Sexual dimorphism in hepatic gene expression and the response to dietary carbohydrate manipulation in the zebrafish (Danio rerio)

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Abstract

In this study, we tested for the presence of sexual dimorphism in the hepatic transcriptome of the adult zebrafish and examined the effect of long term manipulation of dietary carbohydrate on gene expression in both sexes. Zebrafish were fed diets comprised of 0%, 15%, 25%, or 35% carbohydrate from the larval stage through sexual maturity, then sampled for hepatic tissue, growth, proximate body composition, and retention efficiencies. Using Affymetrix microarrays and qRT-PCR, we observed substantial sexual dimorphism in the hepatic transcriptome. Males up-regulated genes associated with oxidative metabolism, carbohydrate metabolism, energy production, and amelioration of oxidative stress, while females had higher expression levels of genes associated with translation. Restriction of dietary carbohydrate (0% diet) significantly affected hepatic gene expression, growth performance, retention efficiencies of protein and energy, and percentages of moisture, lipid, and ash. The response of some genes to dietary manipulation varied by sex; with increased dietary carbohydrate, males up-regulated genes associated with oxidative metabolism (e.g. hadhβ) while females up-regulated genes associated with glucose phosphorylation (e.g. glucokinase). Our data support the use of the zebrafish model for the study of fish nutritional genomics, but highlight the importance of accounting for sexual dimorphism in these studies.

Introduction

The vertebrate liver performs a number of complex functions, including nutrient and vitamin metabolism, synthesis of plasma proteins, inactivation of various substances, and immunity. More specifically, in conjunction with the gastrointestinal tract, the liver plays a major role in glucose metabolism, including gluconeogenesis and glycogen storage. Glucose homeostasis and carbohydrate metabolism are fundamental biological processes that significantly influence growth and development, and define numerous pathological conditions. Most of our knowledge of these processes comes from studies in mammals. Our understanding of these processes in fish species is limited in a number of key areas, including the chronic effects of long term dietary manipulations (i.e. lasting from the larval stage to sexually mature adult) and the potential role of sexual dimorphism in the regulation of nutrient partitioning. These data are important in a variety of contexts, including improving our fundamental understanding of hepatic function and adaptability in fishes, design of alternative diets for aquaculture species (Trushenski et al., 2006) and the use of fish models in biomedical research (Papasani et al., 2006, Elo et al., 2007).

In fish, the genes involved in glucose homeostasis and carbohydrate metabolism respond rapidly to changes in dietary carbohydrate intake (Hemre et al., 2002), although there appears to be considerable interspecific variation in the capacity to utilize dietary carbohydrate (Panserat and Kaushik, 2002, Panserat et al., 2002). For example, carnivorous fish such as the rainbow trout are considered to be glucose-intolerant (Hemre et al., 2002). Rainbow trout suffer prolonged postprandial hyperglycemia, poor utilization of dietary glucose, and reduced growth when dietary carbohydrate levels are above 25–30% (Panserat et al., 2001a, Panserat and Kaushik, 2002). The persistence of high levels of hepatic fructose-1,6-bisphosphatase (FBPase) and glucose-6-phosphatase (G6Pase) gene expression suggests that rainbow trout have reduced transcriptional regulation of gluconeogenic genes by dietary carbohydrates (Panserat et al., 2001a, Panserat and Kaushik, 2002). In contrast, expression of gluconeogenic enzymes is more tightly regulated by dietary carbohydrates in two other fish species, the common carp (Cyprinus carpio) which are relatively tolerant of dietary carbohydrate, and gilthead seabream (Sparus aurata) (Panserat et al., 2002).

While several studies of acute regulation of carbohydrate metabolism have been performed, less work has examined the persistent effect (i.e. in the post-absorptive state) of long term dietary manipulations on hepatic gene expression. Current research indicates striking interspecific variation in metabolic phenotypes. For example, rainbow trout glucokinase activity remains elevated 24 h after a meal, while in common carp glucokinase activity returns to levels similar to those observed in fish fed a diet devoid of carbohydrate (Panserat et al., 2000). In contrast, short term manipulation of dietary carbohydrate in rainbow trout had no effect on the persistent (24 h) expression levels of glucose-6-phosphatase expression (Panserat et al., 1999). While these studies have been important, they have focused on a few genes known to be acutely regulated by carbohydrates, and a detailed genomic approach has not yet been taken in any fish species. Are expression levels of glycolytic and gluconeogenic genes influenced by long term dietary manipulation during post-absorptive periods, or is regulation of these genes only associated with acute, postprandial events? Are genes outside the gluconeogenic and glycolytic pathways affected by long term dietary manipulation?

