Molecular Mechanisms Of Zeaxanthin On Lipid Metabolic Regulation Through AMPK Pathway

With the continuous improvement of human living standards,the proportion of fat in dietary structure is increasing.The long-term high-fat diet intake leads to an imbalance of energy metabolism resulting in excessive lipid accumulation and sharply rising overweight and obesity population.At present,among 1.9 billion overweight people in the world,almost 650 million are obese.The overweight population in China has been almost 300 million,and obese people has reached 89.6 million,ranking first in the world.Overweight and obesity usually result in a variety of metabolic diseases such as hyperlipidemia,fatty liver,hyperglycemia,hypertension,and cardiovascular and cerebrovascular diseases.AMPK signaling pathway,as an energy receptor of cells,plays an important role in the regulation of lipid metabolism and energy metabolism.At present,drugs for treatment of obesity have obvious toxic side effects,thus,the development of safety and natural components for prevention and treatment of obesity attracting more attention.Zeaxanthin is a major carotenoid in cereals,especially yellow corn,which has strong antioxidant activity.Previous studies have reported that zeaxanthin can alleviate lipid-induced oxidative damage in quail and inhibit lipid accumulation in diet-induced obese mice,but the specific mechanism is unclear.In this dissertation,high-fat diet induced obese C57BL/6J mice and 3T3-L1 preadipocyte were used to analyze the regulation of zeaxanthin on lipid metabolism and white adipocyte browning.The molecular mechanism of zeaxanthin on lipid metabolism regulation based on AMPK pathway was elucidated.The research contents and results are as follows:1.Anti-obesity effect of zeaxanthin on high-fat diet induced obese mice.Establishment of obese C57BL/6J mice model induced by high-fat diet and intervention with zeaxanthin at doses of 20 mg/kg and 40 mg/kg for 4 weeks.The results showed that zeaxanthin significantly reduced the body weight gain and epididymal adipocytes size,and improved dyslipidemia by reduced the level of low density lipoprotein(LDL),free fatty acid(FFA),and increased the level of high density lipoprotein(HDL).Zeaxanthin significantly decreased the expression of adipogenic transcription factors Pparg,C/ebpa,Srebp1-c and lipid metabolism related genes Fas,Fabp4 and perilipin in white adipose tissue,significantly decreased the expression of PPAR? protein and increased the phosphorylation level of AMPK and ACC,significantly activated AMPK signaling pathway.Zeaxanthin can regulate the expression of lipid metabolism related factors by activating AMPK pathway in white adipose tissue,result in inhibiting the excessive accumulation of lipid in vivo.2.The effect of zeaxanthin on adipogenic differentiation in 3T3-L1 preadipocytes.Different concentrations of zeaxanthin were supplemented during preadipocytes differentiation.MTT assay showed that zeaxanthin had no toxic effect on cell proliferation at 5-15 ?M concentration,which was selected for subsequent experiments.Zeaxanthin reduced intracellular triglyceride content and inhibited preadipocyte adipogenesis in a dose-dependent manner,significantly downregulated the expression of adipogenic transcription factors Pparg,C/ebpa,Srebp1-c,Fas,Fabp4,perilipin and Acc,significantly increased the levels of p-AMPK?/AMPK? and p-ACC/ACC,then activated AMPK signaling pathway.The results showed that zeaxanthin inhibited adipogenic differentiation of preadipocytes,regulated the expression of lipid metabolism related factors and reduced lipid accumulation by activating AMPK pathway in adipocytes.The results in vitro are agreement with those in vitro.3.The effect of zeaxanthin on white adipocytes browning.Nile red fluorescence staining showed that zeaxanthin significantly reduced the volume of lipid droplets,and promoted the lipid droplets arranged with multi-chambers.It significantly increased the expression of beige adipocytes markers Cd137,Tbx1 and Tmem26,and induced adipocytes browning.Zeaxanthin significantly increased the expression of browning-related factors Ucp1,Ppargc1 a,Sirt1,Prdm16,Ppara,Cidea,Fgf21,Camkk2,Foxo1,Lkb1,Zic 1 and Lhx8,and key browning proteins UCP1,SIRT 1 and PGC-1a in browning adipocytes.The results showed that zeaxanthin could reduce lipid accumulation by promoted adipocytes browning,induce the formation of beige adipocytes,thus increase energy metabolism.4.The effect of zeaxanthin on promoting lipid metabolism and inducing adipocytes browning based on AMPK.Compound C inhibitor used to block AMPK signaling pathway.The results showed that the inhibitor could significantly inhibit the phosphorylation of AMPK after 24 h.Zeaxanthin significantly weakened its inhibition of lipid accumulation,also impaired the regulation of lipid metabolism related factors(Cd36,Acc,Fabp4)and key browning factors(Ppargc1a,Ucp1,Sirt1)under the intervention of AMPK inhibitors.The results indicating that zeaxanthin regulated lipid metabolism and induced browning in adipocytes depended on AMPK signaling pathway activation.Using siRNA transfection respectively silenced AMPK?1 and AMPK?2 targets,zeaxanthin significantly weakened the regulation of lipid metabolism,browning-related genes and proteins in Prkaa 1 gene silenced cells,suggesting that AMPK alpha 1 is a key target for zeaxanthin to inhibit adipogenesis and induce white adipocytes browning.5.Effect of zeaxanthin on mitochondrial biosynthesis in adipocytes.Zeaxanthin significantly increased the number of mitochondria and mitochondrial copies in beige adipocytes,increased the expression of mitochondrial biosynthesis-related genes including Nampt,Tfam and Nrf1,increased the levels of endogenous antioxidant enzymes including superoxide dismutase(SOD),glutathione(GSH)and glutathione peroxidase(GSH-PX),and decreased lipid peroxide malondialdehyde(MDA)and reactive oxygen species(ROS)accumulation.Fluorescence probe staining showed that zeaxanthin significantly increased mitochondrial membrane potential and decreased mitochondrial peroxide levels.These results suggest that zeaxanthin can alleviate oxidative stress in mitochondria,enhance mitochondrial biosynthesis and promote browning of adipocytes.