Identification and localization of Gia3 in the clonal adipocyte cell lines HGFu and Ob17

MONIQUE CADRIN ' Department of Pathology, University of Ottawa, Ottawa, ON KIH 8M5, Canada

AND

NORMA MCFARLANE-ANDERSON~ AND NICOLE B~GIN-HEICK~

Department of Biochemistry, University of Ottawa, Ottawa, ON KIH 8M5, Canada

Received July 5, 1993

CADRIN, M., MCFARLANE-ANDERSON, N., and BBGIN-HEICK, N. 1993. Identification and localization of Gia3 in the clonal adipocyte cell lines HGFu and Ob17. Biochem. Cell Biol. 71: 518-521.

The HGFu and Ob17 cell lines, derived from adipose tissue of lean (+ /?) and ob/ob mice, respectively, express several G-protein peptides. Investigation of the expression and subcellular localization of the Gia3 subunit showed that this peptide is associated with the Golgi apparatus. These findings indicate a role for this subunit in vesicular traffic and are in agreement with the view of the adipocyte as a secretory cell.

Key words: preadipocytes, differentiation, immunofluorescence, confocal microscopy, obesity, Golgi system.

CADRIN, M., MCFARLANE-ANDERSON, N., et BEGIN-HEICK, N. 1993. Identification and localization of Gia3 in the clonal adipocyte cell lines HGFu and Ob17. Biochem. Cell Biol. 71 : 518-521.

Les lignees cellulaires HGFu et Ob17, dkrivtes du tissu adipeux respectivement de souris maigres (+ /?) et de souris obkses (ob/ob), synthetisent plusieurs formes de protiines G. L'etude de l'expression et de la localisation intracellulaire de la sous-unite Gia3 demontre que ce peptide est associt ii I'appareil de Golgi. Ces observations indiquent que cette sous-unite jouerait un rale dans le transport vesiculaire et elles concordent avec le concept selon lequel I'adipocyte serait une cellule sicrttrice.

Mots clks : preadipocyte, diffirenciation, immunofluorescence, microscopie confocale, obisiti, appareil de Golgi. [Traduit par la redaction]

Introduction There is increasing evidence demonstrating that the

heterotrimeric G-proteins, which transduce extracellular signals by interacting with extracellular receptors, are not restricted t o the plasma membrane. This view is supported by the finding that there are intracellular pools of G-proteins, either bound t o intracellular membranes (Codina et al. 1988; Ali et al. 1989) o r free in the cycoplasm (Rasnas et al. 1989; Negishi et al. 1992; Bomsel and Mostov 1992). Immunocytochemical localization has shown subunits to be present in the cytoplasm (Wang et al. 1989; Muntz et al. 1992), G ia subunits in cytoplasm and lysosomes (Lewis et al. 1991), Goa in the cytoplasm (Brabet et al. 1988; Gabrion et al. 1989), and Gia3 in the Golgi apparatus (Ercolani et al. 1990; Wilson et al. 1992). Our own work on the clonal adipocyte cell lines HGFu and Ob17 also shows that the subunits of G,a, Gia , and GP have cytoplasmic localizations and that Gia and GP are redistributed during cell differentiation (McFarlane-Anderson et al. 1993). Changes in expression and cellular distribution of G-proteins

ABBREVIATIONS: HGFu and Ob17 cells, preadipocyte cells derived from epididymal fat pads of the C57BI/6 (+ /?) and ob/ob mice, respectively; G-protein, guanine nucleotide binding regulatory protein; WGA, wheat germ agglutinin; TRITC, tetramethyl rhod- amine isothiocyanate; ConA, concanavalin A; FITC, fluorescein isothiocyanate; DMEM, Dulbecco's modified Eagle medium; T,, 3,3' ,5-triido-L-thyronine; SDS-PAGE, sodium dodecyl sulphate - polyacrylamide gel electrophoresis; ECL, enhanced chemilumines- cence; PBS, phosphate-buffered saline; IgG, immunoglobulin G; kb, kilobase(s); kDa, kilodalton(s).

'current address: Departement de chimie-biologie, Universitk du Quebec a Trois-Rivikres, Trois-Rivitres, PQ G9A 5H7, Canada.

