Identification of functional lipid metabolism biomarkers of brown adipose tissue aging

Figure 1. Age-related decline of lipid-related metabolism in BAT. (A) Analysis of Ucp1 mRNA in interscapular BAT collected from mice of 2.5, 15, and 25 months of age. (B) Analysis of Ucp1 mRNA in iWAT collected from mice of 2.5, 15, and 25 months of age with (+) and without (−) cold exposure. (C, D) UCP1 protein levels normalized to β-actin in BAT (C) and iWAT (D) collected from young (2.5 months) and old (15 months) mice (quantification: Figs. S1B and S1C). (E) Representative immunoblot image of phosphorylated HSL (p-HSL) and total HSL in BAT-explants isolated from young (2.5 months) and old (25 months) mice after normalization to β-actin (quantification: Fig. S1E). (F) Representative western blot image of phosphorylated GSK3β (p-GSK) and total GSK3β in BAT-explants isolated from young (2.5 months), middle aged (15 months), and old (25 months) mice after normalization to β-actin (quantification: Fig. S1F). (G) Representative immunoblot images of phosphorylated pyruvate dehydrogenase (p-PDH) and total PDH in BAT-explants isolated from young (2.5 months), middle aged (15 months) and old (25 months) mice after normalization to β-actin (quantification: Fig. S1G). (H) Glycerol [μmol/l] was…

Figure 2. Aging results in defective adipose tissue lipid metabolism. (A) Pathway clustering for 18O-proteomic analysis of aged BAT. Detected proteins that were reduced in aged mice were identified based on heavy/light ratios (H/L ratio) < 1 to identify panels of potentially age-sensitive protein clusters that were annotated to appropriate pathways based on KEGG classification. Candidates were chosen from two independent experiments executed as forward and reverse experiments, either labeling proteins from young or aged BAT with 18O. Each sample was generated by pooling protein extracts from five animals per age-group (Table S1). Significantly enriched pathway clusters were identified based on p-values < 0.05 and first twenty pathways are depicted in logarithmic scale (candidate protein number in pathway expressed in binary logarithmic scale [log2]) in which light blue indicates total number of genes in pathway and dark blue indicates number of identified proteins in pathway. (B) Pathway clustering for proteins enriched in aged BAT samples by 18O-labeled proteomic analysis. Proteins were identified based on heavy/light ratios (H/L ratio) > 2.5 and were annotated to appropriate pathways…

Figure 3. Metabolomic analysis of BAT reveals age-related changes of several lipid classes. (A) HeatMap of lipids metabolites with relative intensities determined by LC-MS showing significant correlations in BAT as a function of aging in animals aged 2.5, 5, 10, 15, 21, and 25 months. Correlation coefficients (r) for individual lipids were calculated and lipids showing significant (p < 0.05) correlations to age were grouped into lipid classes, i.e. carnitine-bound fatty acids (Carnit.), phospholipids, sphingolipids including ceramides (Sphing.), triacylglycerols (TG), and triacylglycerols containing very short chain fatty acids (VSCF-TG). Heatmap was generated using ClustVis, elevated relative values are depicted in red, low relative values are depicted in blue (Candidate list and data used for heat map generation are summarized in suppl. Table S5). (B–F) Lipids shown in the heatmap (panel A) were clustered into lipid classes to visualize age-dependent correlations of lipid classes: Very short chain fatty acids containing-TGs (B, Tables S6 and S7, VSCFA-TG; p = 0.0081), carnitine-bound fatty acids (C, Carnitine-FA; p = 0.0109), triacylglycerols (D; p = 0.0505), phospholipids (E; p =…

Figure 4. Targeted lipidomic analysis of ceramides and dolichols as potential biomarkers of BAT-age. (A, B) Targeted mass spectrometric analysis of tissue ceramides using QTOF MS in young (2 months; white bars) and aged (15 months; gray bars) BAT (A, n = 10) or iWAT (B, n = 5). Ceramides were identified by their accurate mass-to-charge ratios (m/z) and characteristic MS/MS fragmentations and quantified based on peak area quantification and normalized to samples' protein content. (C, D) HPLC-based analysis of dolichol subspecies with increasing length of isoprenoid side chains from 16 to 20 subunits in young (2 months; white bars) and aged (15 months; grey bars) BAT (C, n = 5) or iWAT (D, n = 5). Individual dolichols were identified after comparison to standards and quantified based on peak area quantification and normalized to samples' protein content. Data are shown as mean ± SEM; *p < 0.05; **p < 0.01 using two-tailed unpaired t-test.

