Angiopoietin-like 4 governs diurnal lipoprotein lipase activity in brown adipose tissue

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  Figure 1. The transcriptome of murine brown adipose tissue consists of four clusters with distinct oscillating expression phases. Interscapular brown adipose tissue samples were collected from chow-fed male C57BL/6J mice at 3-hour intervals throughout a 24-hour period to produce eight time points in total (n = 4 per time point), which were used to perform RNA-sequencing. Oscillation was assessed by JTK, and (A) oscillating (P < 0.05) genes as a subset of all expressed genes were visualized in a Venn diagram. Hierarchical clustering of standardized residuals (Z-scores) of all oscillating genes was visualized in a heat map and (B) expression of genes within the four major clusters was double plotted for visual purposes. Functional enrichment by gene ontology was performed on each cluster, as well as enrichment by transcription factor of which top 10 hits are depicted. ATOH8, atonal BHLH transcription factor 8; CEBPA, CCAAT enhancer binding protein alpha; CHCHD3, coiled-coil-helix-coiled-coil-helix domain containing 3; CREBZF, CREB/ATF BZIP transcription factor; DMTF1, cyclin D binding MYB like transcription factor 1; GLIS2, GLIS family zinc finger 2; GTF2I, general transcription factor IIi;…

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  Figure 2. Enrichment for (lipid) catabolic processes in oscillating genes with oscillating PPARγ binding. Interscapular brown adipose tissue samples were collected from chow-fed male C57BL/6J mice at 3-hour intervals throughout a 24-hour period to produce eight time points in total (n = 8 per time point). On pooled samples, chromatin immunoprecipitation (ChIP)-sequencing was performed for PPARγ, and oscillation of peaks was assessed by JTK. Peaks were annotated, and within each of the four gene clusters with distinct expression phases as identified by RNA-sequencing (Figure 1), (A) proportions of genes with oscillating (P < 0.05) PPARγ binding, non-oscillating PPARγ binding, or no PPARγ binding were visualized in Pie charts. (B) Functional enrichment by gene ontology was performed on the genes with oscillating PPARγ binding, and top 10 hits are depicted. For cluster 1, all four hits are depicted.

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  Figure 3. The lipidome of murine brown adipose tissue consists of three clusters with distinct oscillating phases. Interscapular brown adipose tissue samples were collected from chow-fed male C57BL/6J mice at 3-hour intervals throughout a 24-hour period to produce eight time points in total (n = 4 per time point), which were used to perform ultra-performance liquid chromatography (UPLC)-high-resolution mass spectrometry (HRMS)-based lipidomics. Oscillation was assessed by JTK, and (A) oscillating (P < 0.05) lipid species as a subset of all measured lipids were visualized in a Venn diagram. Hierarchical clustering of standardized residuals (Z-scores) of oscillating lipid species was visualized in a heat map and (B) levels within the three major clusters were double plotted for visual purposes. Within each cluster, the contribution of each lipid category to its composition is presented. AC, acylcarnitine; CE, cholesteryl ester; Cer, ceramide; CL, cardiolipin; DG, diacylglycerol; DLCL, dilysocardiolipin; FA, fatty acid; LPA, lysophosphatic acid; LPE, lysophosphatidylethanolamine; MG, monoacylglycerol; LPG, lysophosphatidylglycerol; LPI, lysophosphatidylinositol; LPS, lysophosphatidylserine;…

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  Figure 4. The lipoprotein lipase pathway follows diurnal oscillations that peak at the onset of the active phase. Interscapular brown adipose tissue (BAT) samples were collected from chow-fed male C57BL/6J mice at 3-hour intervals throughout a 24-hour period to produce eight time points in total (n = 4 per time point), which were used to perform RNA-sequencing. Oscillation was assessed by JTK, and normalized counts of (A) lipoprotein lipase (Lpl), (B) angiopoietin-like (Angptl)4, (C)Angptl8, (D)Angptl3,(E) apolipoprotein C1 (Apoc1), (F) glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1 (Gpihbp1), (G) cluster of differentiation 36 (Cd36), and (H) phospholipid transfer protein (Pltp) were double plotted. (I) A schematic overview of LPL-mediated triglyceride (TG)-rich lipoprotein (TRL) hydrolysis and the autocrine regulation hereof by BAT. FA, fatty acid; HDL, high-density lipoprotein; PL, phospholipid.

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  Figure 5. Angiopoietin-like 4 modulation flattens oscillation of lipoprotein lipase activity and triglyceride-derived fatty acid uptake by brown adipose tissue. Male wild type (WT), angiopoietin-like 4 (Angptl4) knockout (KO) and Angptl4 transgenic overexpression (Tg) mice were examined at the onset of the light (inactive) phase (i.e. Zeitgeber time (ZT)0) and dark (active) phase (i.e. ZT12). Gene expression of Angptl4 and lipoprotein lipase (Lpl) was measured in subscapular brown adipose tissue (sBAT) of (A)Angptl4 KO and (B)Angptl4 Tg mice by quantitative polymerase chain reaction. Protein abundance of LPL was measured in interscapular (i)BAT of (C)Angptl4 KO and (D) Tg mice by Western blot, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. Representative images are shown. In vitro lipoprotein lipase (LPL) activity was measured in iBAT of (E)Angptl4 KO and (G)Angptl4 Tg mice and expressed as triglyceride (TG) hydrolyzing (hydr.) activity in μmol free fatty acids (FFA) per hour (h) per gram (g) tissue. Prior to killing, (F)Angptl4 KO and (H)Angptl4 Tg mice were injected with TG-rich lipoprotein-like particles labeled with glycerol tri[3H]oleate to assess fatty acid…

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  Figure 6. Hypothetical model of diurnal processes in brown adipose tissue. Around Zeitgeber Time (ZT) 0, the onset of the light (inactive) phase, uncoupling protein 1 (Ucp1) expression, which encodes a protein that uncouples ATP production from mitochondrial oxidative phosphorylation (i.e. thermogenesis), and thereby likely brown adipose tissue (BAT) thermogenic activity is lowest. Genes involved in fatty acid (FA) elongation peak throughout the light phase, coinciding with increased abundance of long-acyl chain diacylglycerols (DGs). In the second half of the light phase, expression of mitochondrial complexes, as well as Ucp1 peak, suggesting peak thermogenic activity within the tissue. Genes involved in intracellular lipolysis peak shortly before the highest abundance of FAs and monoacylglycerols (MGs), possibly to supply mitochondria with FAs for β-oxidation and to allosterically activate UCP1 [1]. To replenish intracellular lipid stores, the peak in triglyceride (TG)-rich lipoprotein-derived FA-uptake follows at the onset of the dark (active) phase (i.e. ZT12) [13], driven by lipoprotein lipase (LPL)-mediated hydrolysis, and likely contributing to the FA and MG abundance at the onset…