'current address: Tropical Diseases and Metabolism Research Unit, Kingston, Jamaica.

3 ~ u t h o r to whom all correspondence should be addressed. Printed in Canada / Imprime au Canada

in differentiating 3T3 cells have been described (Lai et al. 1981 ; Watkins et al. 1989) and more recently Wang et al. (1992) have suggested a role for G-proteins in differentiation.

Recent studies have suggested that Gia3 is involved in membrane traffic (Stow et al. 1991; Bomsel and Mostov 1992; Hepler and Gilman 1992; Leyte et al. 1992). This study was undertaken to assess the expression and determine the subcellular localization of G i a 3 in HGFu and Ob17 cells. The data show that the peptide is associated with the Golgi apparatus in both cell lines and that this pattern does not change during differentiation.

Materials and methods Materials

[ a - 3 Z ~ ] d ~ ~ ~ , Hybond nylon membrane, the multiprime label- ling kit, and the ECL Western blotting analysis system were from Amersharn Canada Ltd. (Oakville, Ont.). Salmon sperm DNA and dextran sulphate were from the Sigma Chemical Co. (St. Louis, Mo.). GTP was from PL Biochemicals, (Milwaukee, Wis.). Cell culture reagents were from GIBCO/BRL, Life Technologies Inc. (Burlington, Ont.). The Gia3/Goa (NEI-803) and Goa (NEI-804) antibodies were from NEN, Dupont Canada, (Mississauga, Ont.) WGA-TRITC and ConA-TRITC were from Dimension Lab. Inc. (Mississauga, Ont.). FITC-conjugated goat anti-rabbit antibody was from Bio/Can scientific, (Toronto, Ont.).

Cell lines Ob17 and HGFu cell lines were generously provided by Dr. U.

Kozak, Jackson Laboratories, Bar Harbor, Maine. The cells were grown in DMEM containing 10% fetal calf serum supplemented with penicillin (200 U/mL), streptomycin (100 g/mL), and fungi- zone (0.25 g/mL). Cells were induced to differentiate by adding insulin (17 nM) and T3 (2 nM) to the cultures at confluence. The experiments were conducted with cultures at confluence (undifferentiated) or 6 days after the addition of insulin and T3 (differentiated). For RNA preparation, cells were grown in 100-mm dishes. For the immunocytochemical studies, they were grown on glass cover slips in 35-mm dishes.

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FIG. 1. Immunodetection of G,a3 and G,a in HGFu and Ob17 cells. Samples corresponding to 10, 20, and 40 g protein were processed as described in Materials and methods and detection was done with either NEI-803 (a) or NEI-804 (b). Lanes 1-3, HGFu; lanes 4-6, Ob17 cells; lane 7 (in b only), sample of brain membrane (20 g protein). Bars represent molecular mass markers of 46 and 30 kDa, respectively. Exposure was for 40 s for a and 10 s for b where longer exposures (up to 60 s) did not reveal proteins of the appropriate size in the clonal cells.

SDS-PAGE and irnrnunoblotting The electrophoresis and transfer were done essentially as

described previously (McFarlane-Anderson et al. 1993). The immunodetection was done as follows. The antibodies were diluted 1:8000 and the ECL detection system was used to label the proteins recognized by the antibody.

RNA preparation and cDNA hybridization Total RNA prepared by the guanidinium thiocyanate method

(Chomczynski and Saachi 1987) was used for Northern blots. The mouse G,a3 cDNA, a gift from Dr. R. Reed (Jones and Reed 1987), was used in multiprime DNA labelling reactions as previously described (McFarlane-Anderson et al. 1992).

