Purinergic Receptor Stimulation Increases Membrane Trafficking in Brown Adipocytes PAMELA A. PAPPONE and
SHERWIN C. LEE
From the Section of Neurobiology, Physiology and Behavior, Division of Biological Sciences, University of California, Davis, California 95616
ABSTRACT 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 ~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 --> ATP > ADP > > adenosine, consistent with mediation by a Pzv 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, a n d / o r surface membrane expression of receptors may be regulated in brown adipocytes by P2Ypurinergic receptor activity. KEY WORDS: exocytosis * ATP * P2 purinergic receptor 9 FM1-43 INTRODUCTION
T h e m a j o r role of adipocytes is to store and mobilize body energy reserves. Until recently it was t h o u g h t that adipocytes were essentially passive deposition sites in this process, effecting the net uptake or release of metabolic energy in response to h o r m o n a l c o m m a n d s generated elsewhere. However, a rapidly growing body of evidence indicates that fat cells play an active role in regulating body nutritional energy homeostasis and body weight (Flier, 1995). T h e regulatory mechanisms involved are c o m p l e x a n d incompletely u n d e r s t o o d but are known to be m e d i a t e d by extensive cross-talk between fat and a n u m b e r of o t h e r tissues. Adipocyte proliferation, differentiation, and energy handling are m o d u l a t e d by a wide variety o f n e u r o n a l a n d circulating effectors, and adipocytes in turn secrete a n u m b e r of products that act as autocrine, paracrine, or endocrine factors to affect fat cell properties and energy ho-
Preliminary results of these studies have been previously presented in abstract form (Pappone, P.A., and S.C. Lee. 1994. Biophys.J. 66:429A; Pappone, P.A., S.C. Lee, and S.I. Ortiz-Miranda. 1994.J. Cell. Biochem. Suppl. 18A:176; Pappone, P.A., and S.C. Lee. 1996. Biophys.J. 70:A85). Address correspondence to Pamela Pappone, Section of Neurobiology, Physiology and Behavior, Division of Biological Sciences, University of California, Davis, California 95616. Fax: 916-752-5582; E-mail:
[email protected] 393
meostasis ( H i m m s - H a g e n 1989; Ailhaud et al., 1992; Spiegelman and Hotamisligil, 1993). Awareness of these interactions has fueled growing interest in the receptor responses of fat cells. Two classes of adipocyte are involved in maintaining nutritional energy homeostasis. White adipocytes subserve the classic fat cell role of storing energy in times of plenty and releasing the energy stores in times of need. Brown adipocytes on the o t h e r h a n d can actively waste energy t h r o u g h their capacity to rapidly convert metabolic energy to heat (Nicholls and Locke, 1984). Sympathetic adrenergic n e u r o n a l activity stimulates thermogenesis in brown fat by activating a h o r m o n e sensitive lipase and activating a unique u n c o u p l i n g protein in the inner mitochondrial m e m b r a n e . T h e lipase action mobilizes fatty acids f r o m intracellular lipid droplets to provide substrate for electron transport and the u n c o u p l i n g protein acts to shunt the mitochondrial p r o t o n gradient. Activated brown fat cells are capable of prodigious rates of energy usage, up to 60 times that o f a liver cell (Nicholls, 1974). T h e energy wasting activity o f brown fat is used to b u r n off excess food energy, as well as to generate heat during cold stress and with arousal f r o m hibernation. This action of brown fat is essential for n o r m a l weight regulation, as evidenced by the observations that c o m p r o m i s e d brown fat function is a feature of m o s t animal models o f obesity (Johnson et al., 1991) and that selective genetic knock-
J. GEN. PHYSIOL. 9 The Rockefeller University Press 9 0022-1295/96/11/393/12 $2.00 Volume 108 November 1996 393-404
