Nonalcoholic fatty liver disease (NAFLD) encompasses a continuum, from simple hepatic steatosis with moderate fatty infiltration to NASH with focal inflammation. A small portion of NAFLD may progress to advanced fibrosis, cirrhosis and eventually hepatocellular carcinoma [5]. The prevalence of NAFLD in the general population increases with age: from 1% to 3% in children, 5% in teenagers, 18% between 20 and 40 years, 39% in those aged 40 to 50 years and to over 40% in those older than 70 [6]. Moreover, aging in the liver reportedly accelerates the progression of NAFLD to NASH and fibrosis, thus raising the likelihood of mortality [7], [8]. Aging may promote the development of NAFLD by several mechanisms such as promoting the onset of age-related obesity and diabetes, the cumulative effects of many years of lifestyle factors (e.g., overconsumption of an inadequate diet) or through physiological changes inherent to the process of aging such as altered autophagy or mitochondrial alterations and oxidative stress [9]. There is consistent evidence for a central role of mitochondrial dysfunction in the pathophysiology of NASH [10]. Recent findings suggest that continuous adaptation or “remodeling” of mitochondrial energetics, gene expression, morphology and content is key in the pathogenesis of simple steatosis/NASH [11]. Moreover, reactive oxygen species production by mitochondria has been consistently reported, and a number of studies in experimental models and humans indicate a strong association between the severity of NASH and degree of oxidative stress [12]. Moreover, oxidative stress, mainly at the mitochondrial level, has been associated with aging, and age-increased levels of protein carbonyl have been found in the liver of old mice [3] and rats [13].

Because the liver is the central organ of metabolism, changes in diet greatly affect this organ during aging [14]. Lifelong dietary changes, either in fat quality or in quantity, alter the fatty acid composition and make the liver more prone to damage and to developing NAFLD [8]. Regarding oxidative stress in the liver, the dietary fat source is known to strongly influence the lipid composition of the mitochondrial membrane, affecting the mitochondrial electron transport chain (mtETC) functions, oxidative damage and mtDNA alterations [15], [16]. Thus, altered mitochondrial ultrastructure and function in the liver have been reported in aged rats fed sunflower oil vs. virgin olive oil, together with different levels of oxidative stress and mtDNA alterations [17], [18], [19].

In relation to the aging process in the liver, the present study investigates the effect of lifelong feeding on different dietary fat sources rich in unsaturated fatty acids: virgin olive oil, rich in monounsaturated fatty acids (MUFA); sunflower oil, rich in n6 polyunsaturated fatty acids (n6PUFA); and fish oil, rich in n3PUFA. Young (6 months) vs. old (24 months) rats were studied regarding histopathological and ultrastructural features, telomere length, mtETC function and oxidative stress status. Lastly, the gene expression profile was considered in an attempt to account for the changes observed in the aforementioned markers.

Section snippets

Animals and diets

The rats were treated following the guidelines of the Spanish Society for Laboratory Animals, and the experiment was approved by the Ethics Committee of the University of Granada, Spain (permit number 20-CEA-2004). A total of 72 male Wistar rats (Rattus norvegicus) weighing 80–90 g were housed and maintained in a 12-h light/12-h darkness cycle, with free access to food and water. Individual rats were randomly assigned into three experimental groups and fed from weaning until 24 months of age on

Weight of rats and livers

No differences in body weight were found between treatments at 6 and 24 months (data not shown). Liver weight at 24 months of age was higher (P<.05) in the fish oil group (14.0±1.1 g) than in the virgin olive oil (11.2±0.3 g) or sunflower oil (11.6±0.8 g) groups.

Liver mitochondrial fatty acid profile

The results showed that for C18:1n-9 (oleic acid, the most representative fatty acid found in virgin olive oil), the highest percentage was found for the virgin olive oil group (14.4%±3.9%), values being significantly higher than those

Discussion

Although the liver is one of the organs least affected by aging, it has been found that lifelong changes in diet, either in quality or in quantity of fat, alter the fatty acid composition in liver and make the liver more or less susceptible to damage, leading it to develop NAFLD. The lipid profile was analyzed in the liver mitochondrial membranes since we have previously demonstrated that this is a good marker of dietary fat intervention at different tissue levels, including liver, brain, heart

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    Grants, sponsors and funding sources: This work was supported by I+D grants from the Spanish Ministry of Education and Science (AGL2008-01057), the Government of Andalusia (AGR832) and CEI BioTic Granada (CEI2013-P-20). A. Varela-López and P. Peréz-López were recipients, respectively, of a fellowship of FPU and FPI programs from the Spanish Ministry of Education.

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