Proc. Natl. Acad. Sci. USAVol. 87, pp. 1208-1212, February 1990Cell Biology

Isoproterenol stimulates shift of G proteins from plasma membraneto pinocytotic vesicles in rat adipocytes: A possible meansof signal dissemination

(cAMP/subcellular fractionation/cholera toxin/pertussis toxin/Western blotting)

KAZUTAKA HARAGUCHI AND MARTIN RODBELLLaboratory of Cellular and Molecular Pharmacology, Section on Signal Transduction, National Institute of Environmental Health Sciences,Research Triangle Park, NC 27709

Contributed by Martin Rodbell, November 16, 1989

ABSTRACT Guanine nucleotide-binding regulatory pro-teins (G proteins) are linked to a large number of surfacemembrane receptors and appear to regulate a variety ofeffector systems located both in the plasma membrane and inother parts of the cell. The mechanism of the disseminativeactions of G proteins remains obscure. During an investigationof the fate of two types of G proteins, Gs and G1, in ratadipocytes, we unexpectedly found that isoproterenol, whichstimulates cAMP levels and lipolysis in these cells, inducesparallel increases in both Gs and G, in a low-density microsomalfraction rich in endosomes and Golgi bodies. Two plasmamembrane constitutive enzymes, adenylyl cyclase and 5'-nucleotidase, are also elevated in this fraction. NaF and NaN3,metabolic inhibitors, block the redistribution process. Theisoproterenol-stimulated shifts are completely reversible afterremoval of the hormone, indicating a recycling, endocyticprocess. The endocytic process seems to be fluid phase endocy-tosis, or pinocytosis, since isoproterenol stimulates the uptakeofboth fluorescent-labeled dextran and horseradish peroxidaseinto the same vesicles containing Gs. However, the vesicles thataccumulate in response to isoproterenol seem heterogenous inproperties that may reflect the lipolytic process induced byisoproterenol. It is speculated that the "pinosomes" formed inresponse to lipolytic hormones may continually produce signalswithin the cellular interior during their processing and cycling.Hence, signal production in response to hormones need not beconfined to the cell membrane; circulating pinosomes may beresponsible for some of the disseminative effects of hormones.

GTP-binding proteins share characteristics of binding anddegrading GTP to GDP. One group of these proteins isassociated with the actions of numerous hormones andneurotransmitters at the plasma membrane. These proteins,Ga proteins, are distinct from other groups because whenisolated they are associated with a complex of two otherproteins designated 6//y. The heterotrimeric complexes aretermed G proteins (see ref. 1 for review). An essentialquestion is how these proteins regulate what seems to be amultitude of functions. A related logistical problem stemsfrom that fact that some of these functions are not confinedto the cell membrane where the transduction elements aregenerally localized. One possibility, embodied in the "pro-grammable messenger theory" (2), is that the Ga proteinsdissociate from the l/y subunits when activated by thesynergistic actions of GTP and hormones; the liberatedproteins may diffuse throughout the cell and react witheffectors both at the cell membrane and at various othercompartments in the cell. However, it has not been demon-strated in either cells or with isolated cell membranes that Ga

proteins are released in response to the combined actions ofGTP and hormones or neurotransmitters.We have chosen the rat adipocyte to investigate the fate of

Ga proteins and 8/y complexes under the influence ofhormones. By using cholera toxin (CTx) and pertussis toxin(PTx) labeling and immunoblotting for detection of two typesof Ga proteins (a. and aj) and the pS subunits, it wasunexpectedly found that each of these subunits declined inparallel in subcellular fractions enriched in the plasma mem-brane after activation of 8-adrenergic receptors by isopro-terenol. Similarly 5'-nucleotidase and adenylyl cyclase, en-zymes that are typical plasma membrane (PM) markers,displayed parallel decreases. In all cases, these changes wereaccompanied by the appearance of the marker enzymes, thea proteins, and the ,B subunits (presumably as P//y complexes)in a low-density microsomal (LDM) fraction known to beenriched in endosomes and Golgi membranes. The changes indistribution were energy-dependent and reversible. The pres-ent study documents these findings and provides evidencethat the observed vesiculation of the plasma membrane in fatcells reflects fluid phase endocytosis, or pinocytosis. Dis-cussed is the possibility that the pleiotropic actions of hor-mones acting through G proteins need not be confined to thecell membrane. At least in adipocytes, hormones may gen-erate signals continually during the cycling and processing ofendocytic vesicles containing the transduction elements re-quired for signal production; the "pinosomes" may serve asvehicles for conveying information throughout the cellularinterior.