o u t o f b r o w n fat t h e r m o g e n i c c a p a c i t y causes o b e s i t y in m o u s e m o d e l s (Lowell et al., 1993). T h e i m m e d i a t e b r o w n a d i p o c y t e r e s p o n s e s to a d r e n ergic s t i m u l a t i o n i n c l u d e a n u m b e r o f m e m b r a n e effects in a d d i t i o n to t h e c y t o p l a s m i c e n e r g y - w a s t i n g response, a - a d r e n e r g i c s t i m u l a t i o n i n c r e a s e s cytosolic p H ( H o r w i t z a n d H a m i l t o n , 1993) t h r o u g h activation o f N a / H a n t i p o r t e r s a n d effects o n o t h e r t r a n s p o r t e r s ( G i o v a n n i n i et al., 1988; L e e et al., 1994). e~-adrenergic activation also results in i n c r e a s e s in cytosolic c a l c i u m levels, b o t h t h r o u g h r e l e a s e f r o m i n t r a c e l l u l a r stores a n d i n f l u x across t h e p l a s m a m e m b r a n e (Wilcke a n d N e d e r g a a r d , 1989; L e e et al., 1993). T h e i n c r e a s e in int r a c e l l u l a r c a l c i u m c a n activate two calcium-sensitive c u r r e n t s p r e s e n t in b r o w n fat cells, a h y p e r p o l a r i z i n g p o t a s s i u m c u r r e n t ( L u c e r o a n d P a p p o n e , 1990), a n d a d e p o l a r i z i n g c h l o r i d e c u r r e n t ( P a p p o n e a n d Lee, 1995). [3-adrenergic s t i m u l a t i o n , possibly as a c o n s e q u e n c e o f c h a n g e s in cytosolic r e d o x state (Koivisto et al., 1993; Koivisto a n d N e d e r g a a r d , 1995), activates a d e p o l a r i z i n g n o n s e l e c t i v e c a t i o n c o n d u c t a n c e ( L u c e r o a n d Papp o n e , 1990). T h e a m o u n t o f these c o n d u c t a n c e s p r e s e n t in b r o w n a d i p o c y t e s is h i g h l y v a r i a b l e f r o m cell to cell, a n d m a y b e r e g u l a t e d in r e s p o n s e to c h a n g e s in stimul a t i o n levels o r o t h e r factors in t h e local e n v i r o n m e n t ( P a p p o n e a n d Lee, 1995). T h e variability in m e m b r a n e c o n d u c t a n c e p r o p e r t i e s gives rise to diverse m e m b r a n e p o t e n t i a l responses to a d r e n e r g i c stimulation ( G i r a r d i e r a n d S c h n e i d e r - P i c a r d , 1983; H o r w i t z a n d H a m i l t o n , 1984; S c h n e i d e r - P i c a r d e t al., 1985; L u c e r o a n d P a p p o n e , 1990; P a p p o n e a n d Lee, 1996). T h e f u n c t i o n s o f t h e s e m a n y m e m b r a n e r e s p o n s e s to a d r e n e r g i c activation a r e n o t y e t fully u n d e r s t o o d , b u t t h e y d o n o t s e e m to b e directly involved in g e n e r a t i n g t h e r m o g e n i c r e s p o n s e s ( N e d e r g a a r d a n d L i n d b e r g , 1982; N i c h o l l s a n d L o c k e , 1984; P a p p o n e a n d Lucero, 1992). T h e a m o u n t o f b r o w n a d i p o s e tissue a n d its energy-utilizing capacity a r e highly r e g u l a t e d a n d m o d u l a t e d by a d r e n e r g i c a n d o t h e r stimuli, a n d it is t h o u g h t t h a t t h e m e m b r a n e r e s p o n s e s to a d r e n e r g i c a g e n t s p l a y a r o l e in t h e r e g u l a t i o n o f cell g r o w t h a n d g e n e e x p r e s s i o n ( H i m m s - H a g e n , 1989). It is likely t h a t s y m p a t h e t i c n e u r o n a l s t i m u l a t i o n o f b r o w n fat results in e x p o s u r e o f t h e cells to e x t r a c e l l u l a r A T P a n d t h e p r o d u c t s o f its b r e a k d o w n by e c t o e n zymes. A T P is c o l o c a l i z e d with n o r e p i n e p h r i n e in symp a t h e t i c a d r e n e r g i c n e r v e t e r m i n a l s in m a n y systems (Westfall e t al., 1990). I n a d d i t i o n , r e c e p t o r s f o r extrac e l l u l a r A T P a r e p r e s e n t in m a n y tissues, a n d r e s p o n s e s to e x t r a c e l l u l a r a d e n o s i n e have b e e n f o u n d in b r o w n fat cells (Szillat a n d Bukowiecki, 1983; S c h i m m e l a n d M c C a r t h y , 1984; S c h i m m e l et al., 1987). W e t h e r e f o r e t e s t e d w h e t h e r b r o w n fat cells have m e m b r a n e res p o n s e s to p u r i n e n u c l e o t i d e s . W e f i n d t h a t e x p o s u r e o f b r o w n fat cells to m i c r o m o l a r c o n c e n t r a t i o n s o f ext r a c e l l u l a r A T P raises i n t r a c e l l u l a r c a l c i u m levels, acti394
vates m e m b r a n e i o n c o n d u c t a n c e s , a n d i n c r e a s e s cell m e m b r a n e surface area. T h e size o f t h e surface a r e a inc r e a s e i n d i c a t e s t h a t it m a y r e f l e c t a s u b s t a n t i a l secretory event, s u g g e s t i n g t h a t p u r i n e r g i c s t i m u l a t i o n m a y r e g u l a t e v e s i c l e - m e d i a t e d s e c r e t i o n by a d i p o c y t e s . Alternatively, t h e l o c a l i z a t i o n o f m e m b r a n e t r a n s p o r t proteins or receptors may be under purinergic control.
