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Comparative proteome analysis of brown adipose tissue in obese C57BL/6J mice using iTRAQ-coupled 2D LC-MS/MS.

Li J, Zhao WG, Shen ZF, Yuan T, Liu SN, Liu Q, Fu Y, Sun W - PLoS ONE (2015)

Bottom Line: As compared HFD with ND, we obtained 727 differentially expressed proteins.Functional analysis found that those proteins were mainly assigned to the pathway of mitochondrial function.The results indicated that HFD might induce the apoptosis of brown adipocytes via the up-regulated AIF1.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology, Key Laboratory of Endocrinology of Ministry of Health, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China.

ABSTRACT
High-fat diet (HFD) leads to the development of obesity accompanied by insulin resistance, which increases the risk of type 2 diabetes mellitus and cardiovascular disease. Brown adipose tissue (BAT) plays an essential role in energy metabolism, thus it will give us promising treatment targets through elucidating underlying mechanisms of BAT in obesity. In this study, female C57BL/6J mice were fed HFD or normal diet (ND) for 22 weeks. Hyperinsulinemic-euglycemic clamp was performed to evaluate insulin sensitivity, which was independently correlated with obesity. Using isobaric tag for relative and absolute quantification (iTRAQ) coupled with 2D LC-MS/MS, we quantitated 3048 proteins in BAT. As compared HFD with ND, we obtained 727 differentially expressed proteins. Functional analysis found that those proteins were mainly assigned to the pathway of mitochondrial function. In this pathway, carnitine O-palmitoyltransferase 2 (CPT2), uncoupling protein 1 (UCP1) and apoptosis-inducing factor 1 (AIF1) were up-regulated significantly by HFD, and they were confirmed by western blotting. The results indicated that HFD might induce the apoptosis of brown adipocytes via the up-regulated AIF1. Meanwhile, HFD also stimulated fatty acid β-oxidation and raised compensatory energy consuming through the increases of CPT2 and UCP1, respectively. However, the apoptosis of brown adipocytes might weaken the compensatory energy expenditure, and finally contribute to overweight/obesity. So, preventing the apoptosis of brown adipocytes may be the key target to treat obesity.

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Work flow of establishing obese mice model and proteomic analysis of BAT proteins.Female C57BL/6J mice were fed normal diet (ND) or high-fat diet (HFD) start at 6–8 weeks of age. Glucose tolerance test (OGTT) and hyperinsulinemic-euglycemic clamp were applied to assess the insulin sensitivity in mice after 22 weeks. The levels of blood lipid were tested as the auxiliary evaluation indexes of metabolic state. Proteins of BAT in ND and HFD group were quantified via iTRAQ-coupled 2D LC-MS/MS. After comparing the differential expressed proteins between HFD group and ND group, functional analysis of those differential proteins of BAT indicated the important role of BAT in obesity. Finally, western blot analysis was performed to validate the variation of three key differential proteins.
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pone.0119350.g001: Work flow of establishing obese mice model and proteomic analysis of BAT proteins.Female C57BL/6J mice were fed normal diet (ND) or high-fat diet (HFD) start at 6–8 weeks of age. Glucose tolerance test (OGTT) and hyperinsulinemic-euglycemic clamp were applied to assess the insulin sensitivity in mice after 22 weeks. The levels of blood lipid were tested as the auxiliary evaluation indexes of metabolic state. Proteins of BAT in ND and HFD group were quantified via iTRAQ-coupled 2D LC-MS/MS. After comparing the differential expressed proteins between HFD group and ND group, functional analysis of those differential proteins of BAT indicated the important role of BAT in obesity. Finally, western blot analysis was performed to validate the variation of three key differential proteins.

Mentions: In our study, female C57BL/6J mice were fed normal diet (ND) or high-fat diet (HFD) start at 6–8 weeks of age. At 22 weeks, we applied OGTT and hyperinsulinemic-euglycemic clamp to assess glucose regulation states and insulin sensitivity in mice, respectively. Moreover, the levels of blood lipid were tested as evaluation indexes of metabolic state. In order to obtain the global proteome of BAT in obesity, we identified and quantified the expression of total proteins of BAT via iTRAQ-coupled 2D LC-MS/MS. Then we compared the differential expressed proteins between HFD group and ND group in BAT and furthermore, analyzed their functions. Finally, western blot analysis was performed to validate the variation of three key differential proteins (Fig. 1).


