Optical visualisation of thermogenesis in stimulated single-cell brown adipocytes

Abstract

The identification of brown adipose deposits in adults has led to significant interest in targeting this metabolically active tissue for treatment of obesity and diabetes. Improved methods for the direct measurement of heat production as the signature function of brown adipocytes (BAs), particularly at the single cell level, would be of substantial benefit to these ongoing efforts. Here, we report the first application of a small molecule-type thermosensitive fluorescent dye, ERthermAC, to monitor thermogenesis in BAs derived from murine brown fat precursors and in human brown fat cells differentiated from human neck brown preadipocytes. ERthermAC accumulated in the endoplasmic reticulum of BAs and displayed a marked change in fluorescence intensity in response to adrenergic stimulation of cells, which corresponded to temperature change. ERthermAC fluorescence intensity profiles were congruent with mitochondrial depolarisation events visualised by the JC-1 probe. Moreover, the averaged fluorescence intensity changes across a population of cells correlated well with dynamic changes such as thermal power, oxygen consumption, and extracellular acidification rates. These findings suggest ERthermAC as a promising new tool for studying thermogenic function in brown adipocytes of both murine and human origins.

Figure 1

ERthermAC targets the endoplasmic reticulum in adipocytes, and its intensity is inversely proportional to temperature. (A) ERthermAC (red) co-localises with ER-Tracker Green (green) in WT-1 cells, as evident from the yellow mix colour after superimposition of signals. Scale bar: 10 μm. (B) Chemical structure of ERthermAC. (C) Differentiated WT-1 cells were stained with ERthermAC, fixed with 4% formaldehyde, and imaged at different temperatures ranging between 18 °C and 43 °C. At higher temperatures, cells display lower ERthermAC fluorescence intensity. Scale bar: 20 μm. (D) Calibration curve shows a reversible non-linear relationship between temperature and fluorescence intensity of ERthermAC in fixed WT-1 cells between 18 °C and 43 °C. Temperature sensitivities determined by the linear fit were −1.07%/°C (R² = 0.76, n = 15 cells) between 18.1 °C and 35.0 °C, and −4.76%/°C (R² = 0.83, n = 15 cells) between 37 °C and 43 °C.

Figure 2

ERthermAC shows robust temperature changes after isoproterenol stimulation in WT-1 cells. (A) Individual ISO-stimulated WT-1 cells exhibit decreased ERthermAC fluorescence intensity, suggesting a robust increase in temperature. For time-lapse videos, see Supplementary Video 1. Scale bar: 20 µm.(B) Quantitative analysis of ERthermAC fluorescence intensity after ISO and vehicle stimulation: individual WT-1 cells exhibit a rapid decline in relative intensity after ISO stimulation. The reductions in fluorescence intensity occur at different time points in individual cells. In contrast, vehicle stimulation does not affect ERthermAC intensity. Thick black curves correspond to the mean relative intensities in each group. (C) Scatter plot of ERthermAC relative intensity change shows a significant difference in WT-1 cells. Bars show mean ± SD. WT-1 vehicle: n = 31 cells from 2 cultures; WT-1 ISO: n = 41 cells from 2 cultures.

Figure 3

FCCP stimulation resulted in immediate effects in WT-1 cells. (A) ERthermAC intensity drastically decreases upon FCCP stimulation in all cells, without any lag phase, indicating increased intracellular temperature. The thick black curve corresponds to the mean relative intensity; n = 35 cells from 2 cultures. Scale bar: 20 μm. (B) JC-1 staining shows immediate mitochondrial depolarisation after FCCP treatment. The thick black curve corresponds to the mean relative intensity ratio of green (JC-1 monomers) and red (JC-1 aggregates) signals; n = 50 cells from 2 cultures. Scale bar: 20 μm.

Figure 4

Isoproterenol stimulation induces mitochondrial depolarisation in WT-1 cells. (A) In WT-1 cells, red signal (JC-1 aggregates) corresponding to polarised mitochondria disappears and the intensity of green signal (JC-1 monomers) increases after ISO stimulation, indicating depolarisation. For time-lapse video, see Supplementary Video 3. Scale bar: 20 μm.(B) The relative intensity ratio of green and red signals is rapidly altered following ISO stimulation in WT-1 cells, indicating mitochondrial depolarisation. These sudden changes occur at different time points in individual cells. The thick black curve represents the average relative intensity ratio of green to red signals in all imaged cells, including responding and non-responding cells; n = 47 cells from 2 dishes.

Figure 5

Intensity drop of ERthermAC suggests heat production in human brown adipocytes after forskolin stimulation. (A) Individual forskolin-stimulated human brown adipocytes (BAs) exhibit decreased ERthermAC fluorescence intensity, suggesting a robust increase in temperature. Scale bar: 20 μm. For time-lapse videos, see Supplementary Video 5. (B) Quantitative analysis of ERthermAC fluorescence intensity in human BAs after forskolin and vehicle stimulation: individual cells exhibit a rapid decline in relative intensity after forskolin stimulation. The reductions in fluorescence intensity occur at different time points in individual cells. In contrast, vehicle stimulation does not affect ERthermAC intensity. Thick black curves correspond to the mean relative intensity in each group. (C) Scatter plot of ERthermAC relative intensity change shows a significant difference between forskolin and vehicle stimulated brown adipocytes. Bars show mean ± SD. hBAT vehicle: n = 20 cells from 3 cultures; hBAT forskolin: n = 29 cells from 3 cultures.

Figure 6

Forskolin stimulation induces mitochondrial depolarisation in human brown adipocytes. (A) In human brown adipocytes (BAs), red signal (JC-1 aggregates) corresponding to polarised mitochondria disappears and the intensity of green signal (JC-1 monomers) increases after forskolin stimulation, indicating depolarisation. For time-lapse video, see Supplementary Video 6. Scale bar: 20 μm. (B) The relative intensity ratio of green and red signals is rapidly altered following forskolin stimulation in human BAs, indicating mitochondrial depolarisation. These sudden changes occur at different time points in individual cells. The thick black curve represents the average relative intensity ratio of green to red signals in all imaged cells, including responding and non-responding cells.

Figure 7

ERthermAC forms a contiguous thermometer in the immediate mitochondrial vicinity by targeting the endoplasmic reticulum. Temperature difference (ΔT [K]) is proportional to the power of the heat source (P [W]), and inversely proportional to the distance (L [m]) from the centre of the heat source and the thermal conductivity surrounding the heat source (κ [W m⁻¹ K⁻¹]). Mitochondria-associated ER membrane in adipocytes provides sufficiently close proximity of these two organelles, thus placing ERthermAC into the extensive endoplasmic network and creating an extended contiguous thermometer in the immediate mitochondrial vicinity through targeting of a yet unknown biomolecule. This process is unaffected by radical environmental changes in mitochondria. ER: endoplasmic reticulum; IMM: inner mitochondrial membrane; OMM: outer mitochondrial membrane; UCP1: uncoupling protein 1.