MATERIALS AND METHoDSReagents. [32P]NAD', [a-32P]ATP, uridine diphosphate

galactose, and 5'-[3H]AMP were purchased from New En-gland Nuclear. PTx and CTx were from List BiologicalLaboratories (Campbell, CA). Collagenase (type 1) was fromWorthington. Bovine serum albumin (BSA; type 5, fatty acidfree) was from Armour Research Laboratories. Fluoresceinisothiocyanate-labeled dextran (FITC-dextran) (Mr 70,000)and all other chemicals were either from Sigma or were thehighest quality available.

Antisera. Goat anti-rabbit IgG and rabbit peroxidase-anti-peroxidase were from Organon Teknika-Cappel. Rabbitantisera AS/6 and RM/1 were generous gifts from AllenSpiegel (National Institutes of Health, Bethesda, MD). Rab-bit antiserum U49 was kindly provided by Suzanne Mumby(Department of Pharmacology, Southwestern Medical Cen-

Abbreviations: G proteins, guanine nucleotide-binding regulatoryproteins; Ga proteins, G proteins involved in signal transduction;BSA, bovine serum albumin; LDM, low-density microsomes; PM,plasma membrane; PTx, pertussis toxin; CTx, cholera toxin; FITC-dextran, fluorescein isothiocyanate-labeled dextran.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 87 (1990) 1209

ter, Dallas). Detailed characterizations of AS/6, RM/1, andU49 have been published (3, 4).

Adipocyte Preparation and Subcellular Fractionation. Adi-pocytes were isolated by the method of Rodbell (5) asmodified by Honnor et al. (6), except that minced adipocyteswere incubated with collagenase (type 1) in Krebs-Ringerbicarbonate buffer containing 2 mM glucose and 30 mMHepes (pH 7.4) with 5% (wt/vol) BSA (KRBH5%). Briefly,epididymal fat pads of 150- to 180-g Sprague-Dawley rats ofthe CD strain (Charles River Breeding Laboratories) wereminced with a tissue chopper. Pooled minced fat pads (4 g)were digested in 12 ml of collagenase (0.75 mg/ml) for 45 minin KRBH5% containing 200 nM adenosine at 370C. Afterwashing the cells three times with KRBH5%, 1 vol of packedcells [final concentration = 10-15% (vol/vol)] was incubatedwith or without additives in 4 vol of KRBH5% or Krebs-Ringer bicarbonate buffer containing 30 mM Hepes (pH 7.4)with 1% (wt/vol) BSA (KRBH1%) for various times asindicated in the legends. During this period, adenosine deam-inase (1 unit/ml) was added to the medium. Subcellularfractionation was carried out by the method of Kono et al. (7)with slight modification. Cells were washed once with 0.25 Msucrose in 10 mM Tris-HCI, pH 7.4/5 mM NaCl at 17°C andthen homogenized at 4°C in 2 vol of buffer A (10 mMTris'HCI, pH 7.4/5 mM NaCl/1 mM EDTA/0.1 mM phe-nylmethylsulfonyl fluoride/leupeptin at 50 ,g/ml) containing0.25 M sucrose. The homogenate was kept on ice for 10-15min to compact the fat and was subsequently centrifuged ina Beckman SS34 rotor at 13,000 rpm (20,000 x g) for 1.5 min.The infranatant was centrifuged for 30 min at 200,000 x g.The resultant supernatant was removed, and the pellet wasresuspended in the above homogenizing medium. The thor-oughly dispersed suspension was layered on a sucrose gra-dient (13-45% wt/vol) containing buffer A. After centrifu-gation in a Beckman SW41 rotor at 35,000 rpm for 40 min,0.6-ml aliquots were removed from the top fraction (fraction1) to the bottom fraction. In most experiments, fractions 2-6were consolidated as the LDM fraction; fractions 8-12 wereconsolidated as the PM fraction.

