Rat Brown Fat Cells Research Articles

Protective effects of noradrenaline against tumor necrosis factor-α-induced apoptosis in cultured rat brown adipocytes: role of nitric oxide-induced heat shock protein 70 expression

To elucidate the effects and molecular mechanism(s) by means of which noradrenaline (NA) protects against the tumor necrosis factor (TNF)-alpha-induced apoptosis of brown adipocytes. Brown fat precursor cells were isolated from young rats; 2.5 million cells were added to each 24-well culture plate and cultured in a defined culture medium. On day 8, the cultured cells were exposed to murine recombinant TNF-alpha and/or cycloheximide (CHX; 10 microg/ml) and/or NA and/or nitric oxide (NO) donors and/or the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and/or 10 microM heat shock protein 70 (HSP70) antisense or sense oligomers. Analysis of DNA fragmentation on agarose gel as a marker of apoptosis; reverse transcriptase-polymerase chain reaction analysis of mRNA levels; Western blotting analysis of protein levels. Pretreatment of primary cultures of rat brown fat cells with micromolar concentrations of NA or the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) induced the expression of HSP70 mRNA and protein, which was associated with cytoprotection against TNF-alpha plus CHX-induced apoptosis. The L-NAME inhibitor of NO synthase activity inhibited both NA-stimulated HSP70 expression and cytoprotection. Furthermore, pretreatment of brown adipocytes with an antisense oligonucleotide to HSP70 antagonized both SNAP- and NA-induced cytoprotection. These findings demonstrate that the NO produced by NA stimulation can induce resistance to the TNF-alpha-induced apoptosis of brown adipocytes, possibly by means of the expression of HSP70.

Thermogenesis is beta3- but not beta1-adrenergically mediated in rat brown fat cells, even after cold acclimation.

To examine if acclimation of rats to cold led to alterations in the coupling between different beta-receptor subtypes and thermogenesis in brown fat cells, we investigated the adrenergic response patterns in brown fat cells isolated from warm-acclimated (28 degreesC) and cold-acclimated (4 degreesC) rats. In the cells from warm-acclimated rats, the relative affinities (EC50) for different agonists (isoprenaline, BRL-37344, norepinephrine, CGP-12177, dobutamine, and salbutamol) were those expected from their interaction with a beta3-receptor. The response to norepinephrine was competitively inhibited by propranolol with a pA2 of approximately 6, implying interaction at the beta3-receptor. No evidence for a beta1-receptor-mediated response to the beta1-selective agonist dobutamine could be obtained; the low-affinity response observed was most likely through the beta3-receptor. The beta1-antagonist ICI-89406 could not inhibit a specific fraction of the thermogenic response to norepinephrine. Thus beta3-receptors were the only beta-receptors involved in the control of thermogenesis in brown fat cells from warm-acclimated rats. A modified method of preparation was developed to isolate functional cells from cold-acclimated animals. Also in these cells, the beta-receptor coupled to thermogenesis was the beta3-receptor, although the response was desensitized with an approximately sevenfold shift in EC50 values. The pA2 for propranolol inhibition of norepinephrine-induced thermogenesis was also 6 here, and that for ICI-89406 was 5.5, also implying interaction at the beta3-receptor. Thus acclimation to cold did not alter the beta-adrenergic receptor subtype (beta3) involved in the control of thermogenesis.

Apparent thermogenic effect of injected glucagon is not due to a direct effect on brown fat cells.

To examine the significance of brown adipose tissue for the thermogenic response to glucagon, we injected glucagon intraperitoneally into rats (that have glucagon-sensitive brown fat cells) and into hamsters (that have glucagon-insensitive brown fat cells). Although a thermogenic response to glucagon injection was apparently observed in rats, this response was not augmented by cold acclimation and was not dose dependent. Similar observations were made in hamsters. The thermogenic response could be fully blocked by prior injection of the beta-adrenergic blocker propranolol. Thus no direct thermogenic response to injected glucagon could be demonstrated, and the thermogenic response observed was fully due to vehicle injection. However, glucagon injection was able to unmask mitochondrial [3H]GDP binding. As expected, isolated brown fat cells from rats and mice responded thermogenically to glucagon but brown fat cells from hamsters were unresponsive. The EC50 of the rat brown fat cells was high (5 nM); these cells also responded to secretin, with an EC50 of 22 nM. It was concluded that, in contrast to earlier observations, no thermogenic response to injected glucagon could be observed; this may be related to differences in glucagon preparations. Brown fat cells from certain species are, however, glucagon sensitive. It is uncertain whether glucagon is the endogenous agonist for these receptors, but the presence of the glucagon-responsive receptor indicates alternative means to norepinephrine for stimulation of brown adipose tissue thermogenesis and, probably, of recruitment.

