Fatty acid oxidation is required for active and quiescent brown adipose tissue maintenance and thermogenic programing

Abstract

Objective: To determine the role of fatty acid oxidation on the cellular, molecular, and physiologic response of brown adipose tissue to disparate paradigms of chronic thermogenic stimulation.

Methods: Mice with an adipose-specific loss of Carnitine Palmitoyltransferase 2 (Cpt2^A-/-), that lack mitochondrial long chain fatty acid β-oxidation, were subjected to environmental and pharmacologic interventions known to promote thermogenic programming in adipose tissue.

Results: Chronic administration of β3-adrenergic (CL-316243) or thyroid hormone (GC-1) agonists induced a loss of BAT morphology and UCP1 expression in Cpt2^A-/- mice. Fatty acid oxidation was also required for the browning of white adipose tissue (WAT) and the induction of UCP1 in WAT. In contrast, chronic cold (15 °C) stimulation induced UCP1 and thermogenic programming in both control and Cpt2^A-/- adipose tissue albeit to a lesser extent in Cpt2^A-/- mice. However, thermoneutral housing also induced the loss of UCP1 and BAT morphology in Cpt2^A-/- mice. Therefore, adipose fatty acid oxidation is required for both the acute agonist-induced activation of BAT and the maintenance of quiescent BAT. Consistent with this data, Cpt2^A-/- BAT exhibited increased macrophage infiltration, inflammation and fibrosis irrespective of BAT activation. Finally, obese Cpt2^A-/- mice housed at thermoneutrality exhibited a loss of interscapular BAT and were refractory to β3-adrenergic-induced energy expenditure and weight loss.

Conclusion: Mitochondrial long chain fatty acid β-oxidation is critical for the maintenance of the brown adipocyte phenotype both during times of activation and quiescence.

Keywords: Adipose macrophage; Adrenergic signaling; Brown adipose tissue; Cold induced thermogenesis; Fatty acid oxidation.

Figure 1

Effect of CL-316243 and GC-1 on adipose tissue of Cpt2^A−/−mice. A) H&E staining of BAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice injected with Saline, 0.3 mg/kg GC-1, or 1 mg/kg CL-316243 for 10 days. Scale bar, 200 um. B) mRNA expression in BAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice injected with Saline, 0.3 mg/kg GC-1, or 1 mg/kg CL-316243 for 10 days (n = 6). C) UCP1 protein expression in BAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice injected with Saline, 0.3 mg/kg GC-1, or 1 mg/kg CL-316243 for 10 days. D) H&E staining of iWAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice injected with Saline, 0.3 mg/kg GC-1, or 1 mg/kg CL-316243 for 10 days. Scale bar, 200 um. E) mRNA expression in iWAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice injected with Saline, 0.3 mg/kg GC-1, or 1 mg/kg CL-316243 for 10 days (n = 6). F) UCP1 protein expression in iWAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice injected with Saline, 0.3 mg/kg GC-1, or 1 mg/kg CL-316243 for 10 days. Arrow indicates Ucp1 band. G) H&E staining of gWAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice injected with Saline, 0.3 mg/kg GC-1, or 1 mg/kg CL-316243 for 10 days. Scale bar, 200 μm H) mRNA expression in gWAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice injected with Saline, 0.3 mg/kg GC-1, or 1 mg/kg CL-316243 for 10 days (n = 6). I) UCP1 protein expression in gWAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice injected with Saline, 0.3 mg/kg GC-1, or 1 mg/kg CL-316243 for 10 days. Arrow indicates Ucp1 band. Western blot quantification is expressed as fold change +/− SE (n = 3). Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2

Effect of CL-316243 and GC-1 on mitochondrial proteins in adipose tissue of Cpt2^A−/−mice. A) Western blots of mitochondrial proteins in BAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice injected with Saline, 0.3 mg/kg GC-1, or 1 mg/kg CL-316243 for 10 days. B) Western blots of mitochondrial proteins in iWAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice injected with Saline, 0.3 mg/kg GC-1, or 1 mg/kg CL-316243 for 10 days. C) Western blots of mitochondrial proteins in gWAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice injected with Saline, 0.3 mg/kg GC-1, or 1 mg/kg CL-316243 for 10 days.

Figure 3

Effect of ambient temperature on adipose tissue of Cpt2^A−/−mice. A) H&E staining of BAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice acclimated to 15 °C, 21 °C, or 30 °C for 10 days. Scale bar, 200 um. B) mRNA expression in BAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice acclimated to 15 °C, 21 °C, or 30 °C for 10 days (n = 6). C) UCP1 protein expression in BAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice acclimated to 15 °C, 21 °C, or 30 °C for 10 days. D) H&E staining of iWAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice acclimated to 15 °C, 21 °C, or 30 °C for 10 days. Scale bar, 200 um. E) mRNA expression in iWAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice acclimated to 15 °C, 21 °C, or 30 °C for 10 days (n = 6). F) UCP1 protein expression in iWAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice acclimated to 15 °C, 21 °C, or 30 °C for 10 days. Arrow indicates Ucp1 band. G) H&E staining of gWAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice acclimated to 15 °C, 21 °C, or 30 °C for 10 days. Scale bar, 200 um. H) mRNA expression in gWAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice acclimated to 15 °C, 21 °C, or 30 °C for 10 days (n = 6). I) UCP1 protein expression in gWAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice acclimated to 15 °C, 21 °C, or 30 °C for 10 days. Arrow indicates Ucp1 band. Western blot quantification is expressed as fold change +/− SE (n = 3). Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

Effect of ambient temperature on mitochondrial proteins in adipose tissue of Cpt2^A−/−mice. A) Western blots of mitochondrial proteins in BAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice acclimated to 15 °C, 21 °C, or 30 °C for 10 days. B) Western blots of mitochondrial proteins in iWAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice acclimated to 15 °C, 21 °C, or 30 °C for 10 days. C) Western blots of mitochondrial proteins in gWAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice acclimated to 15 °C, 21 °C, or 30 °C for 10 days.