ADP-Ribosylation by PTx and CTx. By using 5 ,ug ofmembrane protein, PTx-stimulated ADP-ribosylation wasdone at 4°C for 3 hr, as reported previously (8), with the toxinat 50 ,g/ml. ADP-ribosylation by CTx was carried out by theprocedure of Ribeiro-Neto et al. (8). For both PTx- andCTx-stimulated ADP-ribosylation, the [32P]NAD' concen-tration was S ,uM in the presence of 0.005% Lubrol. Thereactions were stopped by addition of Laemmli buffer (9); thesamples were boiled for 3 min and applied to 1o acrylamidegels. Subsequent to SDS/polyacrylamide gel electrophore-sis, the gels were transblotted to nitrocellulose paper andautoradiographed (6-24 hr exposure for PTx; 12-48 hr forCTx).Immunodetection. Immunoblots of the transblotted mate-

rial were prepared as described (10). Only the relevantportions of the autoradiograms or immunoblots are shown inthis paper.Enzyme Assays. Assays for adenylyl cyclase activity were

carried out in a reaction mixture containing 25 mM Tris HCI(pH 7.5), 1 mM EDTA, 5 mM MgCI2, 1 mM cAMP, 0.1 mM[a-32P]ATP, creatine phosphokinase at 0.2 mg/ml, and my-okinase at 20 ug/ml at 30°C for 10 min. The production ofcAMP was measured by the procedure of Salomon et al. (11).5'-Nucleotidase activity was measured by the method ofAvruch et al. (12). Protein concentration was determined bythe method of Bradford (13).

A BP-Tx SUBSTRATE CTx SUBSTR.ATE

,4.0PM~~_1u2 ILI[)N

ISO o

[L NEM

LDMIIs(,)

4 *00.1 1 10 0 0.1 1 10

( CTx SUBSTRATE

0+

-, +*i10 IL %l______I_°

% S 1% I

5;~(/ BSA 1 % BS.A

FIG. 1. Effects of isoproterenol (ISO) and BSA concentrations onPTx and CTx substrates in PM and LDM. (A) Autoradiography ofPTx substrates (40-kDa material) in PM and LDM fractions isolatedfrom adipocytes incubated with indicated concentrations of ISO. (B)The same fractions showing autoradiographs of CTx substrates(42-kDa and 45-kDa bands). Note that both bands are in PM andLDM. (C) Autoradiographs (duplicate samples) ofCTx substrates inPM and LDM fractions from adipocytes incubated with 5% or 1%BSA in the absence or the presence of 10 ,/M isoproterenol.

studies, it was found that incubation of rat adipocytes withisoproterenol led to decreased amounts of CTx-labeled ma-terial in sucrose gradient fractions enriched in plasmamembranes.* The decreases were variable in amount butwere particularly evident when adipocytes were incubatedwith isoproterenol for periods longer than 15 min and inincubation buffers containing 1% (wt/vol) or less BSA (seebelow). In subsequent studies, we found decreased levels notonly of CTx-labeled substrates but also of PTx substrateslabeled with [32P]NAD' in the plasma membrane-enrichedfractions, as shown in Fig. 1 A and B. These unexpectedfindings led to investigations of the subcellular distribution ofthe CTx- and PTx-labeled substrates. As seen in Fig. 1,LDM, a sucrose gradient fraction enriched in Golgi mem-branes and endocytic vesicles (7), showed parallel increasesin CTx- and PTx-labeled substrates when adipocytes werestimulated with as little as 0.1 AuM isoproterenol. The en-hanced levels of these substrates found in the LDM fractionproved more sensitive than the PM fraction for assaying theeffects of agents that induced these changes. For example,note in Fig. 1 that increases in CTx-labeled proteins werereadily detectable with 0.1 ,M isoproterenol in the LDM-enriched fractions, whereas in the corresponding PM frac-tions the decline in CTX-labeled substrates was barely dis-cernible. This sensitivity of the LDM fraction to the effectsof isoproterenol was also noted when the BSA concentrationin the incubation medium was varied. Shown in Fig. 1C is theimportance of albumin and possibly of free fatty acids (seeDiscussion) in the observed phenomena. Cells incubated inthe presence of 1% BSA showed marked isoproterenol-induced decreases and increases, respectively, in the levelsof CTx-labeled substrates in the PM and LDM fractionsrelative to that seen with 5% BSA in the incubation medium.

