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. 2018 Nov 14;1(6):e201800136.
doi: 10.26508/lsa.201800136. eCollection 2018 Dec.

Substrate fluxes in brown adipocytes upon adrenergic stimulation and uncoupling protein 1 ablation

Sabine Schweizer  1 , Josef Oeckl  1   2   3 , Martin Klingenspor  1   2   3 , Tobias Fromme  1   2
Affiliations

Substrate fluxes in brown adipocytes upon adrenergic stimulation and uncoupling protein 1 ablation

Sabine Schweizer et al. Life Sci Alliance. .

Abstract

Brown adipocytes are highly specialized cells with the unique metabolic ability to dissipate chemical energy in the form of heat. We determined and inferred the flux of a number of key catabolic metabolites, their changes in response to adrenergic stimulation, and the dependency on the presence of the thermogenic uncoupling protein 1 and/or oxidative phosphorylation. This study provides reference values to approximate flux rates from a limited set of measured parameters in the future and thereby allows to evaluate the plausibility of claims about the capacity of metabolic adaptations or manipulations. From the resulting model, we delineate that in brown adipocytes (1) free fatty acids are a significant contributor to extracellular acidification, (2) glycogen is the dominant glycolytic substrate source in the acute response to an adrenergic stimulus, and (3) the futile cycling of free fatty acids between lipolysis and re-esterification into triglyceride provides a mechanism for uncoupling protein 1-independent, non-shivering thermogenesis in brown adipocytes.

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Conflict of interest statement

The authors declare no conflicting interests.

Figures

Figure S1.
Figure S1.
Oxygen consumption of cultured brown adipocytes isolated from WT or UCP1-KO mice in response to isoproterenol (OCRiso) as a percentage of basal oxygen consumption rate (OCRbasal). Percentages (2, 3, and 4) refer to BSA content in the medium to buffer fatty acids (n = 2–4). Ucp1-KO cells retained the ability to increase respiration in response to an adrenergic stimulus in all conditions tested. This increase is thus not caused by excessive fatty acid levels and was sensitive to oligomycin (oligo) in Ucp1-KO cells only.
Figure 1.
Figure 1.. Substrate fluxes in WT brown adipocytes.
Substrate fluxes for major catabolic pathways in WT murine brown adipocytes. A white background indicates basal flux rate and a grey background indicates isoproterenol-stimulated rates. All data are provided in nmol × h−1 × cm−2, see the Materials and Methods section for calculations and conversion factors to the per cell and per protein level, and Table 1 for complete dataset. TG, triglyceride mobilized.
Figure 2.
Figure 2.. Contributors to proton production.
PPR as measured in the basal and the isoproterenol (iso)-induced state (uniform light grey bars) and as calculated from three different source molecules (stacked bars). Oligo–oligmycin. All data are mean values ± SD, n = 3.
Figure 3.
Figure 3.. Glycogen content of brown adipocytes.
Altered glycogen content of cultured brown adipocytes in response to isoproterenol (iso) treatment (A) in the absence of glucose and (B) in a medium containing 25 mM glucose.
Figure S2.
Figure S2.
Characterization of primary, brown adipocytes. (A) Western blot–detecting Ucp1 and Actin in brown adipocytes of Ucp1-KO and WT origin. WT cells abundantly express Ucp1 protein, whereas Ucp1-KO cells lack detectable Ucp1 protein. (B) Schematic representation of the genomic Ucp1 locus and mapped reads from three WT (black) and three Ucp1-KO primary brown adipocyte cultures (red); common scale ranging from 0 to 3,516 reads per bin. In Ucp1-KO cells, a remnant transcript is produced that lacks exons 2 and 3 and is thus not translated (compare with panel A). (C) The Ucp1 (remnant) transcript is the only brown adipocyte marker gene of differential expression, n = 3. (D) Transcripts associated with mitochondrial abundance and OXPHOS capacity are unaltered by Ucp1-KO in primary brown adipocytes, n = 3.
Figure 4.
Figure 4.. Substrate fluxes in Ucp1-KO brown adipocytes.
Substrate fluxes for major catabolic pathways in Ucp1-KO murine brown adipocytes. A white background indicates basal flux rate and a grey background indicates isoproterenol-stimulated rates. All data are provided in nmol × h−1 × cm−2, see the Materials and Methods section for calculations and conversion factors to the per cell and per protein level and Table 1 for complete dataset. TG, triglyceride mobilized.
Figure 5.
Figure 5.. Thermogenic respiration in WT and Ucp1-KO brown adipocytes.
(A) Oxygen consumption of cultured brown adipocytes isolated from WT or Ucp1-KO mice. Black arrows indicate injections. Injections 1 and 2 were either oligomycin (oligo) or isoproterenol (iso) (see legend), injection 3 was carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and injection 4 was antimycin A. Shown are mean values ± SD, n = 4–11. (B) FFA re-esterification rate depending on oxidative phosphorylation in cultured brown adipocytes isolated from WT or Ucp1-KO mice in the basal or the isoproterenol (iso)-induced state.
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