In Vitro Effects Of Copper On Lipid Metabolism In Hepatocytes From Grass Carp And Yellow Catfish

As a kind of common heavy metals, copper (Cu) is also an essential element for fish growth. For this reason, many studies have examined the effect of Cu on fish. However, previous studies mainly focused on the oxidative stress, Cu accumulation, histological alterations and so on. The effects of Cu on lipid metabolism, especially studies in vitro, have not been reported yet. In the present study, hepatocytes from grass carp were conducted to evaluate the effects of Cu on lipid metabolism and the protective effects of selenium. Meanwhile, hepatocytes isolated from yellow catfish were also used to study in vitro effects of Cu on lipid metabolism and its possible pathway. The main results were as follows:1. In the present study, three different copper (Cu) concentrations (control,10μM and100μM, respectively) and three incubation times (24h,48h and96h, respectively) were chosen to assess in vitro effect of Cu on lipid metabolism in hepatocytes of grass carp Ctenopharyngodon idellus. Results showed that in vitro effects of Cu on lipid metabolism in hepatocytes indicated in a dose-and time-dependent manner:Cu decreased mRNA levels of several lipogenic and lipolytic genes at24h; Cu down-regulated the process of lipogenesis but up-regulated that of lipolysis at48h; the Cu-driven up-regulation of lipolytic genes was maintained after96h and accompanied by a decreased intracellular triglyceride (TG) accumulation, while no effect on lipogenic genes was shown. Thus,96-hour Cu exposure induced lipid depletion, possibly due to the up-regulation of lipolysis. Although after96-hour Cu exposure, lipogenesis might be up-regulated, it was not enough to compensate lipid consumption. Our study represents the first approach to dose-and time-dependent in vitro effects of Cu on lipid metabolism of fish hepatocytes and provides new insights into Cu toxicity in fish at both enzymatic and molecular levels.2. Hepatocytes cell line from grass carp was conducted to evaluate ability of Se supplementation (5μM,10μM) to prevent Cu-induced changes in lipid metabolism of in vitro hepatocytes from grass carp (Ctenopharyngodon idellus). Results showed that, among three Cu-exposed groups (100μM), increasing Se concentration tended to increase activities of lipogenisis enzymes, but reduced lipolysis enzymes at24h. Compared to control, Cu exposure alone or in combination with Se down-regulated mRNA levels of all the tested genes at24h, down-regulated mRNA levels of lipogenisis genes, but up-regulated mRNA levels of lipolysis genes at48h, which in turn reduced TG content was recorded at48h. Thus, the current study suggested that, although toxic at higher levels, lower levels of Se provide significant protection against Cu-induced changes in lipid metabolism in a time-dependent manner.3. In the present study, a protocol for primary hepatocytes culture of yellow catfish (Pelteobagrus fulvidraco) has been established, demonstrating to be an ideal in vitro model for lipid metabolism. Hepatocytes were successfully isolated with0.25%tripsin with EDTA, resulting in a cell yield of4.03x107cells g-1of liver and viability of95%. For investigation of whether copper can induce lipid metabolism, four different copper (Cu) concentrations (control,5μM,10μM and50μM, respectively) and three incubation times (0,24h and48h) were chosen to assess in vitro effect of Cu on lipid metabolism in the isolated hepatocytes. Results showed that Cu exposure induced changes in lipid metabolism in a concentration specificity manner:10μM Cu exposure induced lipid depletion, possibly due to the up-regulation of lipolysis. However,50μM Cu exposure induced lipid depletion, possibly due to the down-regulation of lipogenesis. What’s more,10μM Cu-induced lipolysis was disrupted by the PPARa antagonist GW6471, suggesting that peroxisome proliferator-activated receptor a pathway involved in the process of Cu-induced lipolysis. Taking together, Cu may regulate lipid metabolism in hepatocytes isolated from yellow catfish with different strategy in a specific concentration.