Figure 5

Loss of fatty acid oxidation induces Fgf21 in BAT. A) Fgf21 expression in BAT of 12-week old Cpt2^A−/− and Cpt2^lox/lox mice acclimated to different ambient temperature or thermogenic agonists for 10 days (n = 6). B) Fgf21 expression in iWAT of 12-week old Cpt2^A−/− and Cpt2^lox/lox mice acclimated to different ambient temperature or thermogenic agonists for 10 days (n = 6). C) Fgf21 expression in gWAT of 12-week old Cpt2^A−/− and Cpt2^lox/lox mice acclimated to different ambient temperature or thermogenic agonists for 10 days (n = 6). Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6

Time course of CL-316243 mediated dysfunction in Cpt2^A−/−BAT. A) H&E staining of BAT from Cpt2^A−/− and Cpt2^lox/lox mice after injection with 1 mg/kg CL-316243 for 0, 2, 4, 6, or 8 days. Scale bar, 200 um. B) Western blots of representative proteins in oxidative phosphorylation complexes I, II, III, and V in BAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice injected with 1 mg/kg CL-316243 for 0, 2, 4, 6, 8, and 10 days. C) Mitochondrial protein expression in BAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice injected with 1 mg/kg CL-316243 for 0, 2, 4, 6, 8, and 10 days.

Figure 7

Loss of adipose fatty acid oxidation causes macrophage infiltration and inflammation in BAT. A) H&E staining of BAT from Cpt2^A−/− and Cpt2^lox/lox mice after injection with 1 mg/kg CL-316243 for 0, 2, 4, 6, or 8 days. B) mRNA expression of macrophage & inflammatory genes in BAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice injected with Saline, 0.3 mg/kg GC-1, or 1 mg/kg CL-316243 for 10 days (n = 6). C) mRNA expression of macrophage & inflammatory genes in BAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice acclimated to 15 °C, 21 °C, or 30 °C for 10 days (n = 6). Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 8

Transmission Electron Micrographs of control and Cpt2^A−/−BAT. TEM of BAT from 16-week old Cpt2^A−/− and Cpt2^lox/lox mice. Scale bars, 2 um and 500 nm, respectively. White arrowheads pointing to dysmorphic mitochondria. Black arrowheads pointing to fibrin deposition. Lipid Droplet (LD), Red Blood Cell (RBC).

Figure 9

Loss of adipose fatty acid oxidation induces genes in extracellular matrix. A) mRNA expression of collagen and extracellular matrix genes in BAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice injected with Saline, 0.3 mg/kg GC-1, or 1 mg/kg CL-316243 for 10 days (n = 6). B) mRNA expression of collagen and extracellular matrix genes in BAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice acclimated to 15 °C, 21 °C, or 30 °C for 10 days (n = 6). C) Mmp-12 mRNA expression in BAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice injected with Saline, 0.3 mg/kg GC-1, or 1 mg/kg CL-316243 for 10 days (n = 6). D) Mmp-12 mRNA expression in BAT from 12-week old Cpt2^A−/− and Cpt2^lox/lox mice acclimated to 15 °C, 21 °C, or 30 °C for 10 days (n = 6). Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 10

Adipose fatty acid oxidation is required for acute β3-adrenergic-induced weight loss. A) Body weights male Cpt2^A−/− and Cpt2^lox/lox littermate control mice on HFD for 6-weeks at 22 °C and 6 weeks at 30 °C. Arrows indicate the start of high-fat diet feeding and injections with 1 mg/kg CL-316243 (n = 10–14). B) Loss in body weight of male Cpt2^A−/− and Cpt2^lox/lox littermate control mice after injections with 1 mg/kg CL-316243 for 10 days at 30 °C (n = 10–14). C) Gross interscapular BAT morphology of male Cpt2^A−/− and Cpt2^lox/lox littermate control mice housed at 30 °C on high-fat diet plus 1 mg/kg CL-316243 injections for 10 days. D) H&E staining of BAT, iWAT, and gWAT from male Cpt2^A−/− and Cpt2^lox/lox littermate control mice housed at 30 °C on high-fat diet plus 1 mg/kg CL-316243 injections for 10 days. Scale bar, 200 um. E) VO₂ consumption and RER of male Cpt2^A−/− and Cpt2^lox/lox littermate control mice housed at 30 °C on high-fat diet plus 1 mg/kg CL-316243 injections for 10 days (n = 9–14). Data were collected during a 96 h ad libitum period, 24 h fast, 24 h reefed, and 3 h after injection with 10 mg/kg CL-316243. F) Energy expenditure of male Cpt2^A−/− and Cpt2^lox/lox littermate control mice housed at 30 °C on high-fat diet plus 1 mg/kg CL-316243 injections for 10 days under fed, fast, and re-fed conditions (n = 9–14). G) VO₂ consumption of male Cpt2^A−/− and Cpt2^lox/lox littermate control mice housed at 30 °C on high-fat diet plus 1 mg/kg CL-316243 injections for 10 days. VO₂ consumption was measured for 3 h after injection with 10 mg/kg CL-316243 (n = 9–14). Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.