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Mol Metab. 2017 May 31;6(8):797-808. doi: 10.1016/j.molmet.2017.05.013. eCollection 2017 Aug.

Ca2+-associated triphasic pH changes in mitochondria during brown adipocyte activation.

Author information

1
WASEDA Bioscience Research Institute in Singapore (WABIOS), 11 Biopolis Way, #05-02 Helios, Singapore 138667, Singapore.
2
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.
3
Department of Biomedical Engineering, National University of Singapore, 117583, Singapore; NUS Tissue Engineering Program, Life Sciences Institute, National University of Singapore, 117510, Singapore; Graduate School for Integrative Sciences and Engineering (NGS), National University of Singapore, 117456, Singapore.
4
Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215, USA.
5
Department of Biomedical Engineering, National University of Singapore, 117583, Singapore; NUS Tissue Engineering Program, Life Sciences Institute, National University of Singapore, 117510, Singapore; Department of Biochemistry, Yong Loo Ling School of Medicine, National University of Singapore, 117597, Singapore.
6
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; PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan. Electronic address: suzu_mado@aoni.waseda.jp.

Abstract

OBJECTIVE:

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.

METHODS:

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 Ca2+ profiles in mitochondria and the intracellular Ca2+ store, the endoplasmic reticulum (ER).

RESULTS:

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 Ca2+. This Ca2+ increase is contributed to by an influx from the ER, and it is further involved in mitochondrial pH regulation.

CONCLUSIONS:

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.

KEYWORDS:

AMA, antimycin A; BAs, brown adipocytes; Brown adipocytes; Ca2+; Confocal microscopy; EGTA, ethylene glycol tetraacetic acid; ER, endoplasmic reticulum; ETC, electron transport chain; Endoplasmic reticulum; FFAs, free fatty acids; Fluorescence imaging; IMS, intermembrane space; ISO, isoproterenol; MAM, mitochondria-associated ER membrane; MCU, mitochondrial calcium uniporter; Mitochondria-associated ER membrane; Rot, rotenone; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; TG, thapsigargin; TMRM, tetramethylrhodamine methyl ester; UCP1, uncoupling protein 1; β-AR, β-adrenergic receptor

PMID:
28752044
PMCID:
PMC5518710
DOI:
10.1016/j.molmet.2017.05.013
[Indexed for MEDLINE]
Free PMC Article

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