Beta-adrenergic modulation of Ca2+ uptake by isolated brown adipocytes. Possible involvement of mitochondria (original) (raw)
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Journal of Bioenergetics and Biomembranes, 1996
The role of the adenine nucleotide translocase on Ca2+ homeostasis in mitochondria from brown adipose tissue was examined. It was found that in mitochondria incubated with 50 μM Ca2+, ADP was not needed to retain the cation, but it was required for strengthening the inhibitory effect of cyclosporin on membrane permeability transition as induced by menadione. In addition, carboxyatractyloside was unable to promote matrix Ca2+ release, even though it inhibits the ADP exchange reaction. However, when the Ca2+ concentration was increased to 150 μM, carboxyatractyloside did induce Ca2+ release, and ADP favored Ca2+ retention. Determination of cardiolipin content in the inner membrane vesicles showed a greater concentration in brown adipose tissue mitochondria than that found in kidney mitochondria. It is suggested that the failure of the adenine nucleotide translocase to influence membrane permeability transition depends on the lipid composition of the inner membrane.
Sodium-induced calcium release from mitochondria in brown adipose tissue
Proceedings of the National Academy of Sciences, 1979
Coupled mitochondria of brown adipose tissue can accumulate Ca2+ if a substrate is present. The Ca2+ is released by addition of 20 mM Na+, but not by addition of K+ or choline +. Energy-dissipating Na+-induced Ca2+ cycling occurs maximally with 20 mM Na+ and 10 microM Ca2+. In brown adipocytes, the Ca2+ ionophore A23187 and the Na+ ionophore monensin increase respiration if substrate is added, and incubation in a low-Na+ buffer decreases norepinephrine-induced respiration. Thus Na+-induced Ca2+ release takes place in brown adipose tissue; released Ca2+ could have a regulatory or thermogenic role or both.
Biochimica et biophysica acta. Molecular cell research, 1985
We have previously demonstrated mobilization of Ca z+ in and efflux ~ff Rb ÷ (K +) from isolated hamster [q'cm'n adipocytes as a consequence of norepinephrine stimulation. We ha~e now investigated the adrenoceptot subtype specificity, of these responses and found them both to he of the aw-sub~pe. Further, we ha~e found that the, Rb+(K *) efflux was dependent upon a primao Ca z+ mobilization event in response to the al.adrenerj~ie stimulati~m, since the Rb + efflux could also he demonstrated b.~ the addition of the Ca z+ hmophore A23187 to the cells. The norepincphrine-and A23187-stimulated Rb ÷ effluxes were both inhibited by the Ca2+-dependent K +.channel blocker apamin. Apemin ",dse significantly attenuated Ca z * mobilization In cells in response to a submaximal concentration of not'epinephrine. We conclude that at-adrenergic stimulation ¢d brown fat cells lea~ to a mobilization of intracellular Ca z+ which, in itself or via other Inechanis~. leads to an iuerease in cytosolic Ca 2+ concentration which, in turn, activates a CaZ+-dependent K ÷ channel, k.ading to a K + relea~ from these cells. A possible role for this channel to sustain and augment the response to ,,t-adrenergic stimulation is discussed. Introduelion The mechanism of action of oq-adrenergic agents in mamlnal~.an cells has not as yet been clarified. Hov*ever. teliular ionic events accompanying stimulation b~ lhe~e agents are being reported. Bro,~n adip,xytes (for general review see Ref. l) have been shown to posse,,s ,r**-adrenergic receptors 12], ot-adrenergically reduced increases in the turnover of phcspbatidylingsitol [3.4 I, and an eqmediated ct)n|~toncl~,* of norepinephrin-stimulated respiration (thermogenc~is) [5-7]. Norepinephrine also leads to a rapid mobilization of Ca:' from intra,:'ellular stores in these cells, and this mobilization is specifically dependent upon the presence of exlracellular Ya + [8]. An observed a-adrenergic dep.larizati~m [9 11] in brown adipocytes after norepinephrine addition seems to be associated with ;in incr.~ased plasma membrane ion permea-0167.4889/85/$03.30 ' 1985 E.Isevter Science Publisher, B.V. bilily {12], probably for Na" [9]. Norepinephrine also induces a K ~ efflux (observable as an Xt'Rb ~ efflux) in brown adipoc?tes [131. 11ere we investigate the relationship between the ('a 2" mobilization and the K" efflux in an at:erupt to define the mechanism by which the potassium ions ate lost and the possible ph)siological function of this horm~me-~,timulated e',ent Some of these results ha~.e r~,..en pre,ented tn preliminary form [14 17]. Materials and Methods (~,1! preparation Ccll~ ~ere prepared from the pooled bro~n adipo.,,c tissue of adult Syrian hamsters (Afe~o-('rt('elus auralll~) by collagenase digestion a~ tiescribed previousl 5 [~l. The aaimals v, ere mainrained at 22°C on an ad libltum rabbit-chow/sunflower-seed diet and water.