MATERIALS
AND METHODS
Cells Brown fat cells were isolated by collagenase digestion of interscapular fat pads from 1--5-d-old rats as described previously (Lucero and Pappone, 1989). The rat pups were fasted for ~12 h and cold-anesthetized at 5~ for ~1 h before killing by decapitation. These procedures were approved by the University of California (Davis) animal care committee. The food-deprivation and cold exposure mobilize fat from the cells and increase their density, so that a significant proportion of the adipocytes sink and adhere to the substrate when plated. The cells were plated on collagen-coated coverslips and incubated at 37~ and 5% CO x in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf/horse serum, 0.2 U/ml insulin, 100 ~g/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 ~g/ml amphotericin B. Most of the experiments were performed on cells maintained 2-10 d in culture, but some experiments used cells immediately (0-1 d) after the isolation procedure. There were no differences apparent in the responses of cells with culture age. All cells used in these experiments contained multiple lipid droplets, identifying them as brown fat cells.
Electrophysiology Whole-cell membrane currents were measured in cultured brown fat cells using our standard perforated patch voltage clamp methods (Lucero and Pappone, 1990). Thick-walled borosilicate glass was used to manufacture pipets with resistances of 1-4 MI~. Membrane currents were recorded, filtered (2-3 kHz), and pipet capacitance was nulled using either an EPC-7 (List Electronic, Darmstadt, Germany) or Dagan 3900 (Dagan Corp., Minneapolis, MN) patch clamp amplifier, connected to a computer via a Basic23 interface (INDEC Systems, Sunnyvale, CA). Pulse protocols were delivered and data collected and analyzed using software developed by R.S. Lewis, Stanford University (Stanford, CA). Unless noted otherwise, the cells were continuously perfused with Krebs' solution consisting of (in mM) 120 NaC1, 4.5 KC1, 2 CaC12, 0.5 MgC12, 25 NaHCO3, 0.7 Na2HPO4, 1.3 NaH2PO 4, 10 glucose, pH 7.4, equilibrated with 95% Oz/5% CO2. 0 Ca/0 Mg Krebs' was identical to normal Krebs' except that calcium and magnesium salts were omitted without substitution. The pipet solution contained (in mM) 115 K aspartate, 25 KC1, 10 NaC1, 10 MOPS, KOH to pH 7.20, 280 mosM, 400 p~g/ml Pluronic F-127 and 250 ~zg/ml nystatin (Fluka Chemical Corp., Ronkonkoma, NY). All voltage clamp measurements were made at room temperature (22-25~ Cell electrical capacitance was determined from the analysis of membrane currents measured during 10-mV depolarizing voltage steps from the holding potential of - 6 0 mV. Voltage steps were of sufficient duration (5-25 ms) for the current level to reach a steady state. Brown fat cells have no voltage-gated currents in this potential range (Lucero & Pappone, 1989), and activation of the calcium-activated K and C1 conductances that can be present is voltage independent (Lucero & Pappone, 1990;
Purinergic Capacitance Increases in Adipocytes
P a p p o n e & Lee, 1995). T h e pipet capacitance was nulled with the clamp compensation circuitry after seat formation, but n o o t h e r on-line capacity or access resistance compensation was used. T h e access resistance, RA, was d e t e r m i n e d before a n d after each set of voltage steps, usually from a single exponential function computer-fit to the capacity c u r r e n t transient a n d the relationship "r = RACM, as d i a g r a m m e d in Fig. 1. Capacity c u r r e n t transients were fit well by a single exponential function b o t h before a n d after exposure to ATP (see Fig. 3 A). Alternatively, RA was d e t e r m i n e d by adjustment of the clamp amplifier compensation circuitry. T h e two methods gave similar values of RA. T h e resulting RA values were used to correct for access resistance errors in the subsequent analysis. RA values were typically 10-50 M ~ a n d generally c h a n g e d by < 1 0 % in the course of the experiments
A
Ecom RA
C
M I!
