Lipid Profiling Reveals Browning Heterogeneity of White Adipose Tissue by Β3-Adrenergic Stimulation

Figure 1. A schematic diagram of experimental design.

Figure 2. Adrenergic stimulation-induced browning heterogeneity of white adipose tissue and ameliorated systemic metabolism. C57BL/6J mice were injected intraperitoneally with CL316,243 at the dose of 1 mg/kg/d for 10 days and the general parameters were measured (n = 5). (A) 24-h food intake; (B) 24-h water intake; (C) Fasting blood glucose; (D) Fed blood glucose; (E) Body weight; (F) Fat coefficient of visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT), and brown adipose tissue (BAT) in vehicle and CL316,243-injected mice; (G) The concentration of blood triglyceride (TG) and free fatty acids (FFA); (H) The shell temperature was spotted by a thermal imaging camera purchased from FLIR when mice were under anesthesia and (I) the temperature was analyzed using FLIR tools. All data are presented as mean ± SEM. ** P < 0.01; *** P < 0.001 compared with Vehicle group; (J) qPCR and (K) immunoblotting analysis of UCP1 expression in VAT, SAT, and BAT of mice injected with CL316,243. qPCR data are normalized to TATA box-binding protein (TBP) and presented as mean ± SEM, n = 5. * P < 0.05; ** P < 0.01 compared with Vehicle group; (L) H&E staining and (M) immunohistochemistry staining of…

Figure 3. Total composition of lipid classes in VAT and SAT altered in response to CL316,243 treatment. (A) Score plot of a multivariate principal component analysis (PCA) of the lipids composition of VAT and SAT from CL316,243-treated C57BL/6J mice; (B) Heatmap visualization of the abundance of 17 lipid classes in VAT and SAT from CL316,243-injected mice. In total, 334 lipid species were quantified, and (C) 160 molecular species were significantly changed in VAT, while (D) 56 molecular species were dramatically altered in SAT.

Figure 4. Exposure to CL316,243 differentially affected the abundance of glycerides in VAT and SAT. The total abundance of (A) triacylglycerol (TAG), (B) diacylglycerol (DAG), (C) free fatty acids (FFA), (D) cholesterol (Cho), and cholesteryl ester (CE) were detected. The concentration of significantly changed TAG molecular species in (E) VAT and (F) SAT of mice after CL316,243 injection. The dotted lines separate the raised and lowered species; (G) The amount of significantly altered DAG subclasses in VAT and SAT from vehicle and CL316,243-treated mice; (H) The abundance of dramatically changed molecular species of FFA and CE in VAT and SAT of mice after CL316,243 treatment. Data are presented as mean ± SEM, n = 5. * P<0.05; ** P < 0.01 compared with VAT Vehicle group. # P < 0.05; ## P < 0.01 compared with SAT Vehicle group. (I) qPCR analysis of adipose triglyceride lipase (Atgl), hormone-sensitive lipase (Hsl), and sterol regulatory element-binding protein 1 (Srebp1) gene expression in VAT and SAT of mice injected with CL316,243. qPCR data are normalized to TBP and presented as mean ± SEM, n = 5. *** P < 0.001 compared with VAT Vehicle group.

Figure 5. Adrenergic stimulation boosted the abundance of more glycerophospholipids species in VAT than in SAT. (A) The overall concentration of classical glycerophospholipids was determined. The abundance of significantly varied (B) phosphatidylcholine (PC), and (C) phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylserines (PS), lyso-PC (LPC) species in VAT; (D) The obviously changed molecular species of PC, PE, PI, LPC, and lyso-PS (LPS) in SAT. Data are presented as mean ± SEM, n = 5. * P < 0.05; ** P < 0.01; *** P < 0.001 compared with VAT Vehicle group. # P < 0.05; ## P < 0.01; ### P < 0.001 compared with SAT Vehicle group. (E) qPCR analysis of carnitine palmitoyltransferase 1a (Cpt1a), carnitine palmitoyltransferase 1b (Cpt1b), phosphatidylserine decarboxylase (Pisd), phospholipase D1 (Pld1), phosphatidylserine synthase 1 (Ptdss1), phosphatidylserine synthase 2 (Ptdss2), phosphatidylinositol synthase (Pis), CDP-diacylglycerol synthase 1 (Cds1), and phospholipase A1 member A (Pla1a) gene expression in VAT and SAT of mice injected with CL316,243. qPCR data are normalized to TBP and presented as mean ± SEM, n = 5. ** P < 0.01 compared with VAT…

Figure 6. CL316,243 increased sphingolipids in VAT without changes in SAT. (A) The total concentration of sphingolipids in VAT and SAT were quantified. Concentration of (B) ceramide (Cer), (C) glucosylceramide (GluCer), galactosylceramide (GalCer), and (D) sphingomyelin (SM) species significantly changed in VAT after 10 days of CL316,243 injection; (E) The molecular species of GluCer and SM changed in VAT by CL316,243. All data are presented as mean ± SEM, n = 5. * P < 0.05; ** P < 0.01; *** P < 0.001 compared with VAT Vehicle group. ## P < 0.01 compared with SAT Vehicle group. (F) qPCR analysis of ceramide synthase 2 (Cers2), serine palmitoyltransferase long chain base subunit 1 (Sptlc1), serine palmitoyltransferase long chain base subunit 2 (Sptlc2), serine palmitoyltransferase small subunit A (Sptssa) and serine palmitoyltransferase small subunit B (Sptssb) gene expression in VAT and SAT of mice injected with CL316,243. qPCR data are normalized to TBP and presented as mean ± SEM, n = 5. * P < 0.05 compared with VAT Vehicle group. # P < 0.05 compared with SAT Vehicle group.

Figure 7. Cardiolipin was elevated by CL316,243 in both VAT and SAT. (A) The total content of cardiolipin was significantly altered in both VAT and SAT in response to CL316,243 treatment. In 35 species detected, (B) 27 molecular species were obviously changed in VAT, and 15 species were greatly altered in SAT. Dramatically changed particular species of cardiolipin are illustrated as circles. Size of circle indicates level of significance. All data are presented as mean ± SEM, n = 5. *** P < 0.001 compared with VAT Vehicle group. # P < 0.05 compared with SAT Vehicle group. (C) qPCR analysis of cardiolipin synthase 1 (Crls1) gene expression in VAT and SAT of mice injected with CL316,243. qPCR data are normalized to TBP and presented as mean ± SEM, n=5. ** P < 0.01 compared with VAT Vehicle group.