| Objective: Obesity is a global health problem.It was reported that maternal cigarette smoke exposure increased obesity susceptibility in adult offspring.Adipose tissue participants in obesity.Current researches focused on the effect of tobacco or nicotine exposure during gestation or lactation on the white adipose tissue in offspring,while the effect on the brown adipose tissue(BAT)in offspring is less concerned about.Our study focused on the morphological and functional changes of BAT in offspring,who exposed to nicotine during gestation and lactation,and its obesity susceptibility,and study on its specific mechanism.At the same time,in vitro,the effect of nicotine on the function related factors of BAT was studied to clarify the underlying mechanism of nicotine on brown adipocyte,and to reveal the mechanisms of nicotine exposure induced obesity from the perspective of the “whitening of brown fat”.Methods: Pregnant rats in nicotine group were subcutaneously administered with nicotine(2.0 mg/kg/day)from gestational day 9 to weaning,and rats in control group were administered with the same volume of vehicle saline.The offspring were killed at 4 and 26 weeks respectively.BAT was collected to detect related indicators.Offspring’s body weight and BAT weight were detected.The morphological changes of BAT were analyzed by HE and transmission electron microscope.The expression of BAT marker protein uncoupling protein 1(UCP1)and vascular marker protein CD34 were observed by immunohistochemistry.Real-time quantitative PCR and Western blot were used to detect the expression of thermogenesis and angiogenesis related pathways genes.In vitro,different doses of nicotine(50n M~50μM)were added into the differentiation medium of C3H10T1/2 cells on day 0 to 8.Results: 1)Male offspring:(1)Body weight and BAT weight: body weight of offspring in the nicotine group was significantly increased from 4 to 26 weeks of age(P<0.05,P<0.01),there was no significant difference in BAT weight and the percentage of the ratio of BAT weight to body weight at 4 weeks of age.At 26 weeks of age,the weight of BAT was heavier in nicotine-exposed offspring(P<0.05),and no significant difference was observed in the percentage of the ratio of BAT weight to body weight.(2)Morphological indicators: at 4 weeks of age,there was no significant difference in the size and number of lipid drops and the cristae of mitochondria;At 26 weeks of age,brown adipocytes in nicotine-exposed offspring presented larger lipid droplets,the number of mitochondria reduced,and impaired mitochondria was observed with a randomly oriented and fractured cristae.At 4 weeks of age,there was no significant difference in the positive area of UCP1 protein.At 26 weeks of age,the positive area of UCP1 protein was significantly decreased(P<0.01)in the nicotine group.No significant difference was observed in the positive area of CD34 protein in both 4 and 26 weeks of age.(3)Gene expression of thermogenic genes: at 4 weeks of age,the gene expression in the nicotine group of UCP1,cytochrome C1(Cyc1)and carnitine palmityltransferase II(Cpt2)showed a decreased trend(P=0.067,P=0.052,P=0.057);At 26 weeks of age,the gene expression of Cidea,UCP1,citrate synthase(Cs),Cyc1 and Cpt2 were decreased in BAT(P<0.01,P<0.05),PR domain-containing 16(Prdm16)had a decreased trend(P=0.059).(4)Gene and protein expression of AMPK-SIRT1-PGC-1α pathway: at 4 weeks of age,the gene expression of PGC-1α was significantly decreased in nicotine-exposed offspring(P<0.05)and the protein expression of PGC-1α had a decreased trend(P=0.086).At 26 weeks of age,the genes and proteins expression of SIRT1,PGC-1α and UCP1 were decreased in BAT of nicotine-exposed male offspring(P<0.05,P<0.01),the phosphorylation level of AMPKα protein was decreased in BAT(P<0.05).