Identification by Immunoblotting of G Proteins. AlthoughADP-ribosylation of Ga proteins by toxins is a sensitiveassay, this procedure has the inherent problem of requiringcofactors that may vary in amounts in the various subcellularfractions. Accordingly, we turned to detection by immuno-

RESULTSEffects of Isoproterenol on Distribution of CTx and PTx

Substrates Between PM and LDM Fractions. During initial

*Adenosine deaminase in the incubation medium also stimulateslipolysis to about the same extent given by isoproterenol. However,the enzyme does not induce the shifts in distribution of G proteins(unpublished observation).

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1210 Cell Biology: Haraguchi and Rodbell

(x 4

30I S()

-

0)0._.

O

o I.

Z E_ XLzu'- ---. -- O- 40 VMMMMW~-_ t- _wo fomq-_Am ~ ao m lw :,I

5 111s;llcrlome ( ;1 tadie ilt Illi-atior'

FIG. 2. Isoproterenol (ISO) induces shifts in the distribution ofsubunits ofG proteins. Adipocytes from 60 rats were incubated with(+) or without (-) 10 AuM isoproterenol for 30 min in KRBH1%. Theisolated membranes were subjected to sucrose gradient fraction-ation. Gas, Gal, and Go were immunodetected on Western blots withantisera RM/1, AS/6, and A5357, respectively.

blotting (Western blots) of the membrane fractions. Fig. 2shows typical results utilizing antisera that specifically reactwith as, ai, and ,3 subunits of G proteins. As was observedwith the toxin labeling studies, incubation of adipocytes withisoproterenol for 30 min resulted in parallel shifts of all threesubunits of G proteins toward the LDM fractions. Note thatthe as antiserum detected both CTx-labeled bands shown inFig. 1; both bands in LDM were increased in response toisoproterenol.The finding that both a and P subunits are affected in a

parallel manner by isoproterenol suggested that intact Gproteins are translocated to the LDM fractions in response toisoproterenol. This finding raised the possibility that isopro-terenol induces a general shift in distribution of plasmamembrane proteins to the LDM. Support for this possibilitywere findings that 5'-nucleotidase and adenylyl cyclase, bothtypical plasma membrane proteins, also shifted to lowerdensity fractions after isoproterenol treatment of adipocytes(Fig. 3). The simplest interpretation of these findings is thatthe hormone induced or enhanced an endocytic process.Energy Dependency. A number of hormones and other

agents that bind to cell surface receptors induce cycling of thereceptors and their ligands through complex energy-dependent processing of endocytic vesicles, as in the case ofinsulin receptors and glucose transporters in adipocytes (14,15). NaN3 and NaF, in combination, reversibly inhibit me-tabolism and lipolysis in rat adipocytes (16). As shown in Fig.4, these agents completely blocked the shift in distribution ofPTx-labeled substrates (and CTx-labeled substrates in otherexperiments) induced by isoproterenol. Moreover, suchtreatment caused effectively all of the labeled material to beconfined to the plasma membrane-containing high-densityfraction. The same treatment also affected the plasma mem-brane-enriched fractions in control cells. Treatment withNaF and NaN3 did not block the ability of isoproterenol tostimulate cAMP production (data not shown).

