Teleost fishes represent a highly diverse group consisting of more than 20,000 species living across all aquatic environments. This group has significant economical, societal and environmental impacts, yet research efforts have concentrated primarily on salmonid and cyprinid species. This review examines carbohydrate/glucose metabolism and its regulation in these model species including the role of hormones and diet. Over the past decade, molecular tools have been used to address some of the downstream components of these processes and these are incorporated to better understand the roles played by carbohydrates and their regulatory paths. Glucose metabolism remains a contentious area as many fish species are traditionally considered glucose intolerant and, therefore, one might expect that the use and storage of glucose would be considered of minor importance. However, the actual picture is not so clear since the apparent intolerance of fish to carbohydrates is not evident in herbivorous and omnivorous species and even in carnivorous species, glucose is important for specific tissues and/or for specific activities. Thus, our aim is to up-date carbohydrate metabolism in fish, placing it to the context of these new experimental tools and its relationship to dietary intake. Finally, we suggest that new research directions ultimately will lead to a better understanding of these processes.

Glucose metabolism in fish: a review

Role of calcium in the hormonal regulation of liver metabolism

Vitellogenin synthesis in cultured hepatocytes; an in vitro test for the estrogenic potency of chemicals

A cell line RTL-W1, has been developed from the normal liver of an adult rainbow trout by proteolytic dissociation of liver fragments. RTL-W1 can be grown routinely in the basal medium, L-15, supplemented with 5% fetal bovine serum. In this medium, the cells have been passaged approximately 100 times over an 8-year period. The cells do not form colonies or grow in soft agar. The cultures are heteroploid. The cell shape was predominantly polygonal or epithelial-like, but as cultures became confluent, bipolar or fibroblast-like cells appeared. Among the prominent ultrastructural features of RTL-W1 were distended endoplasmic reticulum and desmosomes. Benzo[a]pyrene was cytotoxic to RTL-W1. Activity for the enzyme, 7-ethoxyresorufin O-deethylase (EROD), which is a measure of the cytochrome P4501A1 protein, increased dramatically in RTL-W1 upon their exposure to increasing concentrations of either β-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). With these properties, RTL-W1 should be useful for studying the expression of the cytochrome P450 enzymes and as a tool for assessing the toxic potency of environmental contaminants.

Development and characterization of a rainbow trout liver cell line expressing cytochrome P450-dependent monooxygenase activity.

Biochemical assays are not routinely used to assess liver damage in fish, therefore, a histopathological evaluation is usually required to determine the existence or extent of nonneoplastic liver toxicity. Many mammalian pathologists may be uncomfortable when requested to identify and interpret subtle liver changes in these unfamiliar animals. It may be reassuring to note that there are more similarities than differences between fish and mammals in terms of their macro- and microanatomy, physiological and biochemical characteristics, and pathologic responses to hepatotoxic substances. This brief overview addresses several topics pertaining to hepatotoxicity in fish, including: anatomic considerations, that is, how the anatomy of the fish liver may be predictive of its metabolic capacity, and also its microscopic appearance, following exposure to toxins; physiologic considerations, including comparisons between mammalian and fish livers regarding the uptake, elimination, toxification, or detoxification of xenobiotic compounds; morphologic responses to toxicity, in which some of the general types of findings that are most commonly observed in cases or studies of fish hepatotoxicity are highlighted; and last, responses of the fish liver to specific hepatotoxins.

A Brief Overview of Nonneoplastic Hepatic Toxicity in Fish

Adrenergic control of glucose output and adenosine 3':5'-monophosphate levels in hepatocytes from juvenile and adult rats.

