Biological Evaluation Of Artificial Lipid Droplets And The Mechanism Of Them Enhancing Intramuscular Fat Content In Pigs

Intramuscular fat（IMF）deposition is primarily formed by the accumulation of lipid droplets（LDs）in muscle fibers and adipocytes.The LDs are characterized by their unique and conserved monolayer phospholipid membrane structure,which plays a vital role in energy metabolism and protein turnover.However,the lipid and protein composition of LDs is highly dynamic and subject to change based on cellular state.This dynamism,while conferring diverse functions to LDs,poses significant challenges for research.Thus,the connection between the composition and function of LDs remains to be explored,as does the pathway between cellular nutrient perception and lipid droplet response.In this study,we present a novel method for the rapid generation of stable,pure,and artificial lipid droplets（a LDs）.We characterized the structures of these droplets using a range of assays,including particle size analysis and laser confocal detection.Additionally,we incubated proteins with these artificial lipid droplets and investigated their interactions with organelles,demonstrating their potential biological activity.Our findings suggest that a LDs exhibit anti-stress and lipotoxicity alleviation functions and can increase IMF content when injected into muscle tissue.The main findings are as follows.1.Preparation of a LDs and their functional evaluation（1）The prepared nanolipid particles（NLPs）exhibited a spherical shape with a phospholipid monolayer encapsulated.Characterization assays revealed that the NLPs had a particle size of 215 nm and a zeta potential of-11.2 m V,with no evidence of non-spherical structures.The use of a LDs-specific dye yielded positive staining results,further confirming the spherical nature of the particles.The NLPs were prepared by Rhod-PE and laser confocal detection showed that PE was predominantly located in the outer layer of the particles,with no distribution in the interior.Thin-layer chromatography for lipid determination showed a phospholipid/total lipid ratio of 8.5%,indicating the presence of a monolayer phospholipid structure on NLPs.（2）NLPs have good stability and can be made into lyophilized powder.The NLPs were stored at 25°C and 4°C respectively,and no significant changes were observed in terms of concentration or size after 7 days.Furthermore,the morphology of NLPs remained unchanged,and no non-spherical structures were produced upon staining.The addition of5%mannitol acted as as an effective lyophilization protectant,resulting in a white,loose,and porous powder with a smooth,uniform,and fine surface.After adding 2 ml of 0.2%Tween 80 solution,the lyophilized powder could be quickly redispersed and appeared white and creamy after dispersion.（3）The lipid fraction used to prepare NLPs was found to significantly impact their particle size distribution.Specifically,increasing the speed and expanding the ratio of triglycerides and phospholipids resulted in larger NLPs.Other lipid components,such as PE,cholesterol,squalene,phosphatidylinositol,and lysophospholipids were also found to NLPs.However,their presence had varying effects on the resulting particle size distribution.These findings suggest that the selection and composition of lipid components can be manipulated to control the size and distribution of NLPs,thus enabling the production of particles with tailored properties for various applications.（4）ALDs have demonstrated the ability to specifically bind proteins and interact with organelles.In vitro experiments revealed that a LDs could bind purified Plin5-GFP protein with high specificity.Silver staining further confirmed the specific and saturable binding of Apo B and Plin5-GFP protein to NLPs,whereas no binding was observed with Seipin.To investigate their interact with organelles,a LDs were incubated with mitochondria extracted from Hep G2 cells for 2 hours.The findings showed that the a LDs could establish contact with mitochondria,indicating their potential for future in vitro studies on organelle interactions.These results suggest that a LDs offer a promising platform for targeted protein delivery and exploration of organelle biology.（5）ALDs can alleviate the negative effects of H₂O₂-induced ROS and excess fatty acid-induced lipotoxicity.Specifically,the a LDs were found to directly reduce the concentration of H₂O₂ and act as a reservoir for fatty acids,effectively reducing the level of ROS in cells that absorbed them.Additionally,the transfer of Cyto protein was significantly attenuated in these cells,indicating a reduction in lipotoxicity.These findings suggest that a LDs may be a promising therapeutic option for the treatment of oxidative stress and lipotoxicity-related disorders.2.Study on the Mechanism of a LDs Enhancing Intramuscular Fat in Pigs（1）The optimal concentration of oleic acid was 60μM.Oleic acid was selected as an agonist for lipid synthesis and a LDs were prepared with different oleic acid concentration gradients.After incubating the cells separately,the lipid content was significantly increased and was positively correlated with the oleic acid concentration.The oleic acid concentration of 20μM caused a significant increase in cellular ROS,but there was no significant difference between the concentrations of 20-80μM.The optimal concentration of oleic acid was selected as 60μM,taking into account cell ROS,proliferation and lipid content.（2）Injection of OA-loaded a LDs significantly increased the Intramuscular Fat（IMF）content of the longest muscles of the pig dorsum.The IMF content increased from 1.5%to2.1%after injection,and significant positive areas were seen in oil red O staining,with linear lipid deposition between muscle fibers.Tissue fluorescence staining revealed the presence of long strips of lipids,while lipid synthases DGAT1,DGAT2 and PPARγwere up-regulated in expression,indicating that lipid synthesis was activated at this site.（3）ALDs are capable to penetrating cells via two distinct pathways:macropinocytosis and clathrin-mediated endocytosis pathway.ALDs were co-incubated with cells,and fluorescence detection revealed that a LDs were taken up by cells.Treatment with inhibitors of these endocytic pathways led to a significantly inhibit the uptake of a LDs.In contrast,treatment with the MβCD inhibitor did not produce a significant effect on the uptake of a LDs.These results suggest that a LDs can enter cells through specific endocytic pathways（4）a LDs and free-oleic acid cannot stimulate lipid synthesis.To understand how a OA stimulate lipid synthesis,we separately tested the functions of a LDs and free oleic acid.After incubating cells with a LDs for 4 hours,the number of cell LDs reached its maximum and gradually decreased thereafter.The expression of lipolytic genes Lipe and Pnpla2 was up-regulated,and the expression of synthetic gene DGAT1/2 was down regulated,indicating that the a LDs could not stimulate their own lipid synthesis.The experiment in the gastrocnemius muscle muscle of mice verified this result.Free oleic acid cannot be absorbed by cells due to its strong hydrophobicity,and there is no significant change in lipid content after injection into the tissue.（5）Oleic acid can only stimulate lipid synthesis by being loaded into a LDs.Neither a LDs nor free oleic acid can increase the lipid content of cells.We tested the function of60μM a OA.The results showed that the expression of LD structural protein Plin2,lipid synthase genes DGAT1 and DGAT2,fatty acid binding protein Fabp4,and pathway gene PPARγwere significant increase which indicates the enhancement of lipid metabolism activity and activation of lipid synthesis pathways.After injection,TG content was significantly increased and greater than the added amount in gastrocnemius muscle,and lipid synthesis related genes and pathways were activated.In this study,a novel method for the rapid preparation of pure a LDs with a monolayer phospholipid envelope structure has been established.The prepared a LDs can be preserved as lyophilized powder for extended periods.Additionally,the study investigated the impact of lipid composition on the properties of a LDs.In vitro assays demonstrated that the a LDs are biologically active,with the ability to specifically bind proteins and interact with organelles.Cell incubation with a LDs resulted in a reduction in H²O²-induced oxidative stress and the alleviation of lipotoxicity produced by exogenous OA/PA stimulation.Furthermore,the study provides a novel approach to increasing intramuscular fat,with the injection of a OA found to stimulate self-lipid synthesis and increase triglyceride content.These findings offer new avenues for the utilization and in-depth study of LDs,as well as the promotion of intramuscular fat deposition in domestic animals.