Effect Of Apolipoprotein A-I On Adipocyte Phenotype And Autophagy

Background:Obesity and its associated metabolic disorders are risk factors for cardiovascular diseases such as atherosclerosis and hypertension. Epidemiological studies have shown a strong inverse correlation between plasma apolipoprotein A-I (apoA-I) levels and obesity, which was originally attributed to the disturbed metabolism of apoA-I in obese status. Intriguingly, recent studies showed that apoA-I transgenic mice and mice with intra-abdominal injection of apoA-I mimetic peptides were resistant to genetic and diet induced obesity. However, the anti-obesity mechanism of apoA-I is not clear. Certain stimulus such as exposure to cold and (3-adrenergic agonists could induce the appearance of "brown-like adipocyte" in traditional white adipose tissue. Genetic or pharmacological induction of brown phenotype exhibited an anti-obesity effect in animal models. Autophagy is an intracellular degradation pathway. Adipose-specific inhibition of autophagy lead to "browning of WAT" and a lean phenotype. ApoA-I is well known to affect AMPK and PI3K-Akt signaling pathways that are involved in autophagy regulation. However, there is no direct evidence showing that apoA-I modulate autophagy. Recently, it has been reported that apoA-I could increase brown adipose marker gene UCP1expression in scapular regions. Therefore, we speculated that apoA-I may reduce adiposity through modulation of adipocyte autophagy and induction of a "brown-like" phenotype.Objective:The aim of this study is to explore the effect of apoA-I on adipocyte autophagy and phenotype modulation as well as its possible mechanism.Methods:3T3-L1preadipocytes were induced to differentiate into mature adipocytes. ApoA-I was incubated with adipocyte during differentiation. Then following experiments were performed:(1) Oil-red O staining and expression of adipocyte differentiation markers was analyzed by real-time PCR to study the effect of apoA-I on adipocyte differentiation.(2) In order to study the effect of apoA-I on adipocyte morphology, transmission electron microscopy and confocal microscopy were used to visualize mitochondria and lipid droplet.(3) Real-time PCR was used to analyze the expression of thermogenic genes.(4) We examined the effect of apoA-I on TG metabolism by analysis of intracellular TG concentration and supernatant glycerol. Expression of genes related to TG synthesis, lipolysis and fatty acid P-oxidation were also examined through real-time PCR.(5) We examined the effect of apoA-I on autophagy through analysis of the expression of autophagy related protein LC3and Beclinl. Immunofluorescence was also used to detect the LC3dot distribution.(6) To study the effect of apoA-I on UCP1expression, UCP1expression was measured during the process of adipogenesis by real-time PCR.(7) In order to study whether the effect of apoA-I on autophagy and phenotype alteration is dependent on mTORC1signaling, phosphorylation of mTOR, p70S6K and4EBP1was analyzed by western blot with or without mTOR inhibitor rapamycin.Results:1. ApoA-I reduced genes expression of adipocyte differentiation markers:PPAR-y, C/EBP-a and aP2.2. ApoA-I increased cellular mitochondria without inducing obvious morphological changes of lipid droplet.3. ApoA-I significantly elevated expression of thermogenic genes UCP1, PGC-1α, Cox7a and Cox8b.4. ApoA-I reduced cellular TG by24%(P<0.05). TG lipolysis activity was not affected by apoA-I as measured by supernatant glycerol concentration. ApoA-I increased expression of CPT1which is related to fatty acid β-oxidation. However, apoA-I didn’t affect genes expression involved in TG synthesis and lipolysis.5. The expression of autophagy related protein beclinl and LC3was increased during the process of adipocyte differentiation, which was inhibited by apoA-I. 6. A temporary increased expression of UCP1was detected during adipogenesis. ApoA-Ⅰ reduced the decrease of UCP1expression during adipocyte differentiation.7. ApoA-Ⅰ increased phosphorylation of mTOC1catalytic subunit mTOR and the substrate of mTORC1, p70S6K and4EBP1, which indicated activated mTORC1signaling. However, the effect of apoA-Ⅰ on expression of beclinl and LC3was not influenced after blocking mTORC1signaling by rapamycin.Conclusions:1. ApoA-Ⅰ promoted a brown-like phenotype and reduced TG accumulation during adipocyte differentiation.2. ApoA-Ⅰ reduced increased autophagic activity during adipogenesis, promoted UCP1expression and increased the amount of adipocyte mitochonria.3. ApoA-Ⅰ could activate mTORC1signaling. However, the effect of apoA-Ⅰ on beclin-1and UCP1expression was not dependent on mTORC1signaling.