Reversibility of Isoproterenol Action. To test for reversibil-ity, adipocytes were incubated with or without the hormonefor 45 min; the cells were then either washed to remove thehormone and/or incubated with the /3 blocker propranolol toprevent any bound hormone from acting. Adipocytes wereincubated for an additional 45 min in the absence or presenceof isoproterenol. Fig. 5 shows that, when the effects of thehormone on the shift of CTx substrates to the PM fractionwere stopped by the /3 blocker during the subsequent 45-minincubation, the shifts were no longer apparent (Fig. 5, lanes

0 5 10 1 5 20

4-

s_

d. Ec: 0- E

> a._Axs

5 1 0 1 5

Gradient Fraction2 0

FIG. 3. Isoproterenol (ISO) induces shifts in membrane distri-bution of adenylyl cyclase and 5'-nucleotidase activities. Adipocyteswere incubated without or with 10 ,uM isoproterenol for 60 min inKRBH1%. Enzyme activities were measured in response to 10 ,uMforskolin in aliquots of each sucrose gradient fraction. Each datapoint represents the mean of duplicate experiments.

2 and 3), indicating that the endocytic process is completelyreversible during this period of time.

Isoproterenol Stimulates Fluid Phase Endocytosis. UsingFITC-dextran as a probe for monitoring the possible effectsof isoproterenol on fluid phase endocytosis (17), we havereported elsewhere that the hormone increases by 30% theamount of FITC-dextran encapsulated in vesicles in the LDMfraction (18). This finding indicated that the hormone stim-ulates fluid phase pinocytosis. However, FITC uptake alonedoes not provide direct means of identifying the nature of thevesicles present in this fraction. We therefore turned toanother technique (19), which involved incubating the cells inmedium containing both FITC-dextran and horseradish per-

[S. ( )

-IS(). '.T

SIKS)NM.I.

5 tSucrose (Gradient Fraction

15

FIG. 4. Isoproterenol-induced redistribution of PTx substrate isenergy-dependent. Adipocytes were incubated with or without acombination of 0.1% NaN3 and 10 mM NaF (metabolic inhibitors,M.I.) for 10 min in KRBH1%. The cells were then incubated with orwithout 10 ,uM isoproterenol (ISO) for 30 min. Each of the sucrosegradient fractions prepared from isolated membranes was assayedfor PTx substrates by autoradiography ofthe electrophoresed 40-kDaband.

Proc. Natl. Acad. Sci. USA 87 (1990)

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Proc. Natl. Acad. Sci. USA 87 (1990) 1211

A

1 CONTROL I H

2 I0 W1 P,IS0 H

4ISO |p HH

4 1 Iso

0 45 90(min)

BPM

1 2 3

LDM4 1 2 3 4

FIG. 5. Isoproterenol-induced shift in Gas from PM to LDM isreversible. (A) Experimental design: W, washing of cells; P, pro-pranolol (100,uM) added; H, cells homogenized; ISO, isoproterenol(10 ,uM) added. (B) Autoradiographs of CTx substrates found inpooled PM and LDM fractions after fractionation on sucrose gradi-ents. The two bands represent 42-kDa and 45-kDa Gas bands foundin adipocytes.

oxidase followed by incubating the isolated LDM fractionwith diaminobenzidine, which is taken up by the vesicles.Subsequent incubation with H202 allowed those vesiclescontaining peroxidase (and FITC-dextran) to oxidize diami-nobenzidine; the resultant dense particles are contained onlywithin those vesicles representing endosomes. These vesi-cles, being of higher density, could be separated from vesi-cles not containing peroxidase (and FITC) by fractionation onsucrose gradients, as shown in Fig. 6. It is seen that theseparated higher density endosomes contained Gas as amarker for the presence of plasma membrane elements in theFITC-dextran/peroxidase-containing vesicles.