B
presented here. These estimations of RA neglect the contribution of the cell m e m b r a n e resistance, RM, to the m e a s u r e d time constant, resulting in an underestimation of RA of < 5 % for experimental values of R u = 1 G ~ a n d RA = 50 MI). R i was calculated from the steady-state c u r r e n t d u r i n g the last ,-~15% of the voltage step, Iss, using the relation RM = (AECOM -- IssRA)/Iss, where AEcou is the c o m m a n d voltage step amplitude. Capacitative charge moved during the step was measured from the integral of the c u r r e n t transient (AQ) elicited by each voltage step, as shown in Fig. 1 B. This was d o n e by digitally integrating all of the c u r r e n t d u r i n g the step >Iss a n d adding to this a charge c o m p o n e n t equal to Iss'r to correct for the exponential rise of the voltage step. Cell m e m b r a n e capacitance, CM, was calculated from the relation CM = AQ(RM + RA)/(RMAEcoM). W h e n RA = 50 M ~ , a 5% underestimate of R A leads to a ATP > ADP. Prior exposure of the cells to 10-100 ~M of the P ~ antagonist Reactive Blue 2 reversibly blocked the response to 1-5 p~M ATP. The threshold concentration of ATP necessary to elicit a discernible m e m b r a n e response was in the range of 0.1-0.5 ~M in normal Krebs' solution and was similar in solutions with no calcium added or with both calcium and magnesium ions omitted, arguing against involvement of an ATp4--sensitive P2z receptor. Membrane capacitance responses were not seen with exposure to adenosine (100 p~M, n = 2), e,[3-methylene ATP (0.1-1 raM, n = 3), GTP (0.5 mM, n = 4), or UTP (0.25-0.5 mM, n = 3). Insulin (100-200 nM), which can mobilize glucose transporters in brown fat cells (Slot et al., 1991), did not measurably affect m e m b r a n e capacitance in 11 of 11 cells, even when insulin-deprived for 4 (n = 3) or 24 (n = 4) hours p r e c e d i n g exposure to h o r m o n e .
0
A TP May Increase Both Exocytosis and Endocytosis
30 84
Cell m e m b r a n e c o m p o n e n t s cycle rapidly between the cell surface a n d intracellular structures, with some ceils
13,, C.)
2O i00"
2'0
4'0
6'0
Time (rain) IN
P ir-
s1
lE (.)
,OCa ATP
:
ATP
4-
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-T-
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(15)
(4)
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c-
o c
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~d
0
Ca + +
"IJ o o
0
2'0
4'o Time
6'o
(min)
FIGURE 6. ATP stimulates capacitance increases in zero calcium solution. T h e cell was perfused with nominally calcium-free Krebs' solution during the time indicated by the bar. During the times shown, the perfusion solution contained 10 p.M ATP. 399
P A P P O N E AND L E E
FIGURE 7. Agonist a n d calcium d e p e n d e n c e of m e m b r a n e capacitance increases. The values represent the difference between the average m e m b r a n e capacitance before agonist and the maximum capacitance measured in the presence of agonist. M e m b r a n e capacitance was determined for each cell from the current transient during step changes in m e m b r a n e potential, corrected for series resistance errors. Error bars show the standard error of the mean; the n u m b e r of cells is given in parentheses. Measurements were made in normal Krebs' solution or with calcium and magnesium (0 Ca) omitted from the solution. 2-MeSATP is 2-methylthio-ATP. Agonist concentrations were 0.5-100 v.M for ATP, 10 or 100 p~M for ADP, and 1, 2, or 100 ~M for 2-MeSATP.
