Dietary Phosphorus Reduced Hepatic Lipid Deposition By Activating AMPK And Lipophagy In Gift Tilapia Oreochromis Niloticus

Phosphorus is a macro-mineral element essential for growth in fish.Fish mainly obtain phosphorus from the diets.Excess phosphorus in the diet can reduce hepatic lipid deposition and significantly affect lipid metabolism.The AMP-activated protein kinase（AMPK）is an energy sensor,which could be activated when ATP/AMP decreases or the energy demand increases.The AMPK is vital to regulate the metabolism of lipid.Autophagy is a self-defense mechanism that has evolved in animals to maintain normal physiological processes,but excess phosphorus in the feed can activate autophagy,and reduce lipid content.In mammals and fishes,studies have shown that increased phosphorus levels in the diet would inhibit lipid synthesis.However,is the lipid metabolism regulated by the AMPK signaling pathway?And is autophagy involved in this process?What is the specific mechanism of the regulation for phophorus-induced changes of lipid metabolism?These scientific questions are largely unknown.We used tilapia（Oreochromis niloticus）and its primary hepatocytes as the experiment models,to explore whether phosphorus regulates lipid metabolism via the AMPK and autophagy signaling pathway.The results were showed below:1 Effects of dietary phosphorus levels on lipid metabolism in liver of tilapiaWe designed experiment to study the effects of different dietary phosphorus levels on lipid metabolism in liver of tilapia.For our in vivo experiments,three dietary phosphorus levels were included:1.21 g/100g（low-phosphorus diet,LPD）,1.75 g/100g（middle-phosphorus diet,MPD）and 2.66 g/100g（high-phosphorus diet,HPD）.The tilapia were fed for 10 weeks.The results of hematoxylin and eosin（H&E）,oil red O（ORO）and TG content indicated that HPD significantly reduced the lipid content in liver of tilapia,compared to the LPD group.Compared to the LPD group,the HPD group inhibited m RNA levels of lipid synthesis,and promoted m RNA expression of the lipolysis-associated transcription factor.Moreover,the HPD group reduced the activity of lipid synthesis-related enzymes（FAS,G6PD,ICDH,ME）,promoted the activity of the lipolysis-related enzyme（CPTⅠ）.And compared to the LPD group,the HPD group increased autophagosome formation,reduced the number of LDs,and up-regulated the m RNA levels of autophagy-related genes,and increased the protein levels of LC3B-Ⅱand the phosphorylation level of Beclin1,and up-regulated the m RNA expression of many fatty acidβ-oxidation genes and increased NEFA content.These suggested that HPD can activate autophagy.Besides,we found that HPD can increase m RNA levels of the AMPK related genes,and increased the AMPKα1phosphorylation level,which suggested that HPD could activate AMPK signaling pathway.Taken together,these results indicated that dietary phosphorus could reduce hepatic lipid deposition,activate AMPK pathway,and Beclin1 phosphorylation levels,and activate autophagy.2 The mechanism of AMPK/Beclin1 signaling pathway mediated phosphorus（Pi）-induced changes of lipid metabolism in primary hepatocyte of tilapiaHigh-phosphorus diet（HPD）reduced lipid deposition and significantly influences lipid metabolism,activated AMPK signaling pathway and up-regulated the phosphorylation level of Beclin1 in our in vivo studies,which pushed us to conduct further mechanistic research.We isolated the primary hepatocytes from the tilapia for cell experiments.We designed different phosphorus concentrations in the form of Na₂HPO₄·12H₂O and Na H₂PO₄·2H₂O.And we chose 3 m M for our study by MTT assay.Similar to the results of the in vivo experiments,the Pi treatment reduced the lipid by laser confocal microscopy and flow cytometry after BODIPY 493/503 staining and the determination of TG content in the cells.Besides,Pi treatment up-regulated m RNA abundances of AMPK related genes and increased the p-AMPKα1 protein levels.These indicated that Pi activated AMPK pathway in primary hepatocyte of tilapia.Moreover,Pi treatment increased the protein level of the p-Beclin1 and LC3B-II,and the p62 expression was inhibited.Compared to the control,Pi activated autophagy in primary hepatocytes of tilapia.Meanwhile,flow cytometry and laser confocal microscopy confirmed this result.We found that Pi increased the amounts of LDs（green）bound to LC3 puncta（red）and autolysomes（red）.And Pi increased the m RNA levels of fatty acidβ-oxidation genes.These suggested that Pi degraded lipid by activation of specific lipophagy,which was further confirmed by the TEM.CQ（autophagy inhibitor chloroquine）alleviated the phosphorus-induced lipolysis and NEFA content increase,and alleviated the Pi-induced decline of the p62 protein expression and aggravated the expression level of the LC3B-Ⅱprotein.Furthermore,AMPKα1 and Beclin1 knockdown alleviated the Pi-induced increase of the LC3B-Ⅱand p-Beclin1 protein expression,and alleviated the Pi-induced decline of the p62 protein expression.AMPKα1 and Beclin1 knockdown also alleviated the Pi-induced reduction of TG content.Finally,we found that AMPKα1 coprecipitated with Beclin1 in HEK 293T cell and phosphorus aggravated this response when beclin1 and ampkα1 genes were constructed.The S496A mutants of AMPKα1 reduced the Beclin1 phosphorylation level,indicating that tilapia Beclin1 protein could be phosphorylated at the Ser90 site.Similarly,the S90A mutants of Beclin1 reduced the phosphorylation level of AMPKα1.Therefore,these results illustrated that the AMPK/Beclin1 signaling pathway mediated the Pi-induced autophagy and lipolysis.Thus,dietary phosphorus reduced hepatic lipid deposition by activating AMPK pathway and Beclin1 phosphorylation levels to activate lipophagy in tilapia.These results provided new ideas for the occurrence and prevention of fatty liver disease in fish at the molecular level.