Abstract
ObjectiveBrown 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. MethodsA 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). ResultsA 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. ConclusionsWe 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.
Highlights
Brown adipose tissue is capable of burning fat and could potentially counteract obesity-related metabolic diseases through specialized energy expenditure [1]
We revealed a triphasic mitochondrial pH change during ISO-induced Brown adipocytes (BAs) activation
While the phase 3 pH decrease corresponded to thermogenesis, the phase 1 and phase 2 pH increases occurred before thermogenesis, with concurrent increases in mitochondrial Ca2þ
Summary
Brown adipose tissue is capable of burning fat and could potentially counteract obesity-related metabolic diseases through specialized energy expenditure [1]. The discovery of brown adipose tissue in adult humans [2e5] aroused great interest in understanding the regulatory pathways of brown adipocyte (BA) activation in the quest for strategies to promote energy expenditure [6]. Upon binding to b-AR agonists, intracellular cAMP levels are elevated, which leads to the generation of free fatty acids (FFAs) in the cytoplasm [7]. Apart from the known factors in the b-AR signaling pathway, several studies showed that Ca2þ is involved in BA activation [8e10]. The increase in cytosolic Ca2þ levels enhances thermogenesis induced by b-AR signaling [9]. These studies imply regulatory networks of thermogenesis in BAs that are more complex than currently understood.
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