Regulation of the Activity of 27 pS Nonselective Cation Channels in Excised Membrane Patches from Rat Brown-Fat Cells

The regulation of the activity of the ≈30 pS nonselective cation channel (NSC channel) was studied by the patch-clamp technique in inside-out patches obtained from rat brown-fat cells. NSC channel activity was induced by excision; reduced redox state induced by dithiothreitol accelerated the kinetics in the excised state. The NSC channels were inhibited by the fenamates flufenamic acid and mefenamic acid but not by NS-1619 or SKF-96365. The channels were inhibited by purine nucleotides but not by polyamines. No evidence for protein kinase C, CaM kinase or protein kinase A activation of the NSC channel was obtained. NSC-channel activity was stimulated in a concentration-dependent manner by Ca²⁺ but the EC₅₀ was very high (0.81 mM), in comparison to expected cytosolic Ca²⁺ levels. In the presence of ATP, even higher Ca²⁺ levels were necessary for comparable NSC-channel activation. The increase in P_o was not associated with an increase in open-time constants. We conclude that although high Ca²⁺ levels can experimentally activate the NSC channel, a further mediatory step must probably be postulated in order to link α₁-adrenergic stimulation to NSC-channel activation.

Modulation of calcium‐activated non‐selective cation channel activity by nitric oxide in rat brown adipose tissue.

1. Single-channel calcium-activated non-selective cation currents from isolated rat brown fat cells were measured using the inside-out patch configuration of the patch-clamp technique. The existence of a possible modulatory effect of nitric oxide on the putative redox-modulatory site located on the intracellular side of the non-selective cation channel was investigated. 2. The nitric oxide-releasing substances nitroglycerin, sodium nitroprusside, S-nitrosocysteine and S-nitroso-N-acetyl-D,L-penicillamine (all at 100 microM) were able to block channel activity almost completely. 3. In each case the blockade was persistent and could not be washed away. Dithiothreitol (DTT, 2 mM) was able to reverse the blockade to a large extent, whereas oxidized DTT (2 mM) was without effect. 4. It was concluded that nitric oxide can modulate non-selective cation channel activity by oxidizing sulfhydryl groups and that this effect can be reversed by reduction.

Norepinephrine does not stimulate protein and UCP synthesis in brown adipocytes of golden Syrian hamsters

Hamster brown adipocytes were incubated for up to 24 h with or without norepinephrine (NE) in Dulbecco's modified Eagle's medium supplemented with bovine serum albumin, calf serum, and antibiotics. Brown fat cells were viable for 24 h as defined by their ability to respond to NE by a 10-fold increase in oxygen consumption. However, prolonged exposure of the cells to NE led to a decline in NE-stimulated rates of O2 consumption, which was not the result of loss of cell thermogenic capacity. Brown fat cells incubated for 24 h with or without NE showed no significant change in succinate dehydrogenase activity or uncoupling protein (UCP) content. However, cell recovery after 24 h was significantly reduced in the absence of NE. In brown adipocytes isolated from rat, NE increased [35S]methionine incorporation into cell proteins and UCP. In contrast, [35S]methionine incorporation in hamster brown adipocyte proteins and UCP was greater than in rat brown fat cells and was not increased by NE. These results indicate that although NE may be required for cell survival, it does not stimulate protein and UCP synthesis in hamster brown fat cells.

Adrenergically activated Ca2+ increases in brown fat cells: effects of Ca2+, K+, and K channel block.

We measured intracellular calcium concentration ([Ca2+]i) during adrenergic stimulation using fura-2 ratio imaging of individual cultured neonatal rat brown fat cells. One micromolar norepinephrine (NE) increased [Ca2+]i from an average resting value of 105 nM to 555 nM in approximately 30 s. [Ca2+]i remained elevated as long as NE was present but returned to resting levels within 2-3 min after NE removal. The response was half maximal at approximately 50 nM NE and was primarily alpha-adrenergic. The sustained, but not the initial, increase in [Ca2+]i required extracellular calcium. Cells stimulated in high-K media had [Ca2+]i responses like those in 0 Ca2+, suggesting that depolarization abrogates calcium influx. Parallel perforated-patch recordings showed that the increase in [Ca2+]i activates a calcium-activated K conductance. Blocking K channels with moderate concentrations of tetraethylammonium (TEA) had only small effects on NE-induced changes in [Ca2+]i, but high concentrations of TEA significantly reduced the response. We conclude that cytoplasmic calcium is modulated by fluxes from both intracellular and extracellular sources and that K channels may not be required for normal short-term [Ca2+]i responses to hormone.

Potassium channel block does not affect metabolic responses of brown fat cells.

