Effect Of Apolipoprotein A5on Triglyceride Metabolism In Adipocytes And The Possible Mechanisms Investigation

BackgroundObesity is associated with a number of health problems that are often summarized together as the metabolic syndrome and involves the development of type2diabetes, cardiovascular disease and fatty liver disease. Obesity is characterized by increased storage of triglycerides (TG) in an expanded adipose tissue mass and is a result of imbalance between energy intake and expenditure. Numerous studies demonstrated that apoA5exhibited significant association with the prevalence of obesity and related diseases. However, the underlying mechanisms are not fully understood. Besides its apparent effect on plasma TG levels, an intracellular role of apoA5has been recently indicated by others since apoA5is partially retained in cells, targets to lipid droplets and modulates intrahepatic TG accumulation. Since adipocytes provide the largest storage depot for energy in the form of TG, and represent a potential therapeutic target for obesity, we hypothesized that apoA5may also play a role in modulating TG storage in adipocytes, thereby exerting its beneficial effect against obesity. Interestingly, apoA5has been found to serve as a ligand for two members of low density lipoprotein receptor (LDLR) gene family, namely LDLR-related protein1(LRP1) and the mosaic type Ⅰ receptor SorLA. Both free and lipid-bound apoA5can bind to these two receptors. Importantly, binding and internalization studies in human embryonic kidney cells transfected with SorLA showed that apoA5undergoes receptor-mediated endocytosis. Human adipocytes also express LRP1and very low density lipoprotein receptor which is another LDLR family member. Taken together, we speculated that apoA5might be internalized by adipocytes via binding to LDLR family members, and internalized apoA5may participate in the regulation of adipocyte lipid storage by affecting cellular TG metabolism.ObjectiveThe aims of this study were to investigate whether apoA5can be internalized by human adipocytes, its subcellular localization, and whether apoA5exerts its effect on cellular TG storage and the related mechanisms. MethodsHuman adipose-derived mesenchymal stem cells, derived from subcutaneous adipose tissue of patients undergoing abdominal surgery, were differentiated into mature adipocytes. Differentiated adipocytes were incubated with human recombinant apoA5for indicated time. Then the experiments were performed as follows.(1) Pulse-chase experiments and Western blot analysis were performed to test whether apoA5could be internalized by human adipocytes.(2) Heparin and RAP, both of which prevented the apoA5interaction with members of LDLR family, were also used to investigate whether LDLR family members participated in endocytosis of apoA5.(3) To determine the subcellular localization of internalized apoA5, differentiated adipocytes were incubated with Alexa Fluor488-labeled apoA5. Then oil red-O staining and immunofluorescence to visualize known lipid droplet-associated protein perilipin were performed. Images were captured with a confocal microscope.(4) The TG content of adipocytes was determined after incubation with apoA5.(5) We examined the effect of apoA5on the morphological changes of lipid droplets by oil red-O and DAPI staining.(6) The expression of lipid droplet-associated proteins Cidec and perilipin was determined both by quantitative real-time PCR and Western bolt analysis.(7) The effect of apoA5on lipolysis activity of adipocytes was also determined.(8) We finally examined the effects of apoA5on gene expression related to TG metabolism and brown adipose tissue-specific genes.Results1. Pulse-chase experiments revealed that125I-apoA5was internalized into human adipocytes, and～70%of the125I-apoA5internalized during the pulse remained intracellular within a24hours chase, while30%was degraded. Likewise, the uptake of apoA5by human adipocytes was further evidenced by Western blot analysis.2. Preincubation with heparin or RAP markedly reduced the uptake of125I-apoA5by61%and52%, respectively (P<0.001), which were subsequently confirmed by Western blot analysis.3. Confocal microscopy analysis clearly showed that apoA5was distributed in a ring around the lipid droplets, and co-localized with perilipin on the surface of lipid droplets in adipocytes.4. ApoA5dose-dependently decreased TG content in human adipocytes. Intervention with apoA5for48hours significantly decreased intracellular TG levels by29%(P<0.01).5. ApoA5treatment did not affect the pattern of lipid droplets formation in human adipocytes.6. Treatment of adipocytes with apoA5markedly decreased the expression of the lipid droplet-associated proteins Cidec and perilipin.7. ApoA5-treated adipocytes showed an increase in lipolysis activity and expression of brown fat-specific genes Forkhead box C2and uncoupling protein1which is the molecular effector of thermogenesis in brown adipocytes. However, the amounts of mRNAs for genes related to TG synthesis and hydrolysis, and mitochondrial biogenesis or function did not differ between apoA5-treated and control adipocytes.Conclusions1. ApoA5can be internalized by human adipocytes, localize to lipid droplets, and significantly decrease cellular TG storage. Moreover, LDLR family members play important roles in endocytosis of apoA5by human adipocytes.2. The decreased TG accumulation in human adipocytes induced by apoA5intervention is associated with enhanced lipolysis and energy expenditure which may result from reduced expression of lipid droplet-associated proteins Cidec and perilipin.