| Leptin,as a product of the obese gene(ob),plays a various biological functions by binding to leptin receptor(Lep R),especially in regulating energy homeostasis.This study explored the regulation of Chinese perch(Siniperca chuatsi)leptin a and leptin b on glucose and lipid metabolism to analyse the functional differentiation of leptin a and leptin b,and to provid information for elucidating the mechanism of fish leptin on metabolic regulation.In the first experiment,there were six leptin receptor isoforms in mammals,while multiple isoforms were found in a few fish and short subtypes played a role in energy homeostasis.On the basis of Chinese perch genome database,amplification of c DNA 3'ends technology was used to explore whether there were alternatives splicing of Chinese perch lepr and to study the function of leptin a and leptin b in the future.We obtained four alternatives splicing of lepr,long subtypes lepr-L with CDS sequence length of 3474 bp and short subtypes lepr-S1,lepr-S2 and lepr-S3 with CDS sequence length of 1512 bp,945 bp,915 bp respectively.The results of sequence and evolutionary analysis showed that,like other vertebrates,only the long subtypes had various important functional domain,the extracellular LBD binding domain and the intracellular JAK and STAT binding domain,etc.,and the sequence was conservative,while short subtypes had no transmembrane domain and intracellular structure.In the second experiment,to analyze the effect of leptin a and leptin b on hepatocellular adaptive regulation strategies for exogenous glucose,we examined the cell viability,lipid content and the expression of genes related glucose metabolism of primary hepatocyte treated with different concentrations of glucose(5 mmol/L,15 mmol/L,25 mmol/L).In addition,we examined the expression of genes related to glucose metabolism of primary hepatocyte treatmented by leptin a/leptin b protein of Chinese perch with low,middle and high glucose medium.The results showed that:(1)compared with the 5 mmol/L glucose group,15 mmol/L and 25 mmol/L glucose had no significant effect on cell viability andlipid content(P > 0.05),and there was no significant change on the expression of gk,pfk,pk,pepck and g6 pca.1 in the 15 mmol/L glucose group,while the gs level of glycogen synthesis was significantly reduced(P < 0.05);in the 25 mmol/L glucose group,the expression level of gk was significantly increased(P <0.05),while no statistical significantly change on pfk,pk,pepckgs,and g6 pca.1 genes(P > 0.05).It indicated that the glycogen synthesis of primary hepatocyte was inhibited by middle glucose,and high glucose could accelerate the glycolysis,while gluconeogenesis also might be increased.It might be the reason why Chinese perch could not effectively use high-sugar substances.(2)Compared with the 5 mmol/L glucose group,5 mmol/L glucose + leptin a and 5mmol/L glucose + leptin b could significantly increase the expression levels of gk and pfk m RNA of glycolysis(P < 0.05),but no effects on the expression of genes related to gluconeogenesis and glycogen synthesis(P > 0.05);compared with the 15 mmol/L glucose group,the m RNA levels of gk,pfk,pk and g6 pca.1 were significantly up-regulated(P < 0.05),but no significant change on pepck and gs genes in the 15mmol/L glucose + leptin a group(P > 0.05),15 mmol/L glucose + leptin b only up-regulated the expression level of gk gene(P < 0.05);compared with the 25 mmol/L glucose group,the expression of pfk,pk,pepck and g6 pca.1 genes were up-regulated in25 mmol/L glucose + leptin a and 25 mmol/L glucose + leptin b groups.It revealed that leptin a and leptin b were involved in the regulation of glycolysis and gluconeogenesis in primary hepatocyte of Chinese perch,and leptin a might play a major role in regulation.In the third experiment,we tested and analyzed the cell viability and lipid deposition of primary hepatocyte incubated with different concentrations of sodium palmitate(0,0.1,0.12,0.15 and 0.2 mmol/L)for 48 hours,and found that 0.15 mmol/L sodium palmitate could significantly increase the cell activity,triglyceride content and lipid content.It was capable of inducing lipid accumulation in primary hepatocyte,and simulating the physiology condition of obese individuals.Then,the changes of lipid deposition,glucose and lipid metabolism and phosphorylation level of AKT were analyzed on primary hepatocyte treatmented by leptin a/leptin b protein and 0.15 mmol/L sodium palmitate,toexplore the regulation of Chinese perch leptins on lipid metabolism in high fat state.The results showed that leptin a could significantly reduced the lipid deposition of primary hepatocyte(P < 0.05),while leptin b had no significant effect on it(P > 0.05);leptin a and leptin b significantly inhibited the expression of srebp1 and acc1 genes(P < 0.05),while leptin a also up-regulated the expression levels of lepr and hsl genes.In addition,sodium palmitate and leptin protein had no significant effect on the phosphorylation level of AKT of primary hepatocyte(P > 0.05).It suggested that leptin a and leptin b could inhibit the fat synthesis of hepatocytes and participate in lipid metabolism with high lipid medium.In addition,leptin a could also up-regulate the expression of lepr and hsl to promote the lipolysis and reduce lipid deposition in hepatocytes,and the regulation of leptin on glucose and lipid metabolism was not mediated by PI3K/AKT signaling pathway.In summary,leptin a and leptin b could promote the glycolysis and gluconeogenesis of primary hepatocyte,and leptin a could inhibit fat synthesis and promote the lipolysis to reduce lipid deposition,while leptin b colud inhibit fat synthesis but no effect of lipolysis and lipid deposition.In other words,leptin a played a major role in regulating glucose and lipid metabolism in Chinese perch liver.The results of this study would provide information on the molecular mechanism of fish metabolism control network,and were expected to provide a theoretical basis for improving the efficiency of fish feed. |
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