Regulation Of Dhea On Hepatic Lipid Metabolism In Broil Chickens And Its Cellular Molecular Mechanisms Involved

Dehydroepiandrosterone (DHEA) is a precursor of steroid hormone and its sulfated form is released into the circulation. As a precursor of steroid hormone, DHEA can transfer to several steroid hormones by steroidogenic enzymes in peripheral tissues. It is known to have several physiological effects inculding antiobesity. Although some research has been conducted in the area of DHEA regulation and lipid metabolism in rats and mice, there is little information available on the effect of DHEA on the activities of lipid metabolic hormones and parameters in poultry, especially in broiler chickens. Furthermore, the precise mechanisms by which DHEA exerts its biological actions are not fully understood. Therefore, a study was carried out to examine the effects of DHEA on lipid metabolic hormones and hepatic parameters in AA broiler chickens (a fatty meat-type, and excess fat accumulated in the abdomen). We also investigated whether DHEA activates hepatic lipogenic gene mRNA expression and the cAMP-dependent signaling system in cultured primary chicken hepatocytes and, subsequently, whether DHEA protects against fat deposition by regulation of the transcription factors. The purpose of this study is to provide a theoretical and experimental evidence for DHEA regulation on hepatic lipid metabolism in broiler chickens.1 The Effect of DHEA on Growth Performance, Lipid Metabolic Hormones and Parameters in AA Broiler ChickensOne-hundred and eighty (180) commercial broiler chickens (1-day old,38 g) were housed from days 1 to 42 and randomly divided into three equal groups (60 birds per group), and each group was assigned to 1 of 3 treatments. Birds in each group were kept in three pens (20 broilers per pen). All birds were offered the same basal diet with added DHEA at levels of 0 (control),5 and 20 mg/kg. All birds had free access to feed and drinking water. At the conclusion of the experiment, chickens were weighed individually at 42 days to determine final body weight. After the birds were slaughtered, gender was identified, abdominal fat and liver were collected, blotted dry and weighed. Blood samples were taken and retained for subse-quent analysis. The results showed that body weight and abdominal fat content was significantly lower in male and female broiler chickens, as compared with the control groups. In contrast, the relative liver weight was significantly higher than the control liver weight for both male and female broiler chickens. The level of triglyceride (TG) was significantly lower in female broiler chickens fed 5 or 20 mg DHEA/kg than those fed 0 (control), while in male broiler chickens, only those fed 20 mg DHEA/kg were lower. In contrast, male broiler chickens fed with 20 mg DHEA/kg resulted in a pronounced higher level of hepatic lipase (HL) as compared to the control group. However, no significant difference in HL was observed among female birds administered 0, 5, or 20 mg DHEA/kg. The concentration of non-esterified fatty acids (NEFA) was significantly higher in males and females at 20 mg DHEA/kg than at the control level. During the experimental period, DHEA decreased serum concentrations of thyroxine (T4), free triiodothyronine (FT3), free thyroxine (FT4), but significantly increased the serum level of Glucagon and Leptin in male broiler chickens. Meanwhile, female broiler chickens fed DHEA had significantly lower levels of FT3, FT4, and higher concentrations of Leptin as compared with control. These data indicate that the administration of DHEA to poultry improved lipid metabolism by influencing metabolic hormones and their hepatic physiological parameters.2 Regulation of DHEA on Hepatic Lipogenic Gene mRNA Expression and Liver Ultrastructure in AA Broiler ChickensOne-hundred and eighty (180) commercial broiler chickens (1-day old,38 g) were housed from days 1 to 42 and randomly divided into three equal groups (60 birds per group), and each group was assigned to 1 of 3 treatments. Birds in each group were kept in three pens (20 broilers per pen). All birds were offered the same basal diet with added DHEA at levels of 0 (control),5 and 20 mg/kg. All birds had free access to feed and drinking water. At the conclusion of the experiment, chickens were were slaughtered, gender was identified, liver were collected for RT-PCR assays and morphological observations. The result showed that no significant differences were observed in both sterol regulatory element binding protein-lc (SREBP-1) and acetyl Co A