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.
Highlights
Warm-blooded animals have developed a number of metabolic processes for thermogenesis to maintain an optimal body temperature, even under extreme cold conditions[1]
To verify differences in the thermogenic potential of brown adipose tissue (BAT) cell model, we examined the expression of brown fat-specific genes (Fig. S5C)
BAT was assumed to be present only in human newborns and small infants to provide shiver-free thermogenesis, but has been shown to be present in adults[6,7,8,9,10,11]. This discovery has been the subject of substantial interest in both academic and pharmaceutical research[46] as a result of the high level of glucose and free fatty acid consumption by activated murine BAT – it is estimated that 50 g of BAT can burn 250 kcal daily[47]
Summary
Warm-blooded animals (or endotherms) have developed a number of metabolic processes for thermogenesis to maintain an optimal body temperature, even under extreme cold conditions[1]. A number of techniques for the measurement of BAT thermal power have been introduced recently, including infrared thermography of activated human BA cultures[14,15,16], the use of a bimaterial microcantilever, which is able to monitor small temperature variations (0.2 K) in the vicinity of norepinephrine-stimulated primary mouse BAs17, and microcalorimetry[18,19,20] These techniques are not yet widely applied in metabolic research, most likely as a result of the high cell requirements and the inability to measure individual isolated primary cells. We present a novel BODIPY-based thermosensitive dye, ERthermAC, with promising biocompatibility features (small size, excellent cellular uptake, low cytotoxicity, and robust photostability) that is capable of imaging heat production in individual isoproterenol (ISO)-stimulated mouse BAs and in forskolin-stimulated human BAs by confocal microscopy Data obtained using this dye is comparable with that obtained by conventional methods measuring mitochondrial depolarisation, oxygen consumption, and extracellular acidification rates, and with thermal power measurements obtained using the calScreener. We believe our findings will facilitate research on the mechanisms regulating brown fat thermogenesis as well as providing new in vitro tools for testing potential modulators of thermogenesis in this unique tissue
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