Identification of a Ca2+-ATPase in Brown Adipose Tissue Mitochondria

Abstract

In brown adipose tissue (BAT) adrenaline promotes a rise of the cytosolic Ca 2؉ concentration from 0.05 up to 0.70 M. It is not known how the rise of Ca 2؉ concentration activates BAT thermogenesis. In this report we compared the effects of Ca 2؉ in BAT and liver mitochondria. Using electron microscopy and immunolabeling we identified a sarco/endoplasmic reticulum (ER) Ca 2؉ -ATPase bound to the inner membrane of BAT mitochondria. A Ca 2؉ -dependent ATPase activity was detected in BAT mitochondria when the respiratory substrates malate and pyruvate were included in the medium. ATP and Ca 2؉ enhanced the amount of heat produced by BAT mitochondria during respiration. The Ca 2؉ concentration needed for half-maximal activation of the ATPase activity and rate of heat production were the same and varied between 0.1 and 0.2 M. Heat production was partially inhibited by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and abolished by thapsigargin, a specific ER Ca 2؉ -ATPase inhibitor, and by both rotenone and KCN, two substances that inhibit the electron transfer trough the mitochondrial cytochrome chain. In liver mitochondria Ca 2؉ did not stimulate the ATPase activity nor increase the rate of heat production. Thapsigargin had no effect on liver mitochondria. In conclusion, this is the first report of a Ca 2؉ -ATPase in mitochondria that is BAT-specific and can generate heat in the presence of Ca 2؉ concentrations similar to those noted in the cell during adrenergic stimulation. BAT3 is capable of rapidly converting fat stores to heat and has been used as a model system for the understanding of nonshivering heat production and mechanisms of energy wasting to control obesity (1-9). The signal that activates the rate of heat production in BAT cells is the rise of the cytosolic Ca 2ϩ concentration from a basal level of 0.05 M up to the range of 0.2-0.7 M. This is promoted by ␣ 1 -and ␤ 3 -adrenergic receptors located in the cell membrane. Activation of ␣ 1 -adrenoreceptors leads to the release of Ca 2ϩ from intracellular stores into the cytosol, whereas ␤ 3 -adrenergic receptors promote the release of free fatty acids and increase the effect of Ca 2ϩ release induced by ␣ 1 -adrenoreceptors (10, 11). At present we do not know how the rise of the cytosolic Ca 2ϩ concentration activates the rate of heat production in BAT cells.In a previous report (12) we identified a sarco/ER Ca 2ϩ ATPase (SERCA 1) in vesicles derived from BAT ER. In this report we show that the rate of heat produced by BAT mitochondria is enhanced when the Ca 2ϩ concentration in the medium is raised to a level similar to that observed in BAT cells during adrenergic stimulation. This effect is not observed in liver mitochondria, a tissue that is not specialized in heat production. EXPERIMENTAL PROCEDURES Isolation of Mitochondria from Rat BAT and Liver-Adult maleWistar rats were killed by decapitation. Mitochondria were isolated as previously described (13). Briefly, interscapular BAT and liver were removed and homogenized in a mix...

UCP1-independent Thermogenesis in White Adipose Tissue of Cold-acclimatedUcp1-/-Mice

“…It is important to underscore the fact that UCP1-independent thermogenesis will not depend on only one mechanism, but in addition to Ca 2ϩ cycling, other mechanisms such as glycerol phosphate cycling could be important. It is also possible that SERCA activity in the mitochondria of interscapular brown adipocytes could be thermogenic as recently proposed by de Meis et al (29).…”

“…A robust thermogenic mechanism originally described in the heater organ of pelagic fish (22,26,27) was recently proposed to supplement the UCP1 thermogenesis in brown adipose tissue (17,28,29). These authors proposed that Ca 2ϩ -ATPase, normally transporting calcium back to its storage in endoplasmic reticulum, may be a source of heat production, when calcium transport is "uncoupled" from the ATP hydrolysis, thus rendering the functional activity of Ca 2ϩ -ATPase less efficient (28,30).…”

Inactivation of UCP1 and the Glycerol Phosphate Cycle Synergistically Increases Energy Expenditure to Resist Diet-induced Obesity

“…We have previously shown the potential involvement of Ca 2ϩ -ATPase-based thermogenesis for cold adaptation in inguinal fat of Ucp1 Ϫ/Ϫ mice (16) and in the skeletal muscle of Lep ob/ob ⅐Ucp1 Ϫ/Ϫ mice (30). Calcium cycling has been proposed as a heat-generating mechanism in fish thermogenic organs (31), mammalian malignant hyperthermia (32), avian nonshivering thermogenesis (33), and recently as an additional source of heat production contributing to brown adipose tissue thermogenesis (34). The importance of this mechanism in thermogenesis in the inguinal fat of Ucp1 Ϫ/Ϫ mice was shown by the significant changes in the phosphorylation of phospholamban, a Ca 2ϩ cycling regulatory component of SERCA (16).…”

An in vitro study of the ameliorative role of α-tocopherol on methotrexate-induced oxidative stress in rat heart mitochondria

“…Ca 2 + ATPase located within the mitochondria are involved in calcium homeostasis within the organelle. As there are reports of alterations in mitochondrial permeability, we assayed the Ca 2 + ATPase activity in samples incubated with MTX that was enhanced, indicating changes in Ca 2 + homeostasis (33) . Mitochondrial Ca 2 + uptake stimulates O 2 production (34) and Ca 2 + can increase reactive oxygen species production by the mitochondria of hypoxic heart tissue (35) .…”