WASEDA Bioscience Research Institute in Singapore (WABIOS), 11 Biopolis Way, #05-02 Helios, Singapore 138667, Singapore.
WASEDA Bioscience Research Institute in Singapore (WABIOS), 11 Biopolis Way, #05-02 Helios, Singapore 138667, Singapore; Comprehensive Research Organization, Waseda University, Tokyo, 162-0041, Japan.
Mol Metab. 2017 May 31;6(8):797-808. doi: 10.1016/j.molmet.2017.05.013. eCollection 2017 Aug.
Brown adipocytes (BAs) are endowed with a high metabolic capacity for energy expenditure due to their high mitochondria content. While mitochondrial pH is dynamically regulated in response to stimulation and, in return, affects various metabolic processes, how mitochondrial pH is regulated during adrenergic stimulation-induced thermogenesis is unknown. We aimed to reveal the spatial and temporal dynamics of mitochondrial pH in stimulated BAs and the mechanisms behind the dynamic pH changes.
A mitochondrial targeted pH-sensitive protein, mito-pHluorin, was constructed and transfected to BAs. Transfected BAs were stimulated by an adrenergic agonist, isoproterenol. The pH changes in mitochondria were characterized by dual-color imaging with indicators that monitor mitochondrial membrane potential and heat production. The mechanisms of pH changes were studied by examining the involvement of electron transport chain (ETC) activity and Ca profiles in mitochondria and the intracellular Ca store, the endoplasmic reticulum (ER).
A triphasic mitochondrial pH change in BAs upon adrenergic stimulation was revealed. In comparison to a thermosensitive dye, we reveal that phases 1 and 2 of the pH increase precede thermogenesis, while phase 3, characterized by a pH decrease, occurs during thermogenesis. The mechanism of pH increase is partially related to ETC. In addition, the pH increase occurs concurrently with an increase in mitochondrial Ca. This Ca increase is contributed to by an influx from the ER, and it is further involved in mitochondrial pH regulation.
We demonstrate that an increase in mitochondrial pH is implicated as an early event in adrenergically stimulated BAs. We further suggest that this pH increase may play a role in the potentiation of thermogenesis.
棕色脂肪细胞（BAs）由于其高线粒体含量而具有高代谢能力以消耗能量。虽然线粒体 pH 值会根据刺激而动态调节，并且反过来又会影响各种代谢过程，但在肾上腺素能刺激诱导的产热过程中，线粒体 pH 值是如何调节的尚不清楚。我们旨在揭示刺激的 BAs 中线粒体 pH 值的时空动态及其动态 pH 值变化背后的机制。
构建了一种线粒体靶向的 pH 敏感蛋白，mito-pHluorin，并转染到 BAs 中。用肾上腺素能激动剂异丙肾上腺素刺激转染的 BAs。通过使用监测线粒体膜电位和产热的指示剂进行双色成像，来描述线粒体中 pH 值的变化。通过研究电子传递链（ETC）活性和线粒体及细胞内钙库内质网（ER）中 Ca 谱在 pH 值变化中的作用，研究了 pH 值变化的机制。
揭示了肾上腺素刺激下 BAs 中的线粒体 pH 值呈现三相变化。与热敏染料相比，我们发现 pH 值增加的第 1 相和第 2 相先于产热，而第 3 相，其特征是 pH 值下降，发生在产热期间。pH 值增加的机制部分与 ETC 有关。此外，pH 值增加伴随着线粒体 Ca 的增加。这种 Ca 增加是由 ER 内流引起的，并且进一步涉及线粒体 pH 值调节。
我们证明了线粒体 pH 值增加是肾上腺素刺激的 BAs 中的早期事件。我们进一步表明，这种 pH 值增加可能在增强产热中发挥作用。
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