Zrnrnunofluorescence staining Cells were fixed in ethanol for 10 min at - 20°C and processed

for immunofluorescence as previously described in detail (McFarlane-Anderson et al. 1993). Briefly, cover slips were incubated for 45 min with the antibodies, diluted 1:50 in PBS containing 0.5% skim milk. After rinsing in PBS, cover slips were incubated with goat anti-rabbit IgG conjugated to FITC (1:25). For double-labelling experiments, fixed cells were incubated with WGA-TRITC or CO~A-TRITC (1:20) for 20 min at room tem- perature, rinsed in PBS, incubated with the anti Gia3/Go(x anti- body, rinsed in PBS, and then incubated with the FITC-conjugated goat anti-rabbit IgG. After staining, cover slips were mounted (0.1% p-phenylenediamine in 50% glycerol-PBS) on glass slides and viewed by conventional epifluorescence microscopy on a Zeiss Axiophot photomicroscope or with a Leica laser scanning confocal microscope. Controls omitting the first antibody from the labelling reaction were done. In such cases no staining was observed (not shown).

Results and discussion The antibody used for the immunocytochemical locali-

zation studies (NEI-803) recognizes the C-terminal end of

FIG. 2. Expression of Gia3 subunits in HGFu and Ob17 cells. (A) Northern blot analysis of 10 g total RNA. Lanes 1-3, mRNA samples from lung, Ob17, and HGFu cells, respectively. Bars marked 5 and 2 represent 28 and 18s RNA markers, corresponding to 5 and 2 kb, respectively; bar marked 3 corresponds to the size of the Gi.3 mRNA. (B) Slot blots of 0.50, 0.75, 1 .O, 1.7, and 2.0 g of RNA were probed with Gia3 cDNA. (a) HGFu; (b) Ob17 cells. Slots 1-5, undifferentiated; slots 6-10, differentiated cells.

FIG. 3. Distribution pattern of Gia3 in confluent Ob17 cells. Cells were stained by immunofluorescence with anti-Gia3. The Golgi-like staining is indicated by arrows. Diffuse cytoplasmic and some nuclear staining can also be observed.

both Gia3 and Goa. Western blots were done with NEI-803, as well as an antibody that is specific for G,a (NEI-804). Therefore, in HGFu and Ob17 cells, the peptide recognized by NEI-803 corresponds to Gia3, since NEI-804 (specific for Goa) does not detect a peptide in the two cell lines, although it does so in brain (Fig. lb).

Northern blotting with the Gia3 cDNA revealed that the probe hybridized only with a 3.5-kb mRNA (Fig. 2a).

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BIOCHEM. CELL BIOL. VOL. 71, 1993

FIG. 4. Confocal microscopic analysis of G,a3 and Golgi system in confluent Ob17 cells. Cells were double labelled with anti-Gia3 and WGA and confocal imaging of O.Sam sections was used to determine colocalization. To determine if there was colocalization, separate anti-Gia3 (a) and WGA (6) images were obtained and superimposed (c). Green marks Gia3, red marks WGA, and yellow marks colocalization. Some nuclear staining with Gia3 is also evident. Bar, 10 m.

Figure 2b shows that the level of expression of Gia3 was the same in both cell lines (cf. lanes in a with b). Differenti- ation from a fibroblast-like preadipocyte cell type into a lipid-containing adipocyte was not associated with a signif- icant difference in expression of the Gia3 mRNA (cf. slots 1-5 with slots 6-10 in each of the blots), while actin mRNA levels were markedly reduced in differentiating cells (McFarlane-Anderson et al. 1993).

Both cell lines exhibited the same cytoplasmic Gia3 dis- tribution pattern (Fig. 3), which did not change as a result of differentiation. The anti-Gia3 showed diffuse cytoplas- mic distribution with some nuclear staining and a dense pattern that labelled structures located in the perinuclear area, in a Golgi-like distribution. There was little or no label- ling of plasma membranes. To characterize the elements recognized by the antibody, the cells were double stained with the anti-Gia3 and WGA-TRITC or ConA-TRITC. WGA binds to components of the Golgi system (trans- cisternae, endosomes, endosomal tubuloreticular compart- ment and its associated vesicles) (Lipsky and Pagano 1985) and ConA to the rough endoplasmic reticulum (Rabinowitz et al. 1992). The double-labelled cells were analyzed by laser scanning confocal microscopy. Images of 0.5-pm laser- scanned sections were superimposed. Anti-Gia3 staining suggested a nuclear as well as a Golgi distribution (Fig. 4a). WGA staining revealed both tubular and vesicular structures that correspond, respectively, to the trans-Golgi and its associated secretory vesicles (Fig. 4b). Examination of the superimposed images obtained after WGA and anti-Gia3 staining showed a colocalization of the two patterns at the level of the tubular structures stained by WGA and also a localization of Gia3 with nuclear structures (Fig. 4c). Double staining of the cells with ConA and Gia3 demon- strated that the peptide is not associated with the rough endoplasmic reticulum (not shown). These results confirm the association of Gia3 with the Golgi apparatus and indi- cate its localization at the level of the cis- and trans-Golgi system. This suggests that, as is the case in other cell types (Burgoyne 1992; Leyte et al. 1992; Aridor et al. 1993), the adipocyte Gia3 subunit is involved in vesicular traffic.

