Effects of P2 purinergic receptor stimulation in brown adipocytes

1997 ◽  
Vol 273 (2) ◽  
pp. C679-C686 ◽  
Author(s):  
S. C. Lee ◽  
P. A. Pappone
Keyword(s):  
Heat Production ◽  
Purinergic Receptor ◽  
Cytosolic Calcium ◽  
Extracellular Atp ◽  
Brown Fat ◽  
Brown Adipocyte ◽  
Fat Cells ◽  
Sympathetic Stimulation ◽  
Adrenergic Stimulation ◽  
Brown Adipocytes

Sympathetic stimulation of brown adipocytes plays a major role in body energy homeostasis by activating energy-wasting pathways. Sympathetic neuronal input initiates a variety of metabolic, developmental, and membrane responses in brown fat cells. Many of these actions are mediated by adrenergic pathways mobilized by released norepinephrine. However, since sympathetic stimulation may also release vesicular ATP, we tested brown fat cells for ATP responses. Micromolar concentrations of extracellular ATP had a number of effects on brown adipocytes. We have shown previously that ATP elicits substantial (average of approximately 30%) increases in cell membrane capacitance (P. A. Pappone and S. C. Lee, J. Gen. Physiol. 108: 393-404, 1996). Here, we show that cytosolic calcium levels were increased by ATP, both through release from intracellular stores and through influx, as assessed by fura 2 imaging. In addition, ATP indirectly activated a nonselective cation conductance that was independent of cytosolic calcium levels in patch voltage-clamped brown fat cells. Similar calcium, conductance, and capacitance responses could be activated by 2-methylthio-ATP and ADP, consistent with mediation by a P2 type purinergic receptor. Calorimetric measurements from cell suspensions showed that ATP increased basal heat production of isolated brown fat cells by approximately 40% but had no effect on the greater than fivefold increase in heat production seen with maximal adrenergic stimulation. These myriad responses to extracellular ATP suggest that P2 receptor-mediated signaling is important in brown adipocyte physiology and that sympathetic stimulation may normally activate purinergic as well as adrenergic pathways in brown fat.

scholarly journals Purinergic receptor stimulation increases membrane trafficking in brown adipocytes.

1996 ◽  
Vol 108 (5) ◽  
pp. 393-404 ◽  
Author(s):  
P A Pappone ◽  
S C Lee
Keyword(s):  
Membrane Trafficking ◽  
Energy Homeostasis ◽  
Purinergic Receptor ◽  
Sympathetic Innervation ◽  
Surface Membrane ◽  
Cytosolic Calcium ◽  
Membrane Capacitance ◽  
Adrenergic Stimulation ◽  
Membrane Expression ◽  
Brown Adipocytes

Stimulation of brown adipocytes by their sympathetic innervation plays a major role in body energy homeostasis by regulating the energy-wasting activity of the tissue. The norepinephrine released by sympathetic activity acts on adrenergic receptors to activate a variety of metabolic and membrane responses. Since sympathetic stimulation may also release vesicular ATP, we tested brown fat cells for ATP responses. We find that micromolar concentrations of extracellular ATP initiates profound changes in the membrane trafficking of brown adipocytes. ATP elicited substantial increases in total cell membrane capacitance, averaging approximately 30% over basal levels and occurring on a time scale of seconds to minutes. The membrane capacitance increase showed an agonist sensitivity of 2-methylthio-ATP > or = ATP > ADP > > adenosine, consistent with mediation by a P2r type purinergic receptor. Membrane capacitance increases were not seen when cytosolic calcium was increased by adrenergic stimulation, and capacitance responses to ATP were similar in the presence and absence of extracellular calcium. These results indicate that increases in cytosolic calcium alone do not mediate the membrane response to ATP. Photometric assessment of surface-accessible membrane using the dye FM1-43 showed that ATP caused an approximate doubling of the amount of membrane actively trafficking with the cell surface. The discrepancy in the magnitudes of the capacitance and fluorescence changes suggests that ATP both activates exocytosis and alters other aspects of membrane handling. These findings suggest that secretion, mobilization of membrane transporters, and/or surface membrane expression of receptors may be regulated in brown adipocytes by P2r purinergic receptor activity.

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

1993 ◽  
Vol 265 (1) ◽  
pp. R103-R110 ◽  
Author(s):  
M. Desautels ◽  
R. A. Dulos
Keyword(s):  
Prolonged Exposure ◽  
Uncoupling Protein ◽  
Calf Serum ◽  
Fold Increase ◽  
Brown Fat ◽  
Brown Adipocyte ◽  
Fat Cells ◽  
Cell Recovery ◽  
Succinate Dehydrogenase Activity ◽  
Brown Adipocytes

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.