c o m p l e t e l y t u r n i n g over t h e i r surface m e m b r a n e in < 3 0 min. O u r c a p a c i t a n c e m e a s u r e m e n t s r e f l e c t o n l y t h e size o f t h e surface m e m b r a n e , so t h e A T P - i n d u c e d inc r e a s e in m e m b r a n e c a p a c i t a n c e we m e a s u r e c o u l d e q u a l l y well b e d u e to a n i n c r e a s e in exocytosis o r a dec r e a s e in e n d o c y t o s i s . T o d i f f e r e n t i a t e b e t w e e n t h e s e possibilities, we e x a m i n e d t h e effects o f A T P o n FM1-43 f l u o r e s c e n c e . FM1-43 is a m e m b r a n e - i m p e r m e a n t , amp h i p a t h i c f l u o r o p h o r e t h a t shows l a r g e i n c r e a s e s in fluo r e s c e n c e w h e n it is in a m e m b r a n e r a t h e r t h a n a n a q u e o u s e n v i r o n m e n t (Betz et al., 1992; Betz a n d Bewick, 1993). T h e f l u o r e s c e n c e m e a s u r e d with FM1-43 in t h e s o l u t i o n b a t h i n g the cell t h e n reflects t h e a m o u n t o f cell m e m b r a n e accessible to t h e dye o n t h e cell surface, plus t h e a m o u n t o f i n t r a c e l l u l a r m e m b r a n e rapidly e x c h a n g i n g with t h e cell surface m e m b r a n e . T h e m a g n i t u d e o f t h e FM1-43 f l u o r e s c e n c e a p p e a r s to b e a linear function of the amount of stained membrane, a n d its f l u o r e s c e n c e d o e s n o t s e e m to b e m o d i f i e d by i n t e r n a l i z a t i o n ( S m i t h a n d Betz, 1996). A d d i t i o n o f A T P c a u s e d a n i n c r e a s e in FM1-43 f l u o r e s c e n c e in 17 o u t o f 22 e x p e r i m e n t s , s u g g e s t i n g t h a t t h e dye h a d access to n e w m e m b r a n e after t h e s t i m u l a t i o n with ATP, as s h o w n in Fig. 8. T h e newly s t a i n e d m e m b r a n e c o u l d c o m e f r o m f u s i o n o f vesicles with t h e surface m e m b r a n e o r e n d o s o m a l structures. Alternatively, c h a n g e s in e n d o s o m a l p r o p e r t i e s c o u l d a l t e r t h e d y e ' s fluoresc e n c e p r o p e r t i e s . Since b o t h cell m e m b r a n e surface a r e a a n d F M I - 4 3 f l u o r e s c e n c e i n c r e a s e with A T P stimul a t i o n , it is m o s t likely t h a t A T P i n d u c e s t h e exocytosis
,
"8"
E
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o f i n t r a c e l l u l a r m e m b r a n e t h a t p r e v i o u s l y was n o t exc h a n g i n g with t h e surface m e m b r a n e . A d r e n e r g i c s t i m u l a t i o n o f b r o w n fat cells h a d n o effect o n FM1-43 f l u o r e s c e n c e , as s h o w n in Fig. 9. I n five s u c h e x p e r i m e n t s , p e r f u s i o n with txM NE p r o d u c e d n o d i s c e r n i b l e c h a n g e in cell f l u o r e s c e n c e . S u b s e q u e n t exp o s u r e o f t h e cells to A T P m o r e t h a n d o u b l e d t h e fluor e s c e n c e signal in all five o f t h e s e cells. In a d d i t i o n to i n c r e a s i n g exocytosis, A T P s e e m s to affect o t h e r aspects o f m e m b r a n e trafficking in b r o w n fat cells. S i m u l t a n e o u s l y m e a s u r e d CM a n d FM1-43 fluoresc e n c e show e q u i v a l e n t i n c r e a s e s d u r i n g p u r e exocytosis in c h r o m a f f i n cells ( S m i t h a n d Betz, 1996). H o w e v e r , in b r o w n fat cells, FM1-43 r e s p o n s e s to A T P were o n ave r a g e l a r g e r t h a n c a p a c i t a n c e increases. A T P i n d u c e d a n a v e r a g e FM1-43 f l u o r e s c e n c e i n c r e a s e o f 105 _+ 31% (n = 17) c o m p a r e d to t h e a v e r a g e 33% c a p a c i t a n c e increase. T h i s d i f f e r e n c e was also s e e n in e x p e r i m e n t s in w h i c h FM1-43 f l u o r e s c e n c e was m e a s u r e d c o n c u r r e n t l y with m e m b r a n e c a p a c i t a n c e in t h e s a m e cell, as s h o w n in Fig. 10. I n this cell, m e m b r a n e c a p a c i t a n c e i n c r e a s e d ~ 6 5 % in t h e first few m i n u t e s a f t e r ATP, while t h e fluorescence increased ~150% during the same period. In six s u c h e x p e r i m e n t s , t h e dye signal always r e p o r t e d a significantly g r e a t e r i n c r e a s e with A T P s t i m u l a t i o n (ave r a g e 113 -+ 8 % ) t h a n d i d t h e c a p a c i t a n c e m e a s u r e m e n t (average 27 -+ 8 % ) . If, as in c h r o m a f f i n cells, FM1-43's f l u o r e s c e n t p r o p e r t i e s a r e n o t a l t e r e d with endocytosis, this d i f f e r e n c e in t h e two m e a s u r e m e n t s indicates t h a t access o f t h e d y e to i n t r a c e l l u l a r m e m b r a n e s as well as exocytosis a r e i n c r e a s e d by A T P s t i m u l a t i o n . In a d d i t i o n , dye is t a k e n u p i n t o s t r u c t u r e s that d o n o t
