Abstract
Brown adipocytes function to maintain body temperature by heat production. However, direct measurement of heat production at a single cell level remains difficult. Here we developed a method to measure the temperature within primary cultured brown adipocytes using a cationic fluorescent polymeric thermometer. Placement of the thermometer within a matured brown adipocyte and a precursor cell enabled the detection of heat production following uncoupler treatment. The increase in the intracellular temperature due to stimulation with a mitochondrial uncoupler was higher in matured brown adipocytes than in precursor cells. Stimulation with a β-adrenergic receptor (β-AR) agonist, norepinephrine, raised the intracellular temperature of matured brown adipocytes to a level comparable to that observed after stimulation with a β3-AR-specific agonist, CL316.243. In contrast, neither β-AR agonist induced an intracellular temperature increase in precursor cells. Further, pretreatment of brown adipocytes with a β3-AR antagonist inhibited the norepinephrine-stimulated elevation of temperature. These results demonstrate that our novel method successfully determined the difference in intracellular temperature increase between matured brown adipocytes and precursor cells in response to stimulation by an uncoupler and β-AR agonists.
Highlights
Introduction of Rcationic fluorescent polymeric thermometer (CFPT) into pre-BACs and BACs
Browning of primary adipocytes from stromal vascular cells of interscapular BAT can be evaluated by measuring the mRNA expression of uncoupling protein 1 (UCP1) and other gene markers of matured BACs by qRT-PCR
To confirm that the protein level of UCP1 had increased in BACs, we performed western blot analysis and confirmed that UCP1 protein expression was strongly induced by differentiation in primary culture (Figs. 2c and S1a)
Summary
CFPT into pre-BACs and BACs. R-CFPT was incorporated into pre-BACs and BACs without cell detachment. SVF samples isolated from rat interscapular BAT were cultured on a collagen-coated. 35-mm glass-bottom dish (Matsunami Glass Ind., Osaka, Japan) in growth medium. The medium was removed, and the cells were washed with 5% glucose solution and treated with 5% glucose solution containing either 0.05 w/v% (for pre-BACs) or 0.04 w/v% R-CFPT (for BACs). After incubation at 25 °C for 10 min, the cells were. FluoroBrite DMEM (Life Technologies) supplemented with 15 mM HEPES was used for live-cell imaging. To measure the temperature of the culture medium, FluoroBrite DMEM supplemented with 15 mM HEPES containing 0.05 w/v% of R-CFPT was used
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