Characterisation of Ca2+ transport activity by white adipose tissue mitochondria
FEBS Letters, 1983
Ca2' transport in mitochondria isolated from rat white adipocytes has been examined and many of the properties found to be similar to those reported for mitochondria isolated from rat liver. Ca" transport is ruthenium red-sensitive (Ki -5 pmol . mg protein-'), the affinity for free Ca2' is high (Km -3.3 PM) and the V,, is 135 nmol Ca2+ .min-'.mg protein-' at 4°C with 0.2 mM Pi present. Ca" transport is stimulated by increasing the medium [Pi], and is inhibited when ATP or Mg2+ is added to the incubation system and in contrast to brown adipocyte mitochondria, Ca2' efflux is not promoted by Na+. White adipocyte mitochondria may play a role in the regulation of total cell calcium in this tissue.
Mechanism of intracellular calcium ([Ca2+]i) inhibition of lipolysis in human adipocytes
The FASEB Journal, 2001
We investigated the mechanisms responsible for the anti-lipolytic effect of intracellular Ca 2+ ([Ca 2+ ]i) in human adipocytes. Increasing [Ca 2+ ]i inhibited lipolysis induced by -adrenergic receptor activation, A1 adenosine receptor inhibition, adenylate cyclase activation, and phosphodiesterase (PDE) inhibition, as well as by a hydrolyzable cAMP analog, but not by a nonhydrolyzable cAMP analog. This finding indicates that the anti-lipolytic effect of [Ca 2+ ]i may be mediated by the activation of adipocyte PDE. Consistent with this theory, [Ca 2+ ]i inhibition of isoproterenol-stimulated lipolysis was reversed completely by the nonselective PDE inhibitor isobutyl methylxanthine and also by the selective PDE 3B inhibitor cilostamide, but not by selective PDE 1 and 4 inhibitors. In addition, phosphatidylinositol-3 kinase inhibition with wortmannin completely prevented insulin's anti-lipolytic effect but only minimally blocked [Ca 2+ ]i's effect, which suggests that [Ca 2+ ]i and insulin may activate PDE 3B via different mechanisms. In contrast, the antilipolytic effect of [Ca 2+ ]i was not affected by inhibitors of calmodulin, Ca 2+ /calmodulin-dependent kinase, protein phosphatase 2B, and protein kinase C. Finally, [Ca 2+ ]i inhibited significantly isoproterenol-stimulated increases in cAMP levels and hormone-sensitive lipase phosphorylation in human adipocytes. In conclusion, increasing [Ca 2+ ]i exerts an antilipolytic effect mainly by activation of PDE, leading to a decrease in cAMP and HSL phosphorylation and, consequently, inhibition of lipolysis.
