Identification of functional lipid metabolism biomarkers of brown adipose tissue aging

Abstract

Objective: Aging is accompanied by loss of brown adipocytes and a decline in their thermogenic potential, which may exacerbate the development of adiposity and other metabolic disorders. Presently, only limited evidence exists describing the molecular alterations leading to impaired brown adipogenesis with aging and the contribution of these processes to changes of systemic energy metabolism.

Methods: Samples of young and aged murine brown and white adipose tissue were used to compare age-related changes of brown adipogenic gene expression and thermogenesis-related lipid mobilization. To identify potential markers of brown adipose tissue aging, non-targeted proteomic and metabolomic as well as targeted lipid analyses were conducted on young and aged tissue samples. Subsequently, the effects of several candidate lipid classes on brown adipocyte function were examined.

Results: Corroborating previous reports of reduced expression of uncoupling protein-1, we observe impaired signaling required for lipid mobilization in aged brown fat after adrenergic stimulation. Omics analyses additionally confirm the age-related impairment of lipid homeostasis and reveal the accumulation of specific lipid classes, including certain sphingolipids, ceramides, and dolichols in aged brown fat. While ceramides as well as enzymes of dolichol metabolism inhibit brown adipogenesis, inhibition of sphingosine 1-phosphate receptor 2 induces brown adipocyte differentiation.

Conclusions: Our functional analyses show that changes in specific lipid species, as observed during aging, may contribute to reduced thermogenic potential. They thus uncover potential biomarkers of aging as well as molecular mechanisms that could contribute to the degradation of brown adipocytes, thereby providing potential treatment strategies of age-related metabolic conditions.

Keywords: Aging; Brown adipose tissue; Ceramides; Dolichol lipids; Sphingolipids.

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 measured in supernatant of adipose tissue explants using a photometric assay. Explants were treated for 2 h either with 10 μM norepinephrine (NE) or isoproterenol (Iso). The glycerol amount was normalized to explant protein content. Data are shown as mean ± SEM, n = 3–5; *p < 0.05; **p < 0.01; ***p < 0.001 using two tailed unpaired t-test or two way ANOVA.

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 based on KEGG classification. Candidates were chosen from two independent experiments executed as forward and reverse experiment, 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 pathways were identified based on p-values < 0.05 and depicted in logarithmic scale (candidate protein number in pathway expressed in binary logarithmic scale [log2]) in which light red indicates total number of genes in pathway and dark red indicates number of identified proteins in pathway. (C, D) Gene ontology (GO) cluster enrichment analysis was carried out to group redundant GO terms using the top twenty significantly reduced (C) or enriched (D) GO terms using the REVIGO web server. Bubble size and color define frequency (log value) of the GO term and significance levels, respectively (for GO Term list see Tables S2 and S3). Semantic space describes the relatedness degree between two annotation entities. Annotated clusters based on GO term grouping are listed in tables on right. (E) Clusterogram of downregulated individual and overlapping proteins of the different GO pathways listed in panel A (for explanation of gene name abbreviations see Table S4A). Blue arrows indicate candidates belonging to the lipid homeostasis cluster (pathways of fatty acid metabolism and degradation) as well as mitochondrial genes. (F) Clusterogram of upregulated individual and overlapping proteins of the different GO pathways listed in panel B (for explanation of gene name abbreviations see Table S4B). Red arrows indicate candidates belonging to the myogenic gene cluster.

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 = 0.0010) and sphingolipids (F; p = 0.0084). For panels B–F, the average correlation values of all individual candidates' correlations with age were calculated. To this end, individual peak intensities of all age groups were normalized to intensities measured in young control group (age: 2.5 months). Data are depicted as mean ± SEM (n = 3 group); *p < 0.05; **p < 0.01 using two tailed unpaired t-test.

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 (relative fluorescence units, R.F.U.) for quantification (G; n = 6). Basal respiration rate measurements were followed by administration of oligomycin (after third time point) to determine ATP-linked (coupled) respiration. Maximal respiration capacities were measured after FCCP-administration (after sixth time point) and non-mitochondrial respiration was measured by adding rotenone and antimycin A (after ninth time point). Uncoupled, e.g. potentially UCP1-dependent proton leak, was calculated from the relative differences of respiration rates after oligomycin and rotenone/antimycin A. (H, I, J) Primary adipogenic progenitor cells (APCs) were isolated from BAT by flow cytometry. mRNA levels of genes related to brown adipocyte function (H), adipocyte function (I) and mitochondrial function (J) in primary APCs from BAT exposed to control conditions (white bars) or ceramide synthase inhibitor fumonisin B1 (5 μM; black bars) during differentiation. Data are shown as mean ± SEM (n = 3–6); *p < 0.05; **p < 0.01; ***p < 0.001 using two tailed unpaired t-test or one-way ANOVA.

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 release from in vitro differentiated primary brown adipocytes treated either with solvent control (white bar), 1.0 μM S1P (light gray bar), or 1.0 μM JTE (dark gray bar). (I) Ucp1 mRNA expression in in vitro differentiated APCs isolated from wildtype (WT, white bars) or S1pr2-knockout mice (KO, black bars) without treatment (−) or exposed to 1 μM JTE (+) during differentiation. (J) Norepinephrine-stimulated glycerol release from primary adipocytes differentiated after isolation from wildtype mice (WT, white bars) or S1pr2-deficient mice (KO, black bars). Data are shown as mean ± SEM, n = 3; *p < 0.05; **p < 0.01; ***p < 0.001 using two tailed unpaired t-test.

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 a 4 h norepinephrine (+NE) stimulation. (G) Norepinephrine-stimulated glycerol release from Dolk overexpressing immortalized pre-adipocyte cell line differentiated for 10 days (Dolk, black bars) compared to control cells (Ctr, white bars) normalized to total DNA content. (H, I) Analysis of mitochondrial respiration in immortalized control (Ctr) or Dolk-overexpressing (Dolk) brown WT1 pre-adipocytes differentiated under adipogenic conditions for 10 days. Oxygen consumption rates were recorded (H) and normalized to DAPI staining (relative fluorescence units, R.F.U.) for quantification (n = 12) (I). Basal respiration rate measurements were followed by administration of oligomycin (after third time point) to determine ATP-linked (coupled) respiration. Maximal respiration capacities were measured after FCCP-administration (after sixth time point) and non-mitochondrial respiration was measured by adding rotenone and antimycin A (after ninth time point). Uncoupled, e.g. potentially UCP1-dependent proton leak, was calculated from the relative differences of respiration rates after oligomycin and rotenone/antimycin A. (J) DOLK protein in differentiated APCs isolated from BAT of wild type (WT) or heterozygous Dolk knockout (HET) mice after 4 h NE stimulation after normalization to β-actin (Quantification: suppl. Fig. S5B). (K, L) mRNA expression levels of Ucp1 in differentiated APCs isolated from BAT (K) or iWAT (L) tissue of wild type (WT; white bars) or heterozygous Dolk knockout mice (HET; red bars) after 12 days of differentiation without (basal) or after 4 h NE stimulation (+NE). (M) UCP1 protein levels in brown adipocytes of wild type (WT) and heterozygous knockout (HET) mice after normalization to β-actin (Quantification: suppl. Fig. S5C). Data are shown as mean ± SEM, n = 3–12; *p < 0.05; **p < 0.01; ***p < 0.001 using two tailed unpaired t-test or Mann-Whitney-U test.