In addition, most studies conducting nutritional manipulations (particularly those involving carbohydrates) were performed using sexually immature fish (see Hemre et al., 2002). This likely reflects the fact that market sizes of most aquaculture species are dependent on age of maturation, and the cost effective growth phase occurs prior to sexual maturation. While experiments using immature fish have been vital in establishing how key glycolytic and gluconeogenic genes are acutely regulated in response to carbohydrate availability, few data on nutritional effects in sexually mature adults are available in any fish species. This imposes unnecessary limitations on our understanding of genetic, biochemical, and physiological mechanisms that determine hepatic function, growth performance, and overall fish health.

When considering the effects of dietary carbohydrate manipulation in mature fish, one must account for potential sexual dimorphism in the response to dietary manipulation. In many taxa, there is substantial sexual dimorphism in a wide range of physiological phenotypes; however, the extent to which sex differences exist in glucose homeostasis remains unclear. The liver is one of the most sexually dimorphic organs in mammals with respect to patterns of gene expression; thousands of genes show sexual dimorphism in the mouse liver (Yang et al., 2007), and the expression of several commonly measured metabolic genes varies between male and female rats (Verma and Shapiro, 2006). In rats it has been postulated that sex-dependent secretion of steroids (Pankiewicz et al., 2003) and growth hormone (Clodfelter et al., 2006) either directly or indirectly play an important role in the regulation of liver gene expression and enzyme activity. As a result, sexual dimorphism may affect different hepatic functions related to glucose metabolism and metabolic phenotypes, and contribute to sex differences in physiology, homeostasis, and energy metabolism. Most studies of sexually dimorphic gene expression have occurred in mammals, and studies of sexual dimorphism in fish transcriptomes are rare [but see (Aubin-Horth et al., 2005, Wen et al., 2005, Santos et al., 2007)]. The extent of sexual dimorphism in the hepatic transcriptome and its resultant influence on physiological phenotypes are unknown in any fish species.

In this paper, we tested for the presence of sexual dimorphism in the hepatic transcriptome in a model teleost species, the zebrafish (Danio rerio). We then considered the potential effects of this dimorphism on the post-absorptive response to a long term manipulation of dietary carbohydrate. The zebrafish is a well recognized biomedical model system with an emerging new role in the study of glucose metabolism and its associated diseases (Gnugge et al., 2004, Elo et al., 2007). Although the zebrafish is an important biomedical research model, we know surprisingly little about its physiology, metabolic phenotype, and nutritional requirements. These data are important in extending the utility of the zebrafish beyond its traditional role as a model for developmental biology. The goals of this study were therefore to 1) test for sexual dimorphism in the zebrafish hepatic transcriptome, and 2) identify the persistent effects of long term manipulation of dietary carbohydrate on the zebrafish hepatic transcriptome in both males and females.

Section snippets

Zebrafish rearing conditions

Larval zebrafish (N = 2400, mean initial mass 49.7 mg) were obtained from a commercial breeder (Seacrest Farms, FL, USA), randomly assigned to groups of 200 fish, bulk-weighed, and placed into twelve 40-L rectangular glass tanks. Each tank was supplied with constant temperature (28.0 °C) spring water at the Hagerman Fish Culture Experiment Station (University of Idaho). Photoperiod was fixed at 14 h light/10 h dark for the duration of the study. Tanks were randomly assigned to one of four dietary

Chronic effects of dietary carbohydrate on growth and whole body composition

Using the bulk weight data at the 12 week time point, we observed a significant effect of dietary carbohydrate level on specific growth rate (F3,8 = 4.23, p = 0.046), total body mass (F3,8 = 4.68, p = 0.036), and percent weight gain (F3,8 = 4.71, p = 0.035) at the end of the experiment. For each variable, the pattern of response was the same; growth in the 0% carbohydrate diet was significantly lower than in the other three treatments (Fig. 1). Feed conversion ratio followed the same trend, but the effect

Discussion

The goals of our study were to 1) test for sexual dimorphism in hepatic gene expression in zebrafish and 2) identify the effects of long term manipulation of dietary carbohydrate on the zebrafish hepatic transcriptome in sexually mature males and females. We detected substantial sexual dimorphism in the zebrafish hepatic transcriptome. Long term manipulation of dietary carbohydrate influenced growth, body composition, and hepatic gene expression patterns 24 h after a meal. In most cases, these

Acknowledgements

The authors would like to thank Jon Amberg, Jurij Wacyk, Mary Oswald, Joyce Faler, and Mike Casten for their assistance in maintaining and sampling of fish. We would also like to thank Derek Pouchnik of the Washington State University Center for Reproductive Biology Genomics Core for his assistance with microarray hybridization. This work was supported by the NSF-Idaho EPSCoR Program and by the National Science Foundation under award numbers EPS-0132626 and EPS-0447689.

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