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Effects of ATP on the surface accessible membrane area measured using FM1-43 fluorescence. Total cell fluorescence was measured from a single brown fat cell during 200-ms excitations every 10 s. 2 t~M FM1-43 was present during the times shown by the bars. Cell fluorescence was stable in the presence of FMt-43 alone. Addition of 2 ~M ATP in the presence of FM1-43 caused an ~75% increase in fluorescence. Cell fluorescence after washout of ATP and dye decreased to a level somewhat higher than the prestimulation value.
o= I00O e'r~
0
FIGURE 8 .
400
0
2'0
10 Time
3'0
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9. Adrenergic stimulation does not increase surfaceaccessible membrane measured by FM1-43 fluorescence. Shown is total cell fluorescence measured from a single brown fat cell as in Fig. 8. The cell was perfused with 1 ixM norepinephrine (NE) or 5 ixM ATP during the times shown by the bars. FIGURE
Purinergic Capacitance Increases in Adipocytes
seen in the a m o u n t of surface-accessible m e m b r a n e assayed by FM1-43 fluorescence. These changes in m e m b r a n e trafficking are m e d i a t e d by a P2v purinergic receptor, which can increase cytosolic calcium levels. However, the exocytosis elicited by ATP in brown fat does not seem to be mediated by cytosolic calcium alone, since adrenergic stimulation mobilizes calcium without affecting m e m b r a n e trafficking.
FM 1-43
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ib
o
ib
Time (min)
300(>
FM 1-43
I
I
~ m 2500,$= l/I
-~ 20o(> 0
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9 10000 In
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Time (min)
Simultaneous measurement of membrane capacitance and FM1-43 fluorescence in the same cell. 2 IxM FM1-43 was present in the perfusion solution during the time shown by the bar. At the arrow a bolus of 100 IxM ATP was added to the bath. Capacitance measurements were taken every 6 s, and fluorescence measurements were taken every 10 s. Autofluorescence in the absence of dye was subtracted from the record. At 15 min the clamp went whole-cell and the experiment was terminated. FIGURE 10.
exchange rapidly with the surface m e m b r a n e , since the fluorescence level often did not return to baseline levels u p o n washout of the dye. This uptake is presumably responsible for the slow continuous increase in fluorescence seen in Fig. 10. T h e residual intracellular fluorescence was absent f r o m the fat droplets, but otherwise was n o t apparently localized and could not be washed out even when cells were exposed to ATP, NE, insulin, or zero calcium solution to stimulate m e m b r a n e turnover. DISCUSSION
We find that exposure of brown adipocytes to ~M extracellular ATP evokes exocytosis of intracellular m e m brane, resulting in as m u c h as twofold increases in cell m e m b r a n e capacitance. Even greater increases are 401
PAPPONEAND LEE
T h e r e are several possible physiological sources of extracellular ATP that may act on brown adipocyte purinergic receptors. We initially tested for ATP responses because brown adipose tissue has extensive sympathetic innervation (Nechad, 1986) a n d vesicular ATP is usually present in sympathetic nerve terminals (Wesffall et al., 1990). We find that ~ 9 0 % of brown adipocytes tested showed conductance a n d / o r capacitance responses to ATP, a p r o p o r t i o n similar to that showing adrenergic responses ( P a p p o n e and Lee, 1995). Thus, it is likely that sympathetic activation of t h e r m o g e n i c responses in brown adipose tissue normally has b o t h adrenergic a n d purinergic c o m p o n e n t s . Previous experiments in vitro examining activity-dependent changes in brown fat cell n u m b e r and properties using purely adrenergic stimulation to m o d e l sympathetic activity may n e e d to be reevaluated in light of these results. ATP is p r o p o s e d to participate in the regulation of cell proliferation and differentiation in m a n y tissues (Rathb o n e et al., 1992), and in adipocytes these processes are known to be regulated by autocrine a n d / o r paracrine factors (Butterwith, 1994), which may well include extracellular ATP. ATP may be released in addition by o t h e r cell types present in brown adipose tissue. Mast ceils a n d endothelial cells are prevalent in brown adipose tissue (Nechad, 1986) and are also potential sources of exogenous ATP (Dubyak, 1991; Osipchuk and Cahalan, 1992). ATP may be released by these cells in response to inadequate blood flow and mediate vasodilation a n d / o r angiogenesis as has b e e n r e p o r t e d in o t h e r tissues (Motte et al., 1995). In addition, ATP may be released f r o m fat cells themselves. Many o t h e r cell types are known to contain transport proteins such as the multidrug resistance protein, the cystic fibrosis t r a n s m e m b r a n e c o n d u c t a n c e regulator, and the sulfonylurea r e c e p t o r protein that may transport ATP into the extracellular space (Al-Awqati, 1995).