Signal Transduction in the LDM Fraction. To determinewhether the various signal transduction components (G pro-teins and adenylyl cyclase) remained operative in the LDMand PM fractions obtained from isoproterenol-treated adipo-cytes, we measured the response of adenylyl cyclase tovarious activating agents. Shown in Table 1 are comparativeadenylyl cyclase activities in LDM and PM fractions fromadipocytes that had been treated with 10,uM isoproterenolfor 30 min. Activities in response to AIF3 and forskolin weremeasured in the absence and presence of 1 mM GTP as ameans of assessing the functional expression of both Gs andGj; the latter requires concentrations of GTP in excess of 10,M with membranes from rat adipocytes (20). Propranololwas added to control samples to block effects of any endog-enously bound isoproterenol. Isoproterenol-stimulated cy-clase activity in the PM fractions was about 2-fold greaterthan basal activity, whereas in the LDM fraction hormonaleffects were not statistically significant (data not shown).Both AIF3- and forskolin-stimulated cyclase activities were at

-H2°2Sk

+H202

20

10el

1o

1-A

0 5 10 15

FRACTION

FIG. 6. Isoproterenol induces formation of endosomes contain-ing FITC-dextran and horseradish peroxidase. Adipocytes wereincubated without or with isoproterenol (10 AM) for 30 min inKRBH5%. The cells were transferred to fresh KRBH5% containingFITC-dextran (20 mg/ml) and peroxidase (1 mg/ml) and incubatedfor 5 min. After one wash in KRBH5% and three washes in 0.25 mMsucrose/10 mM Tris HCI, pH 7.4/5 mM NaCI, cells were homoge-nized, and LDM was prepared from pooled sucrose gradient frac-tions 3-6. The pooled fractions were incubated with diaminobenzi-dine (0.5 mg/ml) for 30 min at 26°C in the dark. After dilution with2 vol of medium (95 mM NaCI/50 mM KCI/5 mM MgCI2/10 mMTris-HCI, pH 7.4), membranes were incubated with 0.1% H202 for20 min at 26°C in the dark. They were again fractionated on sucrosegradients (15-45%, wt/vol). FITC-dextran was measured (emission515 nm, excitation 485 nm) in aliquots of each fraction. BSA (1mg/ml) and ice-cold trichloracetic acid (16%, vol/vol) were added toother aliquots; the pellets were washed once with cold ether anddissolved with Laemmli buffer followed by SDS/polyacrylamide gelelectrophoresis. Immunodetection on Western blots was carried outwith Gs, antiserum (RM/1).

least 10-fold greater than the basal activity, indicating that Gsis functional in both LDM and PM. GTP significantly (P <0.05) inhibited these activities, suggesting that Gi is presentand functional in both membrane fractions. Thus, both Gi andGs retained their functions when shifted to LDM in responseto isoproterenol.

DISCUSSIONOur initial goal in this study was to investigate whetherhormones that act through receptors coupled to G proteinsstimulate the dissociation and consequent release of their asubunits to the cytosolic compartments of the adipocyte. Ourinvestigations led instead to the conclusion that isoproterenolcauses redistribution of G proteins along with other PMelements (adenylyl cyclase and 5'-nucleotidase) to a low-density membrane fraction that is generally rich in endo-somes and Golgi bodies (7). The characteristics ofthe processinduced by isoproterenol are indicative of an endocytic

Table 1. Adenylyl cyclase activity in LDM and PM from adipocytes treated with10 ,uM isoproterenol

Adenylyl cyclase activity, pmol/min per mg

LDM PM

Addition - GTP + GTP, 1 mM - GTP + GTP, 1 mMNone* 1.9 ± 0.6 18.4 ± 1.2Propanolol (5 AuM) 2.8 ± 0.5 14.1 ± 0.8AIF3 (0.1 mM) 21.2 ± 1.1 17.2 ± 1.2 96.3 ± 3.1 73.8 ± 1.3Forskolin (10 ,uM) 27.1 ± 0.3 22.1 ± 0.3 123.6 ± 3.6 92.5 ± 2.9*AII solutions contained 10 nM GTP.