(5)Gene expression of angiogenesis factors: at 4 weeks of age,there was no significant difference in angiogenesis related genes.At 26 weeks of age,the gene expression of Mmp2 was decreased in the nicotine group(P<0.05).2)Female offspring:(1)Body weight and BAT weight: body weight of female offspring was significant increased from 6 to 26 weeks of age in the nicotine group(P<0.01,P<0.05),but there was no significant difference in BAT weight and the percentage of BAT weight/body weight.At 26 weeks of age,the weight of BAT was heavier in nicotine-exposed offspring(P<0.05),and the percentage of the ratio of BAT weight to body weight in nicotine group had an increased trend.(2)Morphological indicators: at 4 weeks of age,there was no significant difference in the size and number of lipid drops and the cristae of mitochondria.At 26 weeks of age,typical brown adipocytes were observed in control offspring,but brown adipocytes from nicotineexposed offspring presented larger lipid droplets,the number of mitochondria was reduced,and the cristae of mitochondria tended to be randomly oriented.At 4 weeks of age,there was no significant difference in the positive area of UCP1 protein.At 26 weeks of age,the positive area of UCP1 protein was significantly decreased(P<0.05).At 4 and 26 weeks of age,the positive area of vascular marker CD34 protein was decreased(P<0.01,P<0.05).(3)Gene expression of BAT thermogenic genes: at 4 weeks of age in nicotine-exposed offspring,the expression of thermogenic genes showed no significant change.At 26 weeks of age,the gene expression of Prdm16,Cidea and UCP1 were decreased in BAT of nicotine-exposed offspring(P<0.05),Cs had a decreased trend(P=0.072).(4)Gene and protein expression of AMPK-SIRT1-PGC-1α pathway: at 4 weeks of age,there was no significant difference in the expression of pathway.At 26 weeks of age in nicotine-exposed offspring,the gene and protein expression of SIRT1,PGC-1α and UCP1 were decreased in BAT(P<0.01,P<0.05),and the phosphorylation level of AMPKα protein was decreased(P<0.05).(5)Gene expression of angiogenesis genes: at 4 weeks of age,the gene expression of vascular endothelial growth factor receptor 2(Vegfr2),Vegfa,hepatocyte growth factor(Hgf)and Npy were decreased(P<0 05),while the gene expression of Mmp2 and Resistin showed a decreased trend(P=0.078,P=0 070).At 26 weeks of age,the gene expression of Vegfa,Mmp2,platelet derived growth factor(Pdgf)had a decreased trend(P=0.057,P=0.068,P=0.054). 3)Cell experiment:(1)Cell viability: 0~50 μM nicotine did not affect cell viability.(2)Gene expression of thermogenic genes: the gene expression of Cs,Cyc1,Cpt2 and Prdm16 were decreased in 50 μM nicotine group(P<0.05,P<0.01).(3)Gene and protein expression of AMPK-SIRT1-PGC-1α pathway: the gene expression of SIRT1,PGC-1α and UCP1 were decreased in 50 μM nicotine group(P<0.05).No significant difference in the gene expression of AMPKα in 50 μM nicotine group,and the protein phosphorylation level of AMPKα and the protein expression of SIRT1,PGC-1α and UCP1 were decreased in 50 μM nicotine group(P<0.01,P<0.05).(4)Gene expression of angiogenesis factors: compared with the control,the gene expression of Vegfa,Hgf,Mmp2 and Prdm16 were significantly decreased in 50 μM nicotine group(P<0.05),and the gene expression of Angiopoietin 2(Ang2)showed a decreased trend(P=0.058).Conclusion: Gestation and lactation nicotine exposure could lead to the “whitening” of BAT,and increase the susceptibility of obesity in offspring,and its potential mechanisms are as follow: on the one hand,nicotine downregulates the AMPK-SIRT1-PGC-1α signaling pathway,which can lead to an impaired thermogenic function of BAT,further to promote the “whitening” of BAT;on the other hand,nicotine may also decrease the expression of angiogenesis factors and decrease angiogenesis,to promote the “whitening” of BAT in offspring,which lead to adult obesity susceptibility. |
PDF Full Text Request