So far, the association of Gia3 with the Golgi system has been reported mainly in secretory cells (Stow et al. 1991; Aridor et al. 1993). Hitherto, secretion has not been consi- dered a major function of adipose cells, although they have

long been known as a major source of secreted proteins such as lipoprotein lipase. However, it has recently been demon- strated that adipose cells secrete several other proteins, including adipsin (Kitagawa et al. 1989). The finding that clonal adipose cells express Gia3 and that a significant portion of this peptide is localized in the Golgi system supports the emerging recognition of a secretory role for adipose cells (Ailhaud et al. 1992).

Acknowledgments The authors are grateful to the Medical Research Council

of Canada for grant support, to Andrew Vaillant for help with the confocal microscopy, and to Pascale Reinhardt- Poulin for technical assistance with the immunodetection.

Ailhaud, G., Grimaldi, P., and Negrel, R. 1992. Cellular and molecular aspects of adipose tissue development. Annu. Rev. Nutr. 12: 207-233.

Ali, N., Milligan, G., and Evans, W.H. 1989. Distribution of G-proteins in rat liver plasma-membrane domains and endocytotic pathways. Biochem. J. 261: 905-912.

Aridor, M., Rajmilevich, G., Beaven, M.A., and Sagi-Eisdenberg, R. 1993. Activation of exocytosis by the heterotrimeric G protein G,. Science (Washington, D.C.), 262: 1569-1572.

Bomsel, M., and Mostov, K. 1992. Role of heterotrimeric G pro- teins in membrane traffic. Mol. Biol. Cell. 3: 1317-1328.

Brabet, P., Dumuis, A., Sebben, M., Pantaloni, C., Bockaert, J., and Homburger, V. 1988. Immunocytochemical localization of the guanine nucleotide-binding protein Go in primary cultures of neuronal and glial cells. J. Neurosci. 8: 701-708.

Burgoyne, R.D. 1992. Trimeric G proteins in Golgi transport. Trends Biochem. Sci. 17: 87-88.

Chomczynski, P., and Saachi, N. 1987. Single-Step method of RNA isolation by acid guanidinium thiocyanate - phenol - chloroform extraction. Anal. Biochem. 162: 156-159.

Codina, J., Kimura, S., Kraus-Friedman, N., and Higham, S. 1988. Demonstration of the presence of G-proteins in hepatic microsomal fraction. Biochem. Biophys. Res. Commun. 150: 848-852.

Ercolani, L., Stow, J.L., Boyle, J.F., Holtzman, E.J., Lin, H., Grove, J.R., and Ausiello, D.A. 1990. Membrane localization of the pertussis toxin-sensitive G-protein subunits a,, and the expression of a metallothionein-a,.2 fusion gene in LLC-PK, cells. Proc. Natl. Acad. Sci. U.S.A. 87: 4635-4639.

Gabrion, J., Brabet, P., Nguyen Than Dao, B., Homburger, V., Dumuis, A., Sebben, M., Rouot, B., and Bockaert, J. 1989.

Bio

chem

. Cel

l Bio

l. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y C

ON

CO

RD

IA U

NIV

on

11/1

3/14

For

pers

onal

use

onl

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CADRIN ET AL. 521

Ultrastructural localization of the GTP-binding protein Go in neurons. Cell. Signalling, 1: 107-123.

Hepler, J.R., and Gilman, A.G. 1992. G proteins. Trends Biochem. Sci. 17: 383-387.