Figure 5. Ceramides impair adipogenic differentiation of brown pre-adipocytes. (A) mRNA levels of adipocyte specific genes in immortalized brown pre-adipocyte cell line WT1 treated with 10 μM of C16 ceramide (gray bars) or solvent control (white bars) during differentiation. (B) mRNA levels of brown adipogenic genes in immortalized brown pre-adipocyte cell line WT1 treated with solvent control (white bars) or 5 μM of ceramide synthase inhibitor fumonisin B1 (black bars) during adipogenic differentiation. (C, D, E) Primary adipogenic progenitor cells (APCs) were isolated from BAT by flow cytometry. mRNA levels of brown adipocyte marker genes (C), general adipocyte-specific genes (D), and genes of mitochondrial biogenesis (E) in primary APCs treated with control (white bars) or C16 ceramide (10 μM; gray bars) during differentiation. (F, G) Analysis of mitochondrial respiration in immortalized brown WT1 pre-adipocytes differentiated under adipogenic conditions for 10 days comparing control (Ctr; white circles, white bars) to cells treated with C16 ceramide throughout differentiation (C16; gray squares, gray bars). Oxygen consumption rates were recorded (F) and normalized to DAPI staining…

Figure 6. The sphingolipid S1P inhibits brown adipocyte formation and function. (A, B) mRNA levels of Ucp1 (A) and Ppargc1a (B) in primary brown APCs treated with increasing concentrations of S1P during differentiation without (white bars) or after a 4 h treatment with NE (lights gray bars) prior to cell harvest. (C) Comparison of S1pr2 and S1pr4 mRNA expression in APCs isolated from BAT (white bars) and iWAT (black bars) or whole brown and subcutaneous white adipose tissue (Analysis of S1pr1, S1pr3, and S1pr5: Fig. S4). (D) Gene expression analysis in APCs treated with solvent control (white bars) or inhibitors (dark gray bars) targeting S1PR2 (JTE), S1PR4 (Cym), and a combined inhibitor targeting S1PR1 und S1PR3 (VPC) throughout differentiation (all at 1 μM). (E) S1pr2 mRNA expression in undifferentiated APCs (white bars) compared to mature adipocytes (black bars) after 12 days of in vitro differentiation. (F, G) mRNA levels of Ucp1 (F) and Ppargc1a (G) in APCs treated with solvent control (white bars) or increasing concentrations of S1PR2-inhibitor (JTE; gray bars) during differentiation without or with 4 h treatment with NE before harvest. (H) Lipolysis rates as measured by glycerol…

Figure 7. Dolichol metabolism regulates brown adipocyte function. (A, B) Gene expression analysis of enzymes catalyzing dolichol synthesis: HMG-CoA-reductase (Hmgcr), steroid 5 alpha reductase (Srd5a3), dehydrodolichyl diphosphate (Dhdds) and dolichol kinase (Dolk) in BAT (A) and iWAT (B) isolated from young (2.5 months, white bars) and old (15 months, gray bars) mice. (C) Dolk mRNA expression in Dolk-overexpressing immortalized WT1 pre-adipocytes (black bars) differentiated for ten days compared to control cells (Ctr, white bars). (D) DOLK protein detection in undifferentiated cells stably overexpressing Dolk or control cells (Ctr) (upper panel). Quantification of signal intensity of three independent blots with normalization to β-actin (lower panel) of control cells (white bar) compared to Dolk-overexpression (black bars). (E) Oil Red O staining (upper panel, magnifications in Fig. S5A) and quantification after staining of mature control adipocytes (left; white bar) or cells with Dolk-overexpression (right; black bar) differentiated for 10 days. (F) Ucp1 mRNA in Dolk-overexpressing pre-adipocytes (black bars) differentiated 10 days compared to control (white bars) without (basal) or with…