Composition and interactions of cell types in rainbow trout (Oncorhynchus mykiss) liver digested with collagenase and cultured in serum-free media were investigated. Suspensions obtained after digesting trout liver with collagenase contained all the cell types present in the liver, including liver parenchymal cells (hepatocytes), biliary epithelial cells, sinusoidal endothelium, fat-storing cells of Ito, and macrophages. A major cell pellet, mainly hepatocytes but containing significant numbers of biliary epithelial cells, was obtained by centrifuging the cell suspension at 120 X g for 1 min. Cells present in this pellet quantitatively attached to culture plates coated with a trout skin extract and remain attached for 4 to 6 d with good retention of intracellular enzymes and DNA. When in culture, significant changes in and among the cells were observed. Initial preparations were rounded, single cells. Within several hours, however, cellular interactions leading to aggregation became evident and aggregates increased in size for 2 to 3 d. Scanning electron microscopy (EM) showed frequent shaftlike projections from margins of the aggregates. Transmission EM indicated that these projections represent biliary ductules forming in vitro. Adjacent hepatocytes also showed plasma membrane specializations forming junctional complexes and canaliculi characteristics of normal trout liver. After 5 to 6 d in culture, significant numbers of the cell aggregates dislodged from the plate. Analysis showed the dislodged cells were viable but vacuolated. The reestablishment in vitro of morphologic relationships resembling in situ tissue components suggest these culture preparations may have significant utility in cooperative metabolic studies of cell interactions in trout liver.

Isolated trout liver cells: Establishing short-term primary cultures exhibiting cell-to-cell interactions

Acute hormonal control of pyruvate kinase and lactate formation in the isolated rat hepatocyte

The characterization of a recently established system for the short-term culture of rainbow trout (Oncorhynchus mykiss) liver cells in chemically defined medium has been extended to studies on the metabolic competence of the cells and the characterization of their response to hormones. Three areas of metabolism have been addressed: a) the utilization of the exogenously added substrates fructose, lactate, glucose, dihydroxyacetone, and glycerol for glucose and lactate formation; b) the effects of the pancreatic hormones insulin and glucagon on cellular glucose formation, lactate formation, and fatty acid synthesis; and c) the effects of insulin and dexamethasone on the estradiol-dependent production of vitellogenin. Incubation of trout liver cells with fructose, lactate, glucose, dihydroxyacetone, or glycerol resulted in enhanced rates of cellular glucose and lactate production. Substrate-induced effects usually were more clearly expressed after extended (20 h) than after acute (5 h) culture periods. Addition of the hormones insulin or glucagon caused dose-dependent alterations in the flux of substrates to glucose and lactate. Rates of de novo synthesis of fatty acids from [14C]acetate were stimulated by insulin and inhibited by glucagon during acute and extended incubation periods. Treatment of liver cells isolated from male trout for 72 h with estradiol induced vitellogenin production and secretion into the medium. However, the addition of insulin or dexamethasone drastically reduced this estrogen-induced vitellogenesis. These results indicate that trout liver cells cultured in defined medium maintain central metabolic pathways, including glycolysis, gluconeogenesis, lipogenesis, and vitellogenesis as well as their responsiveness to various hormones, for at least 72 h. This cell culture system should provide an excellent model to further characterize metabolic processes in fish liver.

Cultured trout liver cells: utilization of substrates and response to hormones.

Long-term primary cultures of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver have been established. Nearly homogenous (>97%) populations of hepatocytes were placed into primary culture and remained viable and proliferative for at least 70 d. In addition to hepatocytes, proliferative biliary cells persisted in the cultures for at least 30 d. Finally, a third type of epithelial cell, which we have termed a “spindle cell,” consistently appeared and proliferated to confluence in these cultures. The confluent cultures of spindle cells were successfully subcultured and passaged. The initial behavior, growth, and optimization of serum and media requirements for these cells is described. All three cell types proliferated as measured by thymidine incorporation, autoradiography, proliferating cellular nuclear antigen analysis, and propidium iodine staining. Further efforts to characterize the cells included western blotting and immunohistochemical staining with antibodies to cytokeratins previously reported in fish liver. From these data, it appears that all three cell populations are epithelial in nature. Furthermore, significant changes in actin organization, often indicative of transformation or pluripotent cells, were observed with increased time in primary culture.

James B. Blair

Papers

Adrenergic control of glucose output and adenosine 3':5'-monophosphate levels in hepatocytes from juvenile and adult rats.

Isolated trout liver cells: Establishing short-term primary cultures exhibiting cell-to-cell interactions

Acute hormonal control of pyruvate kinase and lactate formation in the isolated rat hepatocyte

Cultured trout liver cells: utilization of substrates and response to hormones.

Long-term primary culture of epithelial cells from rainbow trout Oncorhynchus mykiss) liver.