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process. The process is temperature-dependent, is revers-ible, and is effectively abolished by agents (NaN3 and NaF)that inhibit metabolism and lipolysis in adipocytes (17). Inrecent studies, we have found that procaine or dibucaine,agents that disrupt the cytoskeletal network (21) and thusmay affect endocytic processing, also inhibit the isoprotere-nol-induced process (unpublished observations). Direct evi-dence that the shift in distribution ofPM markers is due to anendocytic process came from findings that isoproterenolstimulates the uptake of FITC-dextran, a marker of fluidphase endocytosis or pinocytosis (17), into the LDM fractionof rat adipocytes (18). Here we show that pinocytotic vesicles(containing FITC-dextran and horseradish peroxidase) in theLDM fraction have Ga, (and probably other transductionelements) in their membranes.Our findings confirm the early observation ofCushman (22)

that epinephrine stimulates pinocytosis, as monitored by theuptake of gold particles, in rat adipocytes. Correlated withincreased pinocytotic activity were increased levels of intra-cellular unesterified fatty acids as reflected by the effects ofalbumin concentration. Because of its fatty acid bindingcapacity, serum albumin controls the egress and thus theaccumulation of fatty acids in adipocytes; in time the accu-mulated intracellular fatty acids retard the lipolytic process(23). In our study, the isoproterenol-dependent shift of Gproteins to the LDM fraction was time-dependent (minimally15 min) and was enhanced at decreased albumin concentra-tions, which again argues for a relationship between pinocy-totic activity and intracellular levels of fatty acids. On thebasis of such correlations, a role for pinocytotic activity in thetransport offatty acids in adipocytes has been suggested (24).We have proposed (18) an alternative, larger role for pinocy-totic activity in adipocytes and possibly many other cell typesthat dynamically control the input and output of storagematerial (fat, protein, and carbohydrate). Briefly, the forma-tion of pinosomes may be a means for conveying to andprocessing information in various compartments in the cell.Thus, for example, hormonally induced signals such ascAMP may be continually generated during the cycling andprocessing of the pinosomes. Fusion (regulated possibly bycAMP and activated protein kinases) of pinosomes withvesicles containing storage fat and subsequent hydrolysis offat by lipases present in or transferred to the fused vesicleswould give rise to fatty acid-rich vesicles. These vesicles maybe destined for fusion with the cell membrane and consequentsecretion of fatty acids to the exterior. At limiting albuminconcentrations, the fatty acid-rich vesicles accumulate, thusaccounting for the increased levels ofLDM vesicles enrichedin PM markers at lower albumin concentrations. We havefound recently that the LDM fraction from isoproterenol-treated adipocytes also contains higher concentrations oftriglycerides (unpublished observations). Heterogeneity ofthe vesicles derived from pinosomes (i.e., vesicles containingPM markers) is further illustrated by the broad distribution ofG proteins over the entire range of sucrose concentrationsduring gradient fractionation (cf. Fig. 2). Such heterogeneityof pinocytotic vesicles is commonly observed and is probablythe consequence of multiple processing of pinosomes as theyprogress through the endocytic transport pathway (25).

In order to understand the role of hormones in the pinocy-totic process, an investigation of the earliest phases of thepinocytotic process prior to multistep processing is neces-sary. Does isoproterenol stimulate the initiation of pinocy-

tosis or does it affect subsequent processing? What factorsdetermine the on/off regulation of adenylyl cyclase in pino-somes: the residency time ofthe hormone within the vesicles,the turnover of GTP, and destruction and/or cycling of thepinosomes back to the cell membrane? Are the poorlyunderstood antilipolytic actions of insulin related to thehormone's actions on various endocytic processes includingthe cycling of glucose transporters and receptors for insulin-like growth factors (26)? Insulin inhibits the stimulatory effectof epinephrine on pinocytosis (22), possibly through itsantilipolytic actions, and exerts a small stimulatory effect onbasal pinocytosis (27). The possibility that signals may begenerated and disseminated continually within the cell duringthe cycling and processing of endocytic vesicles obviouslyraises many additional issues concerning the regulation ofcellular responses to hormones.

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Proc. Natl. Acad. Sci. USA 87 (1990)

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