Jones, D.T., and Reed, R.R. 1987. Molecular cloning of five GTP- binding protein cDNA species from rat olfactory neuro- epithelium. J. Biol. Chem. 262: 14 241 - 14 289.

Kitagawa, K., Rosen, B.S., Spiegelman, B.M., Lienhard, G.E., and Tanner, L.I. 1989. Insulin stimulates the acute release of adipsin from 3T3-L1 adipocytes. Biochim. Biophys. Acta, 1014: 83-89.

Lai, E., Rosen, O.M., and Rubin, C.S. 1981. Differentiation- dependent expression of catecholamine-stimulated adenylate cyclase. J. Biol. Chem. 256: 12 886 - 12 874.

Lewis, J.M., Woolkalis, M.J., Gerton, G.L., Smith, R.M., Jarett, L.T., and Manning, D.R. 1991. Subcellular distribution of the a subunit(s) of Gi: visualization by immunofluorescent and immunogold labelling. Cell Regul. 2: 1097-1 113.

Leyte, A., Barr, F.A., Kehlenbach, R.H., and Huttner, W.B. 1992. Multiple trimeric G-proteins on the trans-Golgi network exert stimulatory and inhibitory effects on secretory vesicle formation. EMBO J. 11: 4795-4804.

Lipsky, N.G., and Pagano, R.E. 1985. A vital stain for the Golgi apparatus. Science (Washington, D.C.), 228: 745-747.

McFarlane-Anderson, N., Bailly, J., and Btgin-Heick, N. 1992. Levels of G-proteins in liver and brain of lean and obese (ob/ob) mice. Biochem. J. 282: 15-23.

McFarlane-Anderson, N., Cadrin, M., and Begin-Heick, N. 1993. Identification and localization of G-proteins in the clonal adipo- cyte cell lines HGFu and Ob17. J. Cell. Biochem. 52: 463-475.

Muntz, K.H., Sternweis, P.C., Gilman, A.G., and Murphy, S.M. 1992. Influence of Y subunit prenylation on association of guanine nucleotide-binding regulatory proteins with membranes. Mol. Biol. Cell 3: 49-61.

Negishi, M., Hashimoto, H., and Ichikawa, A. 1992. Translocation of a subunits of stimulatory guanine nucleotide-binding proteins through stimulation of the prostacyclin receptor in mouse mastocytoma cells. J. Biol. Chem. 267: 2364-2369.

Rabinowitz, S., Horstmann, H., Gordon, S., and Griffiths, G. 1992. Immunocytochemical characterization of the endocytic and phagolysosomal compartments in peritoneal macrophages. J. Cell Biol. 116: 95-1 12.

Rasnas, L.A., Svoboda, P., Jasper, J.R., and Insel, P.A. 1989. Stimulation of @-adrenergic receptors of S49 lymphoma cells redistributes the a subunit of the stimulatorv G rote in between cytosol and membranes. Proc. Natl. cad. ~ c i . U.S.A. 86: 7900-7903.

Stow, J.L., de Almeida, J.B., Narula, N., Holtzman, E.J., Ercolani, L., and Ausiello, D.A. 1991. A heterotrimeric G protein, G,,, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PK, epithelial cells. J. Cell Biol. 114: 1113-1 124.

Wang, H., Berrios, M., and Malbon, C.C. 1989. Indirect immunofluorescence localization of @-adrenergic receptors and G-proteins in human A431 cells. Biochem. J. 263: 519-532.

Wang, H., Watkins, D.C., and Malbon, C.C. 1992. Antisense oligonucleotides to G, protein a-subunit sequence accelerate differentiation of fibroblasts to adipocytes. Nature (London), 358: 334-337.

Watkins, D.C., Rapiejko, P.J., Ros, M., Wang, H.Y., and Malbon, C.C. 1989. G-protein mRNA levels during adipocyte differentiation. Biochem. Biophys. Res. Commun. 165: 929-934.

Wilson, B.S., Hermouet, S., de Mazancourt, P., Spiegel, A.M., and Farquhar, M.G. 1992. Localization of Gi, alpha subunit is dependent on cell type and level of expression: Implications for Golgi function. Mol. Biol. Cell 3: 34a (abstr.).

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