Adipose tissue and liver metabolic responses to different levels of dietary carbohydrates in gilthead sea bream (Sparus aurata)

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Abstract

This study analyzes the effects of replacing dietary lipids by carbohydrates and carbohydrates by fiber on gilthead sea bream growth, as well as lipid and glucose metabolism in adipose tissue and liver over the course of a 15-week feeding trial. Six different diets were formulated and fish were classified into two experimental groups sharing one diet. In the first group (LS), fish were fed four diets where lipids were reduced (23%–17%) by increasing carbohydrates (12%–28%) and, the second group (SF) consisted on three diets where the amount of carbohydrates (28%–11%) was exchanged at expenses of fiber (1%–18%). Differences in growth were not observed; nevertheless, the hepatosomatic index was positively related to dietary starch levels, apparently not due to enhanced hepatic lipogenesis, partly supported by unchanged G6PDH expression. In the LS group, lipogenic activity of adipose tissue was stimulated with low-lipid/high-carbohydrate diets by up-regulating G6PDH expression and a tendency to increase FAS, and promoted carbohydrate utilization versus fatty acid oxidation by modulating the transcription factors LXRα, PPARα and PPARβ expression. In the SF group, PPARs and LXRα increased parallel to fiber levels in adipose tissue. Furthermore, an adaptation of hepatic GK to dietary starch inclusion was observed in both groups; however, the lack of effects on G6Pase expression indicated that gluconeogenesis was not nutritionally regulated under the conditions examined. Overall, metabolic adaptations directed to an efficient use of dietary carbohydrates are present in gilthead sea bream, supporting the possibility of increasing carbohydrate or fiber content in diets for aquaculture sustainability.

Introduction

Aquaculture production has expanded by almost 12-fold in the last three decades (FAO, 2012). Aquafeed relies on fish meal and fish oil due to its high nutritional quality; however, the sustainability of this practice is not well accepted at present, especially over the long term (Watanabe, 2002). Hence, in order to reduce aquafeed costs and alleviate overexploited marine fisheries pressure, research efforts to find suitable alternatives are currently underway.

The use of raw plant materials as protein and lipid sources has been recognized as a sustainable alternative to fish products (Bouraoui et al., 2011, Nasopoulou and Zabetakis, 2012); nevertheless, protein continues to be the most expensive nutrient and in an attempt to spare protein energy in several fish species, lipid content is rising (Company et al., 1999, Watanabe, 2002, Li et al., 2012). The current trend to use high-lipid diets has been shown to induce undesirable increases in fat depots or even physiological alterations such as induction of oxidative stress (Kjaer et al., 2008). Thus, carbohydrates are attractive ingredients as they are considered to supply energy at a low cost; however, the inclusion of high amounts of dietary carbohydrates remains controversial. Carnivorous fish are considered to present a limited ability to use dietary carbohydrates (Moon, 2001, Hemre et al., 2002), and their effects on growth depend upon many factors such as the source, concentration, level of food intake and digestibility (Brauge et al., 1994). Gilthead sea bream has been reported to present excellent starch digestibility coefficients (Enes et al., 2008, Couto et al., 2012) and elevated activity of the main enzymes of the glycolytic pathway (Panserat et al., 2000, Couto et al., 2008, Enes et al., 2008), suggesting a possible efficient utilization of dietary starch for energetic purposes in this species.

Most of the studies in cultured fish species regarding high dietary carbohydrate content have focused on the effects of starch origin and inclusion levels on nutrient digestibility and retention efficiency, as well as growth performance (Capilla et al., 2003, Capilla et al., 2004). The response of glucose, lipid or amino acid metabolism enzymes has been reported mainly in the liver as crucial active metabolic organ (Caseras et al., 2000, Panserat et al., 2000, Caseras et al., 2002, Panserat et al., 2002b, Metón et al., 2004, Enes et al., 2006, Enes et al., 2008, Couto et al., 2008). However, and despite its importance in controlling fish energy balance, scarce information concerning the effects of dietary carbohydrates on adipose tissue is available. It has been reported that the lipogenic activity of adipose tissue is hormonally regulated in rainbow trout and modulated by diet in gilthead sea bream (Bouraoui et al., 2011, Cruz-Garcia et al., 2011, Polakof et al., 2011a), suggesting its possible implication on glucose homeostasis, being glucose one of the main lipogenic precursors. Therefore, the study of both, adipose tissue and liver, as well as their inter-relation may help to better explain how fish use dietary lipids and carbohydrates to grow.

Few studies have analyzed the interactions between dietary lipids and carbohydrates and their effects on glucose metabolism in fish (Polakof et al., 2011b, Figueiredo-Silva et al., 2012). In mammals, it is known that high levels of fatty acids disrupt carbohydrate metabolism, eventually causing impaired glucose tolerance (Randle, 1998). A recent study demonstrated the existence of a similar alteration in rainbow trout, where fish developed high fat-induced persistent hyperglycemia and reduced insulin sensitivity (Figueiredo-Silva et al., 2012). The poor utilization of carbohydrates by rainbow trout was linked, at least to some extent, to the use of high-fat diets (Panserat et al., 2002a). These observations pointed out that a reduction in dietary fat content could improve the glycemic control in carnivorous fish fed high-carbohydrate diets, and highlighted the importance of studying the metabolic interactions between dietary macronutrients.