Extracellular A TP Acts through a P2r Purinergic Receptor Brown fat cell responses to ATP are m e d i a t e d by a P2v type purinergic receptor. T h e p h a r m a c o l o g y of the m e m b r a n e capacitance response, that is a potency order of 2-methylthio-ATP (2-MeSATP) -- ATP > ADP > > adenosine, 0t,[3-methylene ATP, GTP, UTP, and block by Reactive Blue 2, is that of a Pzv r e c e p t o r (Burnstock,
1990). In other cell types Pzv receptors are known to mobilize intracellular calcium through G-protein-mediated generation of IP3 (Harden et al., 1995; Chen et al., 1995). O u r results showing activation of calcium-sensitive m e m b r a n e conductances are consistent with such an action in brown adipocytes as well. Although adenosine-sensitive A1 purinergic receptors are well known in brown and white adipocytes, we are not aware of any previous description of extracellular ATP responses in brown adipocytes. In white adipocytes ATP-induced increases in cytosolic calcium (Blackmore and Augert, 1989; Kelly et al., 1989) and modulations of glucose and pyruvate handling (Kelly et al., 1989; Cheng and Harold, 1990) have been reported. However, in neither tissue is there yet a clearly defined physiological role for ATP receptor stimulation.
Relation between Membrane Exocytosis and Conductances Although purinergic stimulation has a n u m b e r of effects on m e m b r a n e conductances (Pappone and Lee, manuscript in preparation), these seem to be independent of the exocytotic response. The calcium-sensitive potassium and chloride conductances can be activated by other experimental manipulations that raise calcium, such as adrenergic stimulation (Lucero and Pappone, 1990), exposure of cells to calcium ionophores (not shown), and including calcium in the pipet solution in whole cell patch clamp (Pappone and Lee, 1995), without affecting cell m e m b r a n e capacitance. Thus, the activation of these conductances by ATP seems purely a consequence of its action in raising cytosolic calcium levels and does not seem to d e p e n d on ATP's actions in mobilizing intracellular membranes.