Biochemical Journal, 1980
The incorporation of [32p]p1 into phosphatidylinositol by rat fat-cells was markedly increased in the presence of adrenaline. Phosphatidic acid labelling was also increased, but to a lesser extent. These effects are due to a-adrenergic stimulation since they were unaffected by propranolol, blocked by a-blockers in the potency order prazosin < phentolamine < yohimbine and mimicked by methoxamine. The a-adrenergic stimulation of phosphatidylinositol labelling did not require extracellular Ca2 , which supports the hypothesis that an increased turnover of phosphatidylinositol is involved in a-adrenergic activation of Ca2+ entry. Insulin and the ionophore A23187 gave a small increase in 32p labelling of phosphatidylinositol in Ca2+-free medium containing 1mM-EGTA. The increases due to insulin or ionophore A23187 were abolished if 2.5 mM-Ca2+ was added to medium containing EGTA. However, the increases in labelling of phosphatidylinositol due to a-adrenergic amines were still evident in medium containing EGTA and Ca2+. Lipolytic agents such as corticotropin, dibutyryl cyclic AMP, adrenaline in the presence of phentolamine and isoproterenol decreased [32p]p1 incorporation into phosphatidylinositol, phosphatidylethanolamine and phosphatidic acid. This inhibitory effect may be secondary to accumulation of intracellular unesterified fatty acids, since it was decreased by incubating fewer cells in medium with 6 rather than 3% albumin and was restored by the addition of oleate to the medium. The incorporation of [ 32p1p1 into phosphatidylcholine was unaffected by lipolytic agents. The data suggest that there is an inhibition of the synthesis of certain phospholipids in the presence of lipolytic agents, which may be secondary to intracellular accumulation of unesterified fatty acids. In adipocytes ,B-adrenergic stimulators activate adenylate cyclase and lipolysis. The role of cyclic AMP as a second messenger for fl-adrenergic stimulation is well established (Fain, 1979). Much less is known about a-adrenergic effects. There is little effect of a-adrenergic activati;n on glucose oxidation, cyclic AMP accumulation and lipolysis in rat fat-cells (Fain, 1979). However, Lawrence & Larner (1978) reported that activation of rat adipocyte phosphorylase and inactivation of glycogen synthase are produced by stimulation of aadrenergic receptors. The inactivation of glycogen synthase by a-adrenergic catecholamines was dependent on the presence of extracellular Ca2+ and may be secondary to increased entry of Ca2+ (Lawrence & Larner, 1978). Ca2+ has been postulated as a second messenger for a-adrenergic stimulation (Fain, 1979) and has also been implicated in insulin action (Fraser, 1975).
Metabolism, 1991
Previous studies have shown that thyroid status affects the response of brown adipose tissue (BAT) to the sympathetic nervous system. For example, hypothyroidism is associated with the development of a marked synergism between LX,-and p-adrenergic pathways to stimulate type II iodothyronine 5'-deiodinase activity. Hypothyroidism also attenuates the respiratory response (thermogenesis) of isolated brown adipocytes to norepinephrine. To explore the interactions of the sympathetic nervous system and thyroid status in these cells, we compared the thermogenic and 5'-deiodinase responses to adrenergic agonists in isolated brown adipocytes from hypothyroid rats during treatment with 3,5,3'-triiodothyronine (T,). The fivefold synergism of q-and 5-adrenergic catecholamines to increase the deiodinase activity was progressively reduced, reaching a control euthyroid value of unity after 5 days of T, treatment. Hypothyroidism reduced both the 0,max (twofold to threefold) and increased the concentration of agonist required for 50% stimulation (lo-fold) for both norepinephrine and forskolin. In hypothyroid cells, there was a twofold synergism between the o,-agonist cirazoline and forskolin to increase respiration, which was blocked by prazosin and reproduced by the calcium ionophore, A23187. This synergistic effect of the q-agonist was lost within 2 days of T, administration. These studies identify a second Ca'+-dependent intraadrenergic synergism, which functions to ameliorate the reduced cyclic adenosine monophosphate (CAMP) responsiveness of the hypothyroid brown adipocyte.