Comparative proteome analysis of brown adipose tissue in obese C57BL/6J mice using iTRAQ-coupled 2D LC-MS/MS.

Li J, Zhao WG, Shen ZF, Yuan T, Liu SN, Liu Q, Fu Y, Sun W - PLoS ONE (2015)

Work flow of establishing obese mice model and proteomic analysis of BAT proteins.Female C57BL/6J mice were fed normal diet (ND) or high-fat diet (HFD) start at 6–8 weeks of age. Glucose tolerance test (OGTT) and hyperinsulinemic-euglycemic clamp were applied to assess the insulin sensitivity in mice after 22 weeks. The levels of blood lipid were tested as the auxiliary evaluation indexes of metabolic state. Proteins of BAT in ND and HFD group were quantified via iTRAQ-coupled 2D LC-MS/MS. After comparing the differential expressed proteins between HFD group and ND group, functional analysis of those differential proteins of BAT indicated the important role of BAT in obesity. Finally, western blot analysis was performed to validate the variation of three key differential proteins.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352050&req=5

pone.0119350.g001: Work flow of establishing obese mice model and proteomic analysis of BAT proteins.Female C57BL/6J mice were fed normal diet (ND) or high-fat diet (HFD) start at 6–8 weeks of age. Glucose tolerance test (OGTT) and hyperinsulinemic-euglycemic clamp were applied to assess the insulin sensitivity in mice after 22 weeks. The levels of blood lipid were tested as the auxiliary evaluation indexes of metabolic state. Proteins of BAT in ND and HFD group were quantified via iTRAQ-coupled 2D LC-MS/MS. After comparing the differential expressed proteins between HFD group and ND group, functional analysis of those differential proteins of BAT indicated the important role of BAT in obesity. Finally, western blot analysis was performed to validate the variation of three key differential proteins.
Mentions: In our study, female C57BL/6J mice were fed normal diet (ND) or high-fat diet (HFD) start at 6–8 weeks of age. At 22 weeks, we applied OGTT and hyperinsulinemic-euglycemic clamp to assess glucose regulation states and insulin sensitivity in mice, respectively. Moreover, the levels of blood lipid were tested as evaluation indexes of metabolic state. In order to obtain the global proteome of BAT in obesity, we identified and quantified the expression of total proteins of BAT via iTRAQ-coupled 2D LC-MS/MS. Then we compared the differential expressed proteins between HFD group and ND group in BAT and furthermore, analyzed their functions. Finally, western blot analysis was performed to validate the variation of three key differential proteins (Fig. 1).

Bottom Line: As compared HFD with ND, we obtained 727 differentially expressed proteins.Functional analysis found that those proteins were mainly assigned to the pathway of mitochondrial function.The results indicated that HFD might induce the apoptosis of brown adipocytes via the up-regulated AIF1.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology, Key Laboratory of Endocrinology of Ministry of Health, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China.

ABSTRACT
High-fat diet (HFD) leads to the development of obesity accompanied by insulin resistance, which increases the risk of type 2 diabetes mellitus and cardiovascular disease. Brown adipose tissue (BAT) plays an essential role in energy metabolism, thus it will give us promising treatment targets through elucidating underlying mechanisms of BAT in obesity. In this study, female C57BL/6J mice were fed HFD or normal diet (ND) for 22 weeks. Hyperinsulinemic-euglycemic clamp was performed to evaluate insulin sensitivity, which was independently correlated with obesity. Using isobaric tag for relative and absolute quantification (iTRAQ) coupled with 2D LC-MS/MS, we quantitated 3048 proteins in BAT. As compared HFD with ND, we obtained 727 differentially expressed proteins. Functional analysis found that those proteins were mainly assigned to the pathway of mitochondrial function. In this pathway, carnitine O-palmitoyltransferase 2 (CPT2), uncoupling protein 1 (UCP1) and apoptosis-inducing factor 1 (AIF1) were up-regulated significantly by HFD, and they were confirmed by western blotting. The results indicated that HFD might induce the apoptosis of brown adipocytes via the up-regulated AIF1. Meanwhile, HFD also stimulated fatty acid β-oxidation and raised compensatory energy consuming through the increases of CPT2 and UCP1, respectively. However, the apoptosis of brown adipocytes might weaken the compensatory energy expenditure, and finally contribute to overweight/obesity. So, preventing the apoptosis of brown adipocytes may be the key target to treat obesity.

Show MeSH
Related in: MedlinePlus