Although in humans it has been shown that some dietary fibers tend to reduce cholesterolemia and improve glucose tolerance (Johnson, 1990, Kishimoto et al., 1995), the effects of different fiber levels in fish diets are still not clear and some discrepancies between species have been reported. In Atlantic salmon, apparent digestibility of lipids was linearly reduced with the inclusion of cellulose, while starch or protein digestibility was not influenced (Aslaksen et al., 2007). However, no significant effects of cellulose inclusion were found on the digestibility of main nutrients in rainbow trout (Hansen and Storebakken, 2007). Most of the research effort has been focused on the effects of dietary fiber inclusion on growth, digestibility and feces characteristics, whereas little is known about its possible implications on lipid and glucose metabolism. In white sea bream, the use of guar gum in the diet had no effect apparently on glucose utilization but contributed to lower endogenous glucose production (Enes et al., 2013).

In order to characterize the lipogenic potential and glucose utilization capacity of adipose tissue and liver of gilthead sea bream, two experimental groups were established. One group was used to test 4 diets with different lipid-to-carbohydrate ratios, in order to know whether gilthead sea bream is able to efficiently use high levels of starch, and how lowering lipid levels affect the fish energetic status. A second group was used to test 3 diets with different fiber-to-carbohydrate ratios in order to detect specific metabolic changes triggered by modifications in starch levels together with the use of a high content of cellulose as a filler agent. To this end the expression of key enzymes and transcriptional factors involved in glucose and lipid metabolism was evaluated. We chose the enzymes lipoprotein lipase (LPL) and hormone sensitive lipase (HSL) as markers of fatty acid uptake and lipolysis respectively, and enzymes related with lipogenesis, fatty acid synthase (FAS), and one of the main enzymes acting as NADPH donor, glucose-6-phosphate dehydrogenase (G6PDH), in order to assess whether dietary increases in carbohydrate-fiber or carbohydrate-lipid ratios might activate lipogenic processes. The expression of two enzymes, hepatic glucokinase (GK) and glucose 6-phosphatase (G6Pase) involved in glucose uptake and release, respectively, was expected to be regulated by diet composition. The lipid metabolism-related transcription factors determined were: liver X receptor α (LXRα) recently involved in triglyceride breakdown in fish adipose tissue (Cruz-Garcia et al., 2012) and peroxisome proliferator-activated receptors α, β and γ (PPARα, PPARβ, PPARγ), being the α and β isotypes promoters of fatty acids use in mammals (Kota et al., 2005) and PPARγ involved in lipid accumulation and adipogenesis also in fish (Bouraoui et al., 2008). We hypothesize that PPARs transcription levels could be related to the dietary lipid content.

All in all we aimed to elucidate the mechanisms involved in the activation of lipid and carbohydrate metabolic pathways at a transcriptional level, in both, adipose tissue and liver, in response to dietary macronutrient replacements. To generate a complete picture we also studied the effects of these dietary manipulations on growth performance, plasma metabolites and adipocyte size.

Section snippets

Animals and feeding experiment

All animal handling and experimental procedures were conducted in compliance with the experimental research protocol approved by the Committee of Ethic and Animal Experimentation of the University of Barcelona (CEEA 239/09), and the Departament de Medi Ambient i Habitatge (DMAH permit number 5420, Generalitat de Catalunya, Spain) following regulations and procedures established by the European Union, and by the Spanish and Catalan Governments.

A total of 306 juvenile gilthead sea bream (Sparus

Effects of diet on growth performance and feed utilization

Biometric parameters were measured at the end of the feeding experimental trials and values are presented in Table 3. In each group (LS or SF), fish fed with the different diets were comparable and no significant differences due to carbohydrate inclusion levels, changed either by lipids or fiber, were observed after the 15-weeks in terms of growth performance (SGR) or feed utilization (FCR).

In the LS group, dietary effects were reflected in the hepatosomatic index (HSI) of fish, which increased

Discussion

The aim of this study was to gain knowledge on the dietary lipid and carbohydrate use in gilthead sea bream focusing our interest not only in the role of adipose tissue, but also in the liver, both key organs of fat and starch deposition and metabolic turnover. The results demonstrated that in the conditions studied, dietary lipid substitution by carbohydrate up to levels of 28%, and subsequent changes in carbohydrate content by fiber had moderate effects in adipose tissue metabolism

Acknowledgments

We thank M. Monllaó, S. Molas, and E. Hernández from the Institut de Recerca i Tecnologia Agroalimentàries de Sant Carles de la Ràpita (Spain) for the maintenance of the gilthead sea bream as well as their assistance during sampling. We also thank Prof. Mercè Durfort and Jordi Correas from the Department of Cell Biology for their help with the histological studies. E.C. is a “Ramón y Cajal” researcher fellow from the “Ministerio de Ciencia e Innovación” (MICINN) and M.B. is supported by a

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