European Journal of Biochemistry, 1983
The ability of isolated mitochondria from rat brown-adipose tissue to regulate extramitochondrial Ca2+ (measured by arsenazo) was studied in relation to their ability to produce heat (measured polarographically). The energetic state of the mitochondria was expressed as a membrane potential, delta psi (estimated with safranine), and was varied semi-physiologically by the use of different GDP concentrations. In these mitochondria GDP binds to the 32-kDa polypeptide, thermogenin, which regulates coupling. Ca2+ uptake (at 5 microM extramitochondrial Ca2+) was maximal at delta psi greater than 150 mV. Basal Ca2+ release increased from 1 to 2 nmol x min-1 x mg-1 below 150 mV. Na+ -stimulated rate of Ca2+ release was stable within the investigated delta psi span (100-160 mV). Initial Ca2+ levels were maintained below 0.2 microM for 100 mV less than delta psi less than 160 mV. Ca2+ levels maintained after Ca2+ challenge (20 nmol Ca2+ x mg-1) were below 0.4 microM for delta psi greater than 135 mM. Respiration was unstimulated for delta psi greater than 150 mV and was maximal at delta psi less than or equal to 135 mV. In the presence of well-oxidised substrates, the respiration at maximally activated thermogenin was markedly below fully uncoupled respiration and was probably limited by thermogenin activity--i.e. by a limited H+ reentry (OH- exit) and therefore by a membrane potential maintained at about 135 mV. It is concluded that at membrane potentials of 135 mV and above the mitochondria exhibit full Ca2+ control and are able to regulate thermogenic output up to maximum without interfering with this Ca2+ control. Membrane potential probably does not decrease below 135 mV in vivo. Therefore, Ca2+ homeostasis and thermogenesis are non-interfering and can be hormonally independently regulated, e.g. by alpha-adrenergic and beta-adrenergic stimuli, respectively.
α1-Adrenergic stimulation of Cl− efflux in isolated brown adipocytes
FEBS Letters, 1990
Unidirectional 3*Cl-efflux from preloaded isolated brown adipocytes was studied. A norepinephrine-stimulated 36C1-efflux pathway was found which approximately doubled the rate of 36Cl-effl~ from the cells. The response to norepinep~ine was fully i~bit~ by the ar-adrenergic antagonist praxosin, but was una&cted by the padrenergic antagonist propranolol, showing that norepinephrine stimulated the 36CI-efflux pathway via the a,adrenoceptor. The stimulation of 36C1-e8Iux could not be mimicked by the Caz+ ionophore A23187, indicating that the effect was not mediated by elevation of the intracellular Ca 2+ level. It is concluded that brown fat cells possess a specific mechanism for cc,-adrenergic stimulation of Cl-efflux. The possibility is discussed that this Cl-efflux pathway could be the basis for the early a-adrenergic depolarization seen in brown fat cells.
European Journal of Histochemistry, 2014
Mitochondria are key organelles maintaining cellular bioenergetics and integrity, and their regulation of [Ca 2+ ] i homeostasis has been investigated in many cell types. We investigated the short-term Ca-SANDOZ ® treatment on brown adipocyte mitochondria, using imaging and molecular biology techniques. Two-monthold male Wistar rats were divided into two groups: Ca-SANDOZ ® drinking or tap water (control) drinking for three days. Alizarin Red S staining showed increased Ca 2+ level in the brown adipocytes of treated rats, and potassium pyroantimonate staining localized electrondense regions in the cytoplasm, mitochondria and around lipid droplets. Ca-SANDOZ ® decreased mitochondrial number, but increased their size and mitochondrial cristae volume. Transmission electron microscopy revealed numerous enlarged and fusioned-like mitochondria in the Ca-SANDOZ ® treated group compared to the control, and megamitochondria in some brown adipocytes. The Ca 2+ diet affected mitochondrial fusion as mitofusin 1 (MFN1) and mitofusin 2 (MFN2) were increased, and mitochondrial fission as dynamin related protein 1 (DRP1) was decreased. Confocal microscopy showed a higher colocalization rate between functional mitochondria and endoplasmic reticulum (ER). The level of uncoupling protein-1 (UCP1) was elevated, which was confirmed by immunohistochemistry and Western blot analysis. These results suggest that Ca-SANDOZ ® stimulates mitochondrial fusion, increases mitochondrial-ER contacts and the thermogenic capacity of brown adipocytes.