Adrenergic modulation of intracellular pH in isolated brown fat cells from hamster and rat

1994 ◽  
Vol 267 (2) ◽  
pp. C349-C356 ◽  
Author(s):  
S. C. Lee ◽  
J. S. Hamilton ◽  
T. Trammell ◽  
B. A. Horwitz ◽  
P. A. Pappone
Keyword(s):  
Intracellular Ph ◽  
Uncoupling Protein ◽  
Cell Types ◽  
Brown Fat ◽  
Neonatal Rat ◽  
Fat Cells ◽  
Adrenergic Stimulation ◽  
Regulatory Systems ◽  
Ratio Imaging ◽  
Free Media

The activity of the uncoupling protein in brown fat mitochondria is enhanced at alkaline pH, leading to the hypothesis that changes in intracellular pH (pHi) may modulate the thermogenic response to sympathetic stimulation. We employed ratio imaging of the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to measure pHi in acutely isolated single brown fat cells from hamster and neonatal rat and in cultured rat cells. Basal pHi averaged approximately 7.2 in HCO3- media and 0.1-0.15 pH units lower in nominally HCO3(-)-free media in all cell types. In both HCO3- and HCO3(-)-free media, stimulation with norepinephrine (NE) typically caused an alkalinization of approximately 0.05-0.1 pH units, which was followed by a smaller net acidification occurring primarily after NE was removed. Alkalinization seemed to be mediated predominantly by alpha-adrenergic stimulation, while acidification most often followed beta-adrenergic activation. Similar pHi changes were elicited by NE in rat and hamster cells, but responses were more frequent in hamster cells. Assays of recovery from ammonium prepulse-induced acid loads indicated that rat and hamster cells have both Na(+)-H+ and Na(+)- and HCO3(-)-dependent regulatory systems, while hamster cells have, in addition, a Na(+)-independent recovery mechanism activated at acid pHi. We conclude that alpha-adrenergic alkalinization of brown fat may contribute to the control of thermogenesis.

scholarly journals Alpha-adrenergic stimulation activates a calcium-sensitive chloride current in brown fat cells.

1995 ◽  
Vol 106 (2) ◽  
pp. 231-258 ◽  
Author(s):  
P A Pappone ◽  
S C Lee
Keyword(s):  
Intracellular Calcium ◽  
Brown Fat ◽  
Chloride Conductance ◽  
Free Calcium ◽  
Fat Cells ◽  
Adrenergic Stimulation ◽  
Chloride Currents ◽  
Whole Cell ◽  
Perforated Patch ◽  
Whole Cell Recordings

The first response of brown adipocytes to adrenergic stimulation is a rapid depolarizing conductance increase mediated by alpha-adrenergic receptors. We used patch recording techniques on cultured brown fat cells from neonatal rats to characterize this conductance. Measurements in perforated patch clamped cells showed that fast depolarizing responses were frequent in cells maintained in culture for 1 d or less, but were seen less often in cells cultured for longer periods. Ion substitution showed that the depolarization was due to a selective increase in membrane chloride permeability. The reversal potential for the depolarizing current in perforated patch clamped cells indicated that intracellular chloride concentrations were significantly higher than expected if chloride were passively distributed. The chloride conductance could be activated by increases in intracellular calcium, either by exposing intact cells to the ionophore A23187 or by using pipette solutions with free calcium levels of 0.2-1.0 microM in whole-cell configuration. The chloride conductance did not increase monotonically with increases in intracellular calcium, and going whole cell with pipette-free calcium concentrations > or = 10 microM rapidly inactivated the current. The chloride currents ran down in whole-cell recordings using intracellular solutions of various compositions, and were absent in excised patches. These findings imply that cytoplasmic factors in addition to intracellular calcium are involved in regulation of the chloride conductance. The chloride currents could be blocked by niflumic acid or flufenamic acid with IC50s of 3 and 7 microM, or by higher concentrations of SITS (IC50 = 170 microM), DIDS (IC50 = 50 microM), or 9-anthracene carboxylic acid (IC50 = 80 microM). The chloride conductance activated in whole cell by intracellular calcium had the permeability sequence PNOS > PI > PBr > PCl > Paspartate, measured from either reversal potentials or conductances. Instantaneous current-voltage relations for the calcium-activated chloride currents were linear in symmetric chloride solutions. Much of the current was time and voltage independent and active at all membrane potentials between -100 and +100 mV, but an additional component of variable amplitude showed time-dependent activation with depolarization. Volume-sensitive chloride currents were also present in brown fat cells, but differed from the calcium-activated currents in that they responded to cell swelling, required intracellular ATP in whole-cell recordings, showed no sensitivity to intracellular or extracellular calcium levels, and were relatively resistant to block by niflumic and flufenamic acids. (ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Emergence during development of the white-adipocyte cell phenotype is independent of the brown-adipocyte cell phenotype