A TP Activates Exocytosis Purinergic stimulation results in a dramatic increase in cell m e m b r a n e surface area in brown fat cells. We found an average ~ 3 0 % increase in cell m e m b r a n e capacitance and ~ 1 0 0 % increase in FM1-43 fluorescence in these experiments. Membrane responses were not permitted to plateau in most of our experiments, so these values are undoubtedly less than the maximal possible responses. Large increases in membrane capacitance (10100%) such as we see with ATP stimulation of brown fat have been associated in other cell types with the insertion of membrane transporters into the plasma membrane (Lewis and de Moura, 1982) or more commonly with h o r m o n e secretion (Neher and Marty, 1982; Fernandez et al., 1984; Tse et al., 1993). The combined results of our membrane capacitance and FM1-43 fluorescence measurements indicate that, like these processes, the surface membrane increases induced by ATP also involve fusion of intracellular membranes with the surface. Electron microscopic studies have shown numerous submembranous vesicles and plasma membrane invaginations in both brown and white adipocytes (Slavin, 1987), indicating that brown fat cells have sufficient intracellular vesicles available for a substantial exocytotic response. Exocytotic responses to ATP have been reported in other cell types secondary to ATP-induced increases in cytosolic calcium. Raised intracellular calcium alone, however, does not seem sufficient to trigger the m e m b r a n e response of brown fat cells, since NE raised cell calcium to similar levels without activating capacitance increases. This cannot be due to a simultaneous suppression of the m e m b r a n e response by adrenergic pathways because m e m b r a n e capacitance responses to ATP were unaffected by concurrent NE stimulation. In addition, membrane capacitance increases were similar in the presence and nominal absence of extracellular calcium, suggesting that high calcium levels are not required for exocytosis in these cells. 402
Possible Physiological Roles of A TP Responses Membrane responses of brown fat cells to extracellular ATP have not been reported previously, and the role ATP-stimulated exocytosis might play in the physiology of the tissue is not known. One possibility is that ATP regulates the surface expression of a m e m b r a n e protein or proteins (Bradbury and Bridges, 1994). Many studies of m e m b r a n e trafficking in adipocytes have examined the insulin-activated upregulation of glucose transport, which involves significant movement of intracellular membrane containing Glut4-type glucose transporters to the surface in both white and brown adipocytes (Czech, 1995; Slot et al., 1991). These m e m b r a n e events do not seem to be involved in the exocytotic response to purinergic stimulation however. First, insulin did not increase m e m b r a n e capacitance or FM1-43 fluorescence in our experiments, although our cells may have lost insulin sensitivity with time in culture. Second, Glut4 mobilization is also activated by adrenergic stimulation in brown fat cells (Omatsu-Kanbe and Kitasato, 1992), but in our experiments NE evoked minimal capacitance or fluorescence increases. Third, work on an adipocyte model cell line indicates that ATP does not mobilize glucose transporters (Robinson et al., 1992). And finally, extracellular ATP suppresses Glut4 mobilization in response to insulin in white adipocytes (Kelly et al., 1989). Thus, it is unlikely that the membrane surface changes we see in response to ATP result from the upregulation of glucose transport activity. It remains possible however that purinergic m e m b r a n e mobilization mediates the surface expression of some other m e m b r a n e transport protein or receptor, such as fatty acid transporters or insulin receptors. Purinergic m e m b r a n e mobilization may reflect activation of a secretory process. T h e r e is a growing catalog of adipocyte secreted peptides and proteins (reviewed in Ailhaud et al., 1992; Spiegelman et al., 1993; Spiegel-
PurinergicCapacitanceIncreases in Adipocytes
man and Hotamisligil, 1993), which include growth factors (hepatocyte growth factor, insulin-like growth factor, vascular endothelial growth factor, tumor necrosis factor 00, c o m p l e m e n t c o m p o n e n t s (adipsin/factor D, factor B, factor C), and fat handling proteins (cholesterol ester transfer protein, lipoprotein lipase, apolipoprotein E). At least some adipocyte secretory products seem to be involved in regulating the growth and differentiation of adipose tissue (Lau et al., 1990; Ailhaud et al., 1992). In addition, it has b e e n f o u n d recently that the ob gene mutation, which causes obesity in mice, codes for a peptide secreted by fat cells, termed leptin, that can act centrally to suppress appetite, increase metabolism, and reduce body weight (Zhang et al., 1994; Campfield et al., 1995; Halaas et al., 1995; Pelleymounter et al., 1995). While regulated secretion of fat cell products has not been reported, release of one
or more or these products may be controlled by extracellular ATP levels. It is likely that in many biochemical preparations sufficient ATP could be released from only a few damaged cells to elicit the exocytotic responses we report, or there may be sufficient endogenous ATP release to trigger secretion. Fat cells are fragile, and even nanomolar concentrations of ATP are sufficient to elicit cell responses (Barnard et al., 1994). Thus it is possible that secretion that has been considered constitutive may have in fact been stimulated by ATP released from broken cells. Secretion a n d / o r expression of many adipocyte products correlates with the metabolic a n d / o r differentiative state of the tissue and the metabolic status of the organism, and so are altered in obesity or with development of the adipocyte phenotype. Given these phenomena, it seems likely that purinergic stimulation plays an important role in adipocyte physiology.
We wish to t h a n k Dr. Martin Wilson for c o m m e n t s o n the work a n d manuscript, Jeffrey Phan for technical assistance, a n d Michael G u i n a n for help with the figures. This work was supported by the National Institutes of Health, grant GM-44840.
Original version received 11 April 1996 and accepted version received 15July 1996.
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Purinergic Capacitance Increases in Adipocytes
