Regulation Of Adipocyte Insulin Sensitivity By Macrophage-derived Exosomes

The prevalence of obesity in developed countries leads to increased risk of many diseases including cardiovascular disease,metabolic syndrome,type 2 diabetes and some types of cancers.Adipose tissue(AT)was once thought to be an inert mass whose sole function was the storage of fat.However,it is now recognized that adipose tissue is an active endocrine organ that secretes numerous adipokines,cytokines and chemokines.With the onset of obesity,adipocytes become hypertrophic and store lipid with reduced efficiency.Simultaneously,adipose tissue is infiltrated by immune cells,most of which are adipose tissue macrophages(ATMs).ATMs exert inflammatory actions that can contribute to the adverse effects of obesity on local and systemic metabolism.Inflammatory pathways are activated in tissues of obese animals and humans and play an important role in obesity-associated insulin resistance.However,the origin of inflammation during obesity and the underlying molecular mechanisms are not fully understood.Macrophages,which are widely distributed in lymphoid and hematopoietic tissues and organs,is also able to eliminate foreign bodies through phagocytosis.Lipopolysaccharide(LPS),an endotoxin,is the component of cell wall of gram-negative bacteria.LPS can cause inflammatory reaction in immune cells.LPS is toxic when it is released from death bacteria.LPS is a gram-negative bacterium pathogen-associated molecular pattern(PAMP)and could interactions with macrophage surface pattern recognition receptor(PRR)to initiate innate immunity.Upon recognition,macrophage will release a variety of inflammatory factors and kill bacteria and could trigger autophagy.CD36 is a very important PRR,expressed in macrophages.It is still unclear how CD36 affects autophagy.Originally thought that autophagy is a nonspecific process,but in fact autophagosomes are selective for bacteria carried to the lysosomal for degradation and plays an important role in cellular antibacterial reactions.But the specific mechanism remains unclear.Exosomes are small membranous vesicles diameter of 40 ~ 100 nm whose density is 1.10 ~ 1.18 g/ml.It is generated by inward budding of late endosomes,which resulting in the formation of multi vesicular bodies in the cell cytosol.In general,exosomes act as molecular carriers during immune cell-cell communication.Exosomes can be derived from macrophages play pivotal roles in both physiological crosstalk between cells and disease pathogenesis.In order to explore the relationship between obesity and inflammation,we examined the roles of exosome.We used LPS treated macrophages to stimulate the low-grade inflammation in adipocytes.In this case,we compared the mass and contend of exosomes derived from LPS treated and non treated macrophages.It was found that LPS treated macrophages released more exosomes.And mature mouse fibroblast cells(3T3-L1 cells)became insulin resistance(IR)upon treated with pro-inflammatory macrophages derived exosomes.Furthermore,we used Western blot(WB),real-time fluorescence quantitative PCR(Q-PCR),and other methods to explored the molecular mechanism underling the exosome induced IR in 3T3-L1 cells.Our study also found that inhibiting the process of autophagy,exosomes from proinflammatory macrophages cannot cause insulin resistance in 3T3-L1 cells.In order to explore the relationship between autophagy and exosome we first investigated the influence of LPS on RAW264.7 cell lines and the influence of autophagy.RAW264.7 cells were stimulated by LPS and the effect on autophagy were explored by Western blot.Further,we explored the molecular mechanism of LPS-induced autophagy in macrophages.Mitogen-activated Protein Kinase(MAPK)signaling pathway has an important role on the cellular immunity of pathogens.So,we investigate the influence of LPS on macrophage MAPK signaling pathways.We used Extracellular regulated protein kinase(ERK),c-Jun N-terminal kinase(JNK)kinase corresponding inhibitors to test the effect of MAPK signaling pathways on regulation of autophagy affected by LPS.Chinese hamster ovary(CHO)cells express no CD36,but have the capability to over express exogenous proteins.Therefore,we used the immunofluorescence to examine autophagy activity and Western blot to compare the MAPK signaling pathways in three stably transfected cell lines including CHO cells transfected with vector plasmid(CHO/Vector),upon stimulating by LPS to investigate the effect of CD36 on autophagy.The main research results are:(1)LPS-treated macrophages released more exosomes,and 3T3-L1 adipocytes became insulin resistance(IR),upon treated with pro-inflammatory macrophages derived exosomes.(2)After the process of autophagy being inhibited,exosomes from pro-inflammatory macrophages could not cause insulin resistance in 3T3-L1 cells.(3)MAPK pathway has an important role in LPS-induced autophagy in macrophages,especially the ERK and JNK pathways.(4)After LPS stimulation,it was discovered that CD36 could activate ERK and JNK,which was essential for LPS induced autophagy process in CHO cell.