2001 ◽  
Vol 356 (2) ◽  
pp. 659-664 ◽  
Author(s):  
Karine MOULIN ◽  
Nathalie TRUEL ◽  
Mireille ANDRÉ ◽  
Emmanuelle ARNAULD ◽  
Maryse NIBBELINK ◽  
...  
Keyword(s):  
Brown Fat ◽  
Late Gestation ◽  
Brown Adipocyte ◽  
Cell Phenotype ◽  
Lacz Gene ◽  
Experimental Proof ◽  
Brown Adipocytes ◽  
White Adipocytes ◽  
Adipocyte Cell ◽  
White Fat

In mammals, two types of adipose tissue are present, brown and white. They develop sequentially, as brown fat occurs during late gestation whereas white fat grows mainly after birth. However, both tissues have been shown to have great plasticity. Thus an apparent transformation of brown fat into white fat takes place during post-natal development. This observation raises questions about a possible conversion of brown into white adipocytes during development, although indirect data argue against this hypothesis. To investigate such questions in vivo, we generated two types of transgenic line. The first carried a transgene expressing Cre recombinase specifically in brown adipocytes under the control of the rat UCP1 promoter. The second corresponded to an inactive lacZ gene under the control of the human cytomegalovirus promoter. This dormant gene is inducible by Cre because it contains a Stop sequence between two loxP sequences, separating the promoter from the coding sequence. Adipose tissues of progeny derived by crossing independent lines established from both constructs were investigated. LacZ mRNA corresponding to the activated reporter gene was easily detected in brown fat and not typically in white fat, even by reverse transcriptase PCR experiments. These data represent the first direct experimental proof that, during normal development, most white adipocytes do not derive from brown adipocytes.

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

1992 ◽  
Vol 262 (3) ◽  
pp. C678-C681 ◽  
Author(s):  
P. A. Pappone ◽  
M. T. Lucero
Keyword(s):  
Potassium Channels ◽  
Metabolic Response ◽  
Brown Fat ◽  
Neonatal Rat ◽  
K Channel ◽  
Fat Cells ◽  
Adrenergic Stimulation ◽  
K Channels ◽  
Voltage Gated ◽  
Calcium Activated Potassium Channels

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.

Thermogenically competent nonadrenergic recruitment in brown preadipocytes by a PPARγ agonist

2008 ◽  
Vol 295 (2) ◽  
pp. E287-E296 ◽  
Author(s):  
Natasa Petrovic ◽  
Irina G. Shabalina ◽  
James A. Timmons ◽  
Barbara Cannon ◽  
Jan Nedergaard
Keyword(s):  
Primary Cultures ◽  
Brown Adipocyte ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Sympathetic Stimulation ◽  
Promoting Effect ◽  
Brown Adipocytes ◽  
Peroxisome Proliferator Activated Receptor ◽  
Pparγ Agonist ◽  
Brown Adipose

Most physiologically induced examples of recruitment of brown adipose tissue (BAT) occur as a consequence of chronic sympathetic stimulation (norepinephrine release within the tissue). However, in some physiological contexts (e.g., prenatal and prehibernation recruitment), this pathway is functionally contraindicated. Thus a nonsympathetically mediated mechanism of BAT recruitment must exist. Here we have tested whether a PPARγ activation pathway could competently recruit BAT, independently of sympathetic stimulation. We continuously treated primary cultures of mouse brown (pre)adipocytes with the potent peroxisome proliferator-activated receptor-γ (PPARγ) agonist rosiglitazone. In rosiglitazone-treated cultures, morphological signs of adipose differentiation and expression levels of the general adipogenic marker aP2 were manifested much earlier than in control cultures. Importantly, in the presence of the PPARγ agonist the brown adipocyte phenotype was significantly enhanced: UCP1 was expressed even in the absence of norepinephrine, and PPARα expression and norepinephrine-induced PGC-1α mRNA levels were significantly increased. However, the augmented levels of PPARα could not explain the brown-fat promoting effect of rosiglitazone, as this effect was still evident in PPARα-null cells. In continuously rosiglitazone-treated brown adipocytes, mitochondriogenesis, an essential part of BAT recruitment, was significantly enhanced. Most importantly, these mitochondria were capable of thermogenesis, as rosiglitazone-treated brown adipocytes responded to the addition of norepinephrine with a large increase in oxygen consumption. This thermogenic response was not observable in rosiglitazone-treated brown adipocytes originating from UCP1-ablated mice; hence, it was UCP1 dependent. Thus the PPARγ pathway represents an alternative, potent, and fully competent mechanism for BAT recruitment, which may be the cellular explanation for the enigmatic recruitment in prehibernation and prenatal states.

