Fig 1.
Characterization of eWAT, iWAT, and BAT derived adipocytes.
(A) Expression profile of genes that have been linked previously to white or brown adipocytes, WAT or BAT, or AT browning. Shown are means ± SD of 3 mice; annotation of “a, b” indicates that group "a" is statistically different from "b"; annotation of “a, b, c” indicates that all 3 groups are significantly different from each other; significant difference was tested using a one-way ANOVA (Post Hoc: Tukey Test; p < 0.05). (B–D) Representative images show eWAT, iWAT, and BAT derived adipocytes; the “bright dots” are lipid droplets; images are scaled equally; the red line equals 100 μm. The underlying data of (A) can be found in S1 Data. BAT, brown adipose tissue; iBAT, intrascapular brown adipose tissue; eWAT, epididymal white adipose tissue; iWAT, inguinal white adipose tissue; WAT, white adipose tissue.
Fig 2.
Lipid class composition of white, brite, and brown adipocytes.
(A) Common membrane lipids, (B) PE/PE P, (C) GPL/FC, (D) GPL/SL, (E) CL, (F) TG, (G) DG, (H) CE. Shown are means ± SD of 3 independent experiments, each performed in triplicates with AT pooled from 3 mice; annotation of “a, b” indicates that group "a" is statistically different from "b"; annotation of “a, b, c” indicates that all 3 groups are significantly different from each other; significant difference was tested using a one-way ANOVA (Post Hoc: Tukey Test; p < 0.05). The underlying data of (A–H) can be found in S1 Data. CE, cholesterylester; Cer, ceramide; CL, cardiolipin; DG, diacylglycerol; FC, free cholesterol; GPL, glycerophospholipid; HexCer, hexosylceramide; LPC, lyso-PC; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PE P, PE-based plasmalogen; PI, phosphatidylinositol; PS, phosphatidylserine; SL, sphingolipid; SM, sphingomyelin; TG, triacylglycerol.
Fig 3.
Lipid species profiles of major GPL from white, brite, and brown adipocytes.
(A) PC, (B) PE, (C) PI, (D) CL. Panels are (I) species composition, (II) chain length as the number of carbons in the sum of FA moieties, and (III) saturation as the number of double bonds in the sum of FA moieties. Shown are means ± SD of 3 independent experiments, each performed in triplicates with AT pooled from 3 mice; annotation of “a, b” indicates that group "a" is statistically different from "b"; annotation of “a, b, c” indicates that all 3 groups are significantly different from each other; significant difference was tested using a one-way ANOVA (Post Hoc: Tukey Test; p < 0.05). The underlying data of (A–D) can be found in S1 Data. AT, adipose tissue; CL, cardiolipin; FA, fatty acid; GPL, glycerophospholipid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol.
Fig 4.
PS (GPL), Cer and HexCer (SL), TG and DG (GL) species composition of white, brite, and brown adipocytes; Cer (SL) profile of BAT at different browning degrees.
(A) PS, (B) Cer in adipocytes; (C) Cer in BAT samples from mice housed for 7 days at 4°C, 23°C, or 30°C; (D) HexCer, (E) TG, (F) DG in adipocytes. Panels show (I) species composition, (II) chain length as the number of carbons in the sum of FA moieties, (III) saturation as the number of double bonds in the sum of FA moieties. For adipocytes (A–B, D–F) means ± SD of 3 independent experiments are shown, each performed in triplicates with AT pooled from 3 mice; for BAT (C), the means ± SD of n = 9 (4°C), n = 9 (23°C), and n = 12 (30°C) mice are shown; annotation of “a, b” indicates that group "a" is statistically different from "b"; annotation of “a, b, c” indicates that all 3 groups are significantly different from each other; significant difference was tested using a one-way ANOVA (Post Hoc: Tukey Test; p < 0.05). The underlying data of (A–F) can be found in S1 Data. Cer, ceramide; BAT, brown adipose tissue; DG, diacylglycerol; FA, fatty acid; GL, glycerolipid; GPL, glycerophospholipid; HexCer, hexosylceramide; PS, phosphatidylserine; SL, sphingolipid; TG, triacylglycerol.
Fig 5.
Effects of β-adrenergic stimulation in brite adipocytes.
(A) LPC, (B) LPC/PC, (C) LPC composition, (D) LPC distribution, (E) LPC 16:0, (F) LPC 18:0, (G) LPC 16:1, (H) LPC 18:1; primary cells were treated with 0.5 μM ISO for 1 hour, 2 hours, or 4 hours. (I) U-13C-palmitate to–palmitoleate desaturation, (J) U-13C-palmitate to–stearate elongation; primary cells were simultaneously treated with 0.5 μM ISO and 100 μM U-13C-pamitate for 1 hour. (K) LPC, (L) LPC/PC; primary cells originating from UCP1 KO mice and WT littermates stimulated for 1 hour with 0.5 μM ISO. (M) D9-PC (N) D9-PC/total PC, (O) D9-PC composition, (P) D9-PC distribution, (Q) D9-PC 32:0, (R) D9-PC O-32:0; cells were simultaneously incubated with D9-choline and 0.5 μM ISO for 1 hour. Shown are means ± SD of 3 mice, the p-value indicates a significant difference between the treatment groups “Co” and “ISO”; annotation of “a, b” indicates that group "a" is statistically different from "b," which was determined for panels (A–B) and (E–H) using a two-way ANOVA (Post Hoc: Tukey Test) and was determined for (C–D) and (I–R) using a Student t test. The underlying data of (A–R) can be found in S1 Data. Co, control; ISO, isoproterenol; KO, knock out; LPC, lyso-PC; MUFA, monosaturated fatty acid; PC, phosphatidylcholine; PUFA, polyunsaturated fatty acid; SAFA, saturated fatty acid; UCP, uncoupling protein; WT, wild type.
Fig 6.
Mitochondrial bioenergetics in brown adipocytes after treatment with LPC 16:0 or BEL.
(A) LDH release after stimulation with 5 μM and 25 μM LPC 16:0 for 1 hour. Shown are means ± SD of triplicates with AT pooled from 3 mice. (B) Mitochondrial bioenergetics profile (OCR in pmol O2/min), including basal respiration, basal leak respiration, UCP1-mediated leak respiration, maximal oxygen consumption, and nonmitochondrial respiration of primary brown adipocytes after stimulation with 5 μM and 25 μM LPC 16:0 for 1 h. Shown are means ± SEM of n = 7 (Co, 5 μM LPC) and n = 8 (25 μM LPC) with AT pooled from 3 mice. Mitochondrial bioenergetics—(C) OCR shown in pmol O2/min and (D) OCR baselined to basal leak respiration—of brown adipocytes pretreated with 5 μM BEL for 1 hour. Shown are means ± SEM of n = 14 (Co) and n = 13 (BEL) with AT pooled from n = 3 mice. The underlying data of (A–D) can be found in S1 Data. Anti A, antimycin A; AT, adipose tissue; BEL, bromoenollactone; Co, control; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; ISO, isoproterenol; LDH, lactate dehydrogenase; LPC, lyso-PC; Mito,; OCR, oxygen consumption rate; Oligo, oligomycin; PLA2, phospholipase A2; UCP, uncoupling protein.