carboxylase (ACC) mRNA expression in broiler livers treated with DHEA. However, peroxisome proliferators-activated receptor a (PPARa), carnitine palmitoyl transferase I (CPT I) and acyl-Coenzyme A oxidase 1 (ACOX1) mRNA expression showed a trend toward enhancement as the treatment concentration of DHEA increased in the absence of a gender difference. The increase in the number of peroxisomes was observated in both male and female birds. These data indicate that DHEA accelerates lipid catabolism by induction of relevant gene expression and by induction of changes in hepatocyte peroxisome.3 Regulation of DHEA on Hepatic Lipogenic Gene mRNA Expression and Celluar Ultrastructure in Cultured Primary Chicken HepatocytesCultured primary chicken hepatocytes were equilibrated to culture conditions for 24h and then exposed to DHEA (0μmol/L,1μmol/L,10μmol/L and 100μmol/L) dissolved in DMSO. Control and treated cells were left for either 1 h,2 h,4 h,6 h,12 h,24 h,48 h,72 h or 6 d for RT-PCR or MTT assay and morphological observations. The results demonstrated that the expression of SREBP-1 mRNA in cultured primary hepatocytes did not vary with time, except for a significant decrease in gene expression when cells were treated with 100μmol/L or 10μmol/L DHEA for either 1 h or 2 h. A similar tendency toward decreased gene expression was evident for ACC. The expression of PPARa and CPT I mRNA was dramatically enhanced subsequent to treatment with 100μmol/L or 10μmol/L DHEA for periods of time from 6 to 48 h, while there was a decrease in the expression of PPARa in the presence of 10μmol/L DHEA after 1 h and 2 h of exposure, and in the presence of 100μmol/L DHEA after 2 h of exposure. Hepatocytes treated with 100μmol/L or 10μmol/L DHEA contained more mitochondria and mitochondria with a higher electron density than did untreated hepatocytes. Furthermore, cell survival was significantly inhibited by treatment with 100μmol/L DHEA at 24 h,48 h and 72 h. In contrast,1μmol/L DHEA administration significantly increased cell survival after 72 h, However,10μmol/L DHEA treatment had no pronounced effect on cell survival. Overall, the results reported here indicate that 10μmol/L DHEA accelerates lipid catabolism by direct regulation of hepatic gene expression and by induction of changes in hepatocyte mitochondria without adversely affecting cell survival.4 Regulation of DHEA on cAMP/PKA Signalling and its Transcription Factor Involved in Cultured Primary Chicken HepatocytesCultured primary chicken hepatocytes were equilibrated to culture conditions for 24 h and then exposed to DHEA (0μmol/L,0.01μmol/L,0.1μmol/L,1μmol/L,10μmol/L and 100μmol/L) dissolved in DMSO. Control and treated cells were left for 20 min for cyclic adenosine monophosphate (cAMP) determination by RIA kit. The results showed that a marked increase in cAMP was found in 0.1-100μmol/L DHEA treated groups. A DHEA concentration of 0.1μmol/L caused the maximum stimulation of intracellular cAMP accumulation when compared with the control group. Thus, we used 0.1μmol/L DHEA to culture chicken hepatocytes for various time periods. Cells incubated with 0.1μM DHEA for varying lengths of time (5 min,10 min,20 min,30 min and 60 min) were collected for cAMP, adenylate cyclase (AC), cAMP-specific phosphodiesterase (PDE) and cAMP-dependent kinase A (PKA) assay, and others were scraped for the subsequent determination of protein levels by western blot. The levels of cAMP were significantly higher during the experimental period from 5 min to 30 min as compared to the control group, but then decreased, with no further marked increase after 60 min of DHEA incubation. Furthermore, no significant differences were observed in AC activity in both the DHEA-treated and the control groups at different time periods. However, DHEA markedly suppressed PDE activity at 10 min and 20 min. In contrast, cells incubated with 0.1μmol/L DHEA for 20 min or 30 min exhibited a pronounced increase in PKA activity. Moreover, incubation of 0.1μmol/L DHEA for 1 h caused a significant decrease in the level of SREBP-1 protein and a marked enhance in cAMP response element binding protein (CREB) phosphorylation. No significant differences was found in cells pretreated with PKA inhibitors. The results here indicate that direct action by DHEA leads to activation of the cAMP/PKA signaling system in the modulation of lipid metabolism by repressing SREBP-1 protein and increasing CREB phosphorylation, thereby providing a novel explanation for some of the underlying effects proposed for DHEA in the prevention of fat deposition in poultry.