Norepinephrine-induced sustained inward current in brown fat cells: α1-mediated by nonselective cation channels

2000 ◽  
Vol 279 (5) ◽  
pp. E963-E977 ◽  
Author(s):  
Ari Koivisto ◽  
Detlef Siemen ◽  
Jan Nedergaard
Keyword(s):  
Fold Increase ◽  
Brown Fat ◽  
Cation Channels ◽  
Current Response ◽  
Fat Cells ◽  
Adrenergic Stimulation ◽  
Open Probability ◽  
Inward Current ◽  
Nonselective Cation Channels ◽  
Sustained Inward Current

The nature of the sustained norepinephrine-induced depolarization in brown fat cells was examined by patch-clamp techniques. Norepinephrine (NE) stimulation led to a whole cell current response consisting of two phases: a first inward current, lasting for only 1 min, and a sustained inward current, lasting as long as the adrenergic stimulation was maintained. The nature of the sustained current was here investigated. It could be induced by the α1-agonist cirazoline but not by the β3-agonist CGP-12177A. Reduction of extracellular Cl− concentration had no effect, but omission of extracellular Ca2+ or Na+ totally eliminated it. When unstimulated cells were studied in the cell-attached mode, some activity of ≈30 pS nonselective cation channels was observed. NE perfusion led to a 10-fold increase in their open probability (from ≈0.002 to ≈0.017), which persisted as long as the perfusion was maintained. The activation was much stronger with the α1-agonist phenylephrine than with the β3-agonist CGP-12177A, and with the Ca2+ionophore A-23187 than with the adenylyl cyclase activator forskolin. We conclude that the sustained inward current was due to activation of ≈30 pS nonselective cation channels via α1-adrenergic receptors and that the effect may be mediated via an increase in intracellular free Ca2+ concentration.

White, brite, and brown adipocytes: the evolution and function of a heater organ in mammals

2014 ◽  
Vol 92 (7) ◽  
pp. 615-626 ◽  
Author(s):  
Yongguo Li ◽  
David Lasar ◽  
Tobias Fromme ◽  
Martin Klingenspor
Keyword(s):  
Adipose Tissue ◽  
Molecular Mechanisms ◽  
Uncoupling Protein ◽  
Brown Fat ◽  
Brown Adipocyte ◽  
Nonshivering Thermogenesis ◽  
Inner Mitochondrial Membrane ◽  
Brown Adipocytes ◽  
Mitochondrial Density ◽  
Beige Adipocytes

Brown fat is a specialized heater organ in eutherian mammals. In contrast to the energy storage function of white adipocytes, brown adipocytes dissipate nutrient energy by uncoupling of mitochondrial oxidative phosphorylation, which depends on uncoupling protein 1 (UCP1). UCP1, as well as UCP2 and UCP3, belong to the family of mitochondrial carriers inserted into the inner mitochondrial membrane for metabolite trafficking between the matrix and the intermembrane space. UCP1 transports protons into the mitochondrial matrix when activated by a rise in free fatty acid levels in the cell. This UCP1-dependant proton leak drives high oxygen consumption rates in the absence of ATP synthesis and dissipates proton motive force as heat. The enormous heating capacity of brown fat is supported by dense vascularization, high rates of tissue perfusion, and high mitochondrial density in brown adipocytes. It has been known for more than 50 years that nonshivering thermogenesis in brown fat serves to maintain body temperature of neonates and small mammals in cold environments, and is used by hibernators for arousal from torpor. It has been speculated that the development of brown fat as a new source for nonshivering thermogenesis provided mammals with a unique advantage for survival in the cold. Indeed brown fat and UCP1 is found in ancient groups of mammals, like the afrotherians and marsupials. In the latter, however, the thermogenic function of UCP1 and brown fat has not been demonstrated as of yet. Notably, orthologs of all three mammalian UCP genes are also present in the genomes of bony fishes and in amphibians. Molecular phylogeny reveals a striking increase in the substitution rate of UCP1 between marsupial and eutherian lineages. At present, it seems that UCP1 only gained thermogenic function in brown adipocytes of eutherian mammals, whereas the function of UCP1 and that of the other UCPs in ectotherms remains to be identified. Evolution of thermogenic function required expression of UCP1 in a brown-adipocyte-like cell equipped with high mitochondrial density embedded in a well-vascularized tissue. Brown-adipocyte-like cells in white adipose tissue, called “brite” (brown-in-white) or “beige” adipocytes, emerge during adipogenesis and in response to cold exposure in anatomically distinct adipose tissue depots of juvenile and adult rodents. These brite adipocytes may resemble the archetypical brown adipocyte in vertebrate evolution. It is therefore of interest to elucidate the molecular mechanisms of brite adipocyte differentiation, study the bioenergetic properties of these cells, and search for the presence of related brown-adipocyte-like cells in nonmammalian vertebrates.

Sign in / Sign up

Export Citation Format

Share Document