Hormonally stimulated brown fat cells are capable of extremely high metabolic rates, making them an excellent system in which to examine the role of plasma membrane ion channels in cell metabolism. We have previously shown that brown fat cell membranes have both voltage-gated and calcium-activated potassium channels (Voltage-gated potassium channels in brown fat cells. J. Gen. Physiol. 93: 451-472, 1989; Membrane responses to norepinephrine in cultured brown fat cells. J. Gen. Physiol. 95: 523-544, 1990). Currents through both the voltage-activated potassium channels, IK,V, and the calcium-activated potassium channels, IK,Ca, can be blocked by the membrane-impermeant K channel blocker tetraethylammonium (TEA). We used microcalorimetric measurements from isolated neonatal rat brown fat cells to assess the role these potassium conductances play in the metabolic response of brown fat cells to adrenergic stimulation. Concentrations of TEA as high as 50 mM, sufficient to block approximately 95% of IK,V and 100% of IK,Ca, had no effect on norepinephrine-stimulated heat production. These results show that neither voltage-gated nor calcium-activated K channels are necessary for a maximal thermogenic response in brown fat cells and suggest that K channels are not involved in maintaining cellular homeostasis during periods of high metabolic activity.

Immunocytochemistry of the Microtubules of fat-laden cells. Brown fat cells and adrenocortical cells in primary monolayer culture.

Cytoplasmic microtubules of fat-laden cells containing fine lipid droplets, such as brown fat cells and adrenocortical cells, were studied in relation to the metabolism of intracellular lipid. In these cells, the amount and distribution of lipid droplets reflect the state of inherent cellular function. Materials used were primary monolayer culture of fetal rat brown fat cells and that of bovine adrenocortical cells. The method was the immunocytochemistry with anti-tubulin antibody. When brown fat cells were being lipolyzed or the steroidogenesis of adrenocortical cells were being stimulated, the cytoplasmic microtubules in the cells were organized in a radial pattern in response to the behavior of the lipid droplets. It is assumed that the microtubules were in the regulation of cellular function in terms of the metabolism of lipid droplets in these cells. We have devised, in the course of the current study, a double fluorescence technique as an observational method whereby microtubules were observed immunocytochemically and lipid droplets by a secondary fluorescence with phosphine E staining.

Oligomycin effects on lipolysis and the oxidative metabolism of brown fat cells

1. Oligomycin inhibited the respiration of rat brown fat cells incubated in the absence but not in the presence of 10 mM glucose. 2. Oligomycin increased the conversion of glucose to CO 2 in brown fat cells. In white fat cells oligomycin diminished the conversion of glucose to CO 2. 3. m-Chloro(carbonyl cyanide)phenylhydrazone doubled respiration by brown fat cells and markedly increased the conversion of glucose to CO 2 over a 2-h period in the presence of glucose. In the absence of glucose, m-chloro(carbonyl cyanide)phenylhydrazone reduced respiration of brown fat cells by over 50%. 4. Octanoic acid (0.9 mM) increased O 2 consumption of brown fat cells over 10-fold in the presence of glucose and 4-fold in the absence of glucose. Oligomycin had only a small effect on octanoate conversion to CO 2 or octanoate-induced respiration of brown fat cells in the presence of glucose, but in the absence of glucose oligomycin markedly inhibited these parameters. 5. Oligomycin blocked the lipolytic response of both brown and white fat cells to epinephrine and theophylline if the antibiotic was added prior to the lipolytic agents. 6. These data indicate that in the presence of glucose both basal and fatty acid-stimulated respiration in brown fat cells is largely oligomycin insensitive under conditions in which hormonal activation of lipolysis is blocked by oligomycin.

Potassium-dependent Stimulation of Respiration in Brown Fat Cells by Fatty Acids and Lipolytic Agents

Abstract The omission of K+ from the buffer used for isolation and incubation of rat brown fat cells blocked the large increases in respiration seen after the addition of lipolytic agents. The effect of K+ was on the activation of respiration by free fatty acids rather than upon the lipolytic action of agents such as theophylline. Rb+ and Cs+, but not NH4+, could partially substitute for K+ in the activation of respiration by theophylline. Valinomycin, an antibiotic which activates the uptake of K+ by isolated mitochondria, markedly stimulated respiration in brown fat cells in the presence but not in the absence of K+. Nigericin, another antibiotic which blocks K+ uptake by mitochondria, also blocked the increase in respiration due to valinomycin and theophylline. The effects of lipolytic agents on respiration could be mimicked by addition of albumin-bound free fatty acids to brown fat cells, an effect which was also K+-dependent. The omission of Ca++ and Mg++ reduced the activation of respiration by fatty acids or lipolytic agents. These findings indicate that the activation of energy metabolism by fatty acids involves a K+-dependent process which is influenced by divalent cations. The increased oxygen consumption may be secondary to utilization of high energy intermediates of oxidative phosphorylation for increased K+ flux across membranes.