Dye-based mito-thermometry and its application in thermogenesis of brown adipocytes
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Mitochondrion is the main intracellular site for thermogenesis and attractive energy expenditure targeting for obesity therapy. Here, we develop a method of mitochondrial thermometry based on Rhodamine B methyl ester, which equilibrates as a thermosensitive mixture of nonfluorescent and fluorescent resonance forms. Using this approach, we are able to demonstrate that the efficacy of norepinephrine-induced thermogenesis is low, and measure the maximum transient rate of temperature increase in brown adipocytes.
KeywordsMitochondrial thermometry Nanothermometry Thermogenesis Brown adipocytes
Temperature probing for live cells is challenging, and a lot of efforts have been made to develop nanothermometry to monitor temperatures of living cells (Ye et al. 2011; Jaque and Vetrone 2012; Li and Liu 2012; Kiyonaka et al. 2013; Kucsko et al. 2013; Arai et al. 2014, 2015; Homma et al. 2015). Recently, all such works have been challenged and criticized by Baffou et al., who have claimed no detectable temperature heterogeneities in living cells (Baffou et al. 2014). Apparently, Baffou et al. have neglected well-known facts in biology, such as the thermogenesis of brown adipocytes (BA) and mitochondrial role in thermogenesis (Cannon and Nedergaard 2004).
Sympathetic neurotransmitter norepinephrine (NE) can mobilize free fatty acids stored in lipid droplets of BA, and dissipate electrochemical potential energy stored in mitochondrial proton gradient to product heat (Cannon and Nedergaard 2004; Fedorenko et al. 2012). Not only as the energy factory of the cells, mitochondrion is the main intracellular site for thermogenesis, which has been targeted for therapy to reduce obesity (Lowell and Spiegelman 2000; Tseng et al. 2010). Here, we demonstrate a method of mitochondrial thermometry (mito-thermometry) based on the thermosensitive characteristics of Rhodamine B methyl ester (RhB-ME). With this mito-thermometry, we revealed the low efficacy of NE-induced thermogenesis and the maximum transient rate of temperature increase in BA, and indicated the improper critique of Baffou both practically and theoretically.
Evaluation of RhB-ME-based mito-thermometry in HeLa cells
The thermochromic transformation of RhB-ME in aqueous solution results in a simple temperature profile, which can be fitted with Arrhenius equation, a single exponential model for the temperature dependence of reaction rates (Fig. 1F). The Arrhenius plot indicates that activation energy of RhB-ME thermochromic transformation is about −4.4 kcal/mol. In living cells, to cancel out the influence of mitochondrial membrane potentials on RhB-ME concentration, the fluorescent intensity ratio of Rh800 to RhB-ME is used to represent mitochondrial thermal profile (Fig. 1G). Both RhB-ME and Rh800 are insensitive to pH, Ca2+ or Mg2+ (Supplementary Fig. S2). This RhB-ME-based mito-thermometry enables us to acquire the mitochondrial thermal map of HeLa cells at room temperature (RT). The image in Fig. 1G shows mitochondrial temperature gradients in HeLa cells with higher temperature at the center, which can be explained by the geometry of the cells (Fig. 1C).
The mechanism of RhB-ME thermochromic transformation
Study the thermogenesis of BA with RhB-ME-based mito-thermometry
To evaluate and make use of RhB-ME based mito-thermometry, we applied it to study the thermogenesis of BA. For thermogenic studies of BAT or BA, calorimeter and oxygen consumption rate (OCR) have been frequently used to evaluate heat production (Clark et al. 1986; Wikstrom et al. 2014), but both are indirect and might be cumbersome (Cannon and Nedergaard 2004). Although the genetically coded thermometry is versatile for organelle targeting, due to low transfection efficiency of BA, it is difficult to be used for collecting large-scale datasets, which are usually necessary for experiments with large variations. Since there is no need for transfection, injection, or elaborate equipment, dyes (Arai et al. 2015; Homma et al. 2015) and RhB-ME-based mito-thermometry demonstrated in this study make it easier for large-scale data acquisition, and are capable of detecting thermogenic responses at mitochondrial level.
Baffou et al. have criticized all methods for temperature imaging in living cells (Baffou et al. 2014), which have based on a conclusion “temperature increase should be on the order of ΔT ~ 10−5 K” governed by the heat diffusion equation at one-dimensional steady-state conditions for cell. However, “ΔT” is a spatial temperature gradient independent of time in Baffou’s model rather than “temperature increase” (over time), so it is inappropriate to apply a spatial gradient “ΔT” to discuss thermogenesis in living cells, a temporal process in time-variant systems. In addition, such small spatial temperature gradient (10−5 K) would result in negligible heat flows within cell.
In summary, we have practically demonstrated uneven mitochondrial thermal maps in living cells, theoretically inferred detectable heat sources (mitochondria), and also pointed out the error of Baffou’s critique. RhB-ME-based mito-thermometry makes it easier for large-scale data acquisition, especially for primary cultured cells, such as BA. Our current observations raise open questions about diversely thermogenic responses of individual BA evoked by 0.1 μmol/L NE, for instance, what is the in vivo regulatory mechanism to increase the efficacy of NE-induced thermogenesis in BA?
A mixture of RhB (500 mg, 1.04 mmol) and thionyl chloride (2 ml) in chloroform (20 ml) was heated to 60 °C and stirred for 10 min. After cooling to room temperature, the mixture was quenched with methanol. The solvent was removed under reduced pressure and purified by prep-HPLC to give compound 375 mg, yield 73%. 1H-NMR (600 MHz, CDCl3) δ 8.30 (d, J = 7.2 Hz, 1H), 8.23 (brs, 2H), 7.79–7.82 (m, 1H), 7.73–7.76 (m, 1H), 7.31 (d, J = 7.2 Hz, 1H), 7.06 (d, J = 9.6 Hz, 2H), 6.82–6.83 (m, 4H), 3.68 (s, 3H), 3.60 (q, J = 7.2 Hz, 8H), 1.32 (t, J = 7.2 Hz, 12H); ESI-HRMS exact mass calcd for [M]+ requires m/z 457.2486; found m/z 457.2484.
The change rate of normalized intensity ratio (Rh800 to RhB-ME) at a temperature
Full Methods and any associated references are available in the online version of the paper.
We thank the following people for their help: S.-L. You and X.-W. Liu for 1H-NMR and discussions; Y. Chen, H.-B. Cai, Z.-H. Sheng, D.-S. Li, Q.-W. Zhai, H. Ying and H.-X. Qi for critical reading of the manuscript; S.-L. You, X.-W. Liu, K. Hou, C. Chen and J.-J. Hao for chemical synthesis. Y.-Y. Le for the gift of HeLa cell line. This work was partially supported by National Basic Research Program of China (2011CB910903 and 2010CB912001), National Natural Science Foundation of China (31171369), Chinese Academy of Sciences (Hundred Talents Program and 2009OHTP10).
Compliance with Ethical Standards
Conflict of interest
Tao-Rong Xie, Chun-Feng Liu, Jian-Sheng Kang declare that they have no conflict of interest.
Human and Animal Rights and Informed Consent
All institutional and national guidelines for the care and use of laboratory animals were followed.
- Wikstrom JD, Mahdaviani K, Liesa M, Sereda SB, Si Y, Las G, Twig G, Petrovic N, Zingaretti C, Graham A, Cinti S, Corkey BE, Cannon B, Nedergaard J, Shirihai OS (2014) Hormone-induced mitochondrial fission is utilized by brown adipocytes as an amplification pathway for energy expenditure. EMBO J 33:418–436PubMedPubMedCentralGoogle Scholar
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