gift from Dr. Jack Wagner at Eli Lilly (Indianapolis. IN). collaqenase ..... ['"I)-labeled goat anti-rabbit IgG as described by Humby (441, following a 3 h incubation ...
THEJOURNALOF BIOLOGICAL CHEMISTRY
Vol. 266, No. 24, Issue of August 25, pp. 15949-15955, 1991 Printed in U.S.A.
Alterations in mRNA Levels, Expression, and Function of GTP-binding RegulatoryProteins inAdipocytes fromObese Mice (CS7BL/GJ-ob/ob)* (Received for publication, February 28, 1991)
Tom W. GettysS, Vickram Ramkumar, Ronald J. Uhings, Lucy Seger, andIan L. Taylor From the Departments of Medicine and SPatholoKy, Duke Uniuersity and Veterans Administration Medical Centers, Durham, North Carolina 27710 . ”
Messenger RNA levels for thea subunit of G-proteins expressed in adipocytes of lean and obese (obfob)mice were compared with relative levels of the encoded proteins. Using both toxin labeling andWestern blots, expression of G,, Gia-l, and Gia-3was decreased by approximately 2-fold in adipocytes of obese mice, while levels of Gi,.z did not differ between the phenotypes. The decreases inGia-land G, in the obese mouse were attributed to decreased mRNA levels for these proteins. Similar mRNA levels for Gia-3were noted in both phenotypes, but Gia.z message was increased 2fold in the obese mouse. Inhibitory regulation of adipocyte adenylylcyclase through G-proteins was evaluated by comparing the abilityof R-PIA to inhibitisoproterenol-stimulated responses between the phenotypes. In spite of the decrease in Gia-l and Gia-3 in adipocytes from obese mice,R-PIA inhibited adenylylcyclase, CAMP-dependentprotein kinase,and lipolysis in similar fashion in both phenotypes. The GTPanalog, Gpp(NH)p also inhibited forskolin-stimulated adenylylcyclase in a comparable manner, but themagnitude of the inhibition was slightly less in adipocyte membranes from obese mice. In contrast, the decrease in expression of G,was translatedintosubstantially poorer activation of isoproterenol-stimulated responses in the obese mouse. The concentration of isoproterenol producing half-maximal activation of adenylylcyclase, protein kinase, andlipolysis did not differ between the phenotypes, but themaximal responses were much lower in cells from obese mice. Similar lipolytic potential in isolated adipocytes from eachphenotype and similar total forskolin-stimulated cyclase activity in adipocyte membranes from each phenotype suggest that decreased expression of G, may contribute to the characteristic alteration in mobilization of triglycerides noted in adipocytes from obese mice.
In mice, inheritance of the gene for obesity from both parents produces offspring (ob/ob) which are hyperglycemic, hyperinsulinemic, insulin-resistant, and hyperphagic. Hypertrophy of adipocytes and deposition of excess fat is observed * This work was supported by Research Grants DK 42486 and DK 38216 from the National Institutes of Health, a small instrument grant from the Duke Hospital Auxiliary, and the Veterans Administration. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $Author to whom correspondence should be addressed: Div. of Gastroenterology, P. 0.Box 3913, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-6119; Fax: 919-684-8857.
well before the onset of hyperphagia (1) and occurs in obese mice (ob/ob) even when they are pair-fed with lean littermates (2). This model of non-insulin-dependent diabetes is also characterized by resistance to mobilization of free fatty acids by adrenergic stimulation (3-5). The potential of altered hormonal signaling in several of the metabolic disturbances seems likely, but the underlying mechanisms have not been clearly established. The original suggestion by Gawler and coworkers (6) that chemically induced diabetes abolished expression and function of pertussis toxin-sensitive G-proteins in hepatocytes has been controversial (7). Potential alterations of adipocyte G-proteins in models of non-insulindependent diabetes have also been debated (8) with an eye toward understanding whether these changes result from or contribute to the etiology of the disease (9-12). Such studies are motivated by indications that the obese, hyperglycemic mouse suffers from hypercorticoidism and hypothyroidism (13) and by the knowledge that alterations in adrenal (14-16) or thyroid (17-20) function have profound effects on Gprotein expression in a number of tissues. Our incomplete understanding of the complexity and scope of G-proteincoupled signaling mechanisms has made it difficult to assess the functional consequences of altered expression of particular G-proteins. The scope of this task is underscored by the recent appreciation of possible promiscuity in interactions among Gproteins and signaling pathways (21). These points are illustrated in several cases where disease-associated changes in expression of G-proteins have not been correlated with a coordinate change in function of pathways known to be regulated by the affected G-proteins (11, 15, 19,22). In contrast, there are reported examples of coordinate changes in expression and function of G-proteins (10,16,17,23).We have used adipocytes from the obese, hyperglycemic mouse to examine the relationship between mRNA levels and G-protein expression and evaluated the functional significance of any detected changes in their expression. Evidence is presented to suggest regulation of G-proteins at both transcriptional and posttranscriptional levels, and it is shown that altered G-protein expression may be translated into defective propagation of hormonal signals. MATERIALS AND METHODS A N D RESULTS’ DISCUSSION
The failure of adipose tissue from the obese-hyperglycemic mouse to effectively mobilize triglycerides in response to
’ Portions of this paper (including “Materialsand Methods,” “Results,” Figs. 1-13, and Table 1) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal thatis available from Waverly Press.
15949
15950
Adipocytes G-proteins in
epinephrine was recognized 30 years ago (3), and numerous studies have addressed the mechanisms and potential importance of this defect in the interim. Adrenergic activation of adenylylcyclase (4, 24) and elevation of intracellular cAMP ( 5 )were lowerin obese micethan theirlean littermates. Taken together, these studies suggest a deficiency in thetransmission of hormonal signal from receptor to adenylylcyclase but do not exclude phenotypic differences downstream from cAMP elevation. The present studies demonstratepoor activation of adenylylcyclase by isoproterenol in purified adipocyte membranes from obese mice, whichparallels equally poor mobilization of triglycerides by the same agonist from intact cells. However, phenotypic differences in lipolytic capacity were discounted in previous work (4) and by the present finding that 8-bromo-CAMPelicited comparable mobilization of glycerol from adipocytes of the two phenotypes. Since this experimental approach bypasses the regulatory steps prior to protein kinase activation, the results argue that thedefect in the signaling pathway in the obese mouse resides upstream from protein kinase activation. The present finding that CAMPdependent protein kinase activation by isoproterenol is deficient in the same cells suggests that thedefect in thepathway must exist between the p-adrenergic receptor and the adenylylcyclase enzyme. Nearly identical responses of adenylylcyclase to forskolin in membranes from the two phenotypes indicate that thedeficit is not at thelevel of adenylylcyclase. In addition, previous studies have found similar levels of padrenergic receptors in adipocytes (25) and hepatocytes (26) from the two phenotypes, thereby ruling out a reduction in preceptors as the cause of diminished isoproterenol-elicited responses. The present results showing diminished adenylylcyclase activation by ACTH’in adipocyte membranes of obese mice, and the previous report of poor stimulation of lipolysis by ACTH in tissuefragments of obesemice ( 5 ) are also consistent with the above conclusion that a reduction in G,, is translated into attenuated, receptor-mediated adenylylcyclase activation. In contrast, Begin-Heick (4, 25, 27) found similar adenylylcyclase activation by sodium fluoride in adipocyte membranes from each phenotype. One of these reports (27) also showed that isoproterenol plus Gpp(NH)p produced similar cyclase activation in adipocytes of both phenotypes, while another (25) showed substantially lower cyclase activation in obese membranes using the same conditions. These results differ quantitatively, but Begin-Heick reported in a later study (10) that G., was reduced in adipocytes of the obese mouse. Our results suggest that the decrease in G,, noted in adipocytes of obese mice may be due in part to a defect in gene expression. A decrease in both mRNA and protein levels may explain the attenuated ability of CAMPelevating agonists to mobilize triglycerides in obese mice. The present work has shown that Gim.land Giu.3are lower in adipocyte membranes from obese mice. Gi,,.z appeared to be expressed at much lower levels than Gicv.land Gim-ain both groups of mice but at levels which apparently did not differ between the phenotypes. Previous work using membranes from rat adipocytes concluded that thelower band migrating at 39-40 kDa was Go (28), while others concluded that Go was not present in adipocyte membranes (29). Recent work using
of Obese foblob) Mice
antisera against the C-terminal sequence ofGia.‘ also concluded that thelower band was Gi,.’ (30,31). We believethat the lower 40-kDa band labeled by pertussis toxin, recognized by antiseraagainst Gia.P, but not recognizedby antisera against Go, is in fact Gi,.z. This conclusion is also supported by observations concerning the relative mobility ofGi,.z on SDS gels (32, 33). It is interesting that the upper and lower bands are present in similar amounts in rat adipocyte membranes (34, 35) while the lower 40-kDa band (Gi,.J is expressed at lower levels in mouse adipocytes. Adenylylcyclase in adipocytes from both species is inhibited by R-PIA, but the concentrations required to produce these effects are substantially higher in mouse adipocyte membranes (8).Whether this difference is related to the relative expression of Girr.*in adipocytes remains to be established. The potential significance of the observed decline in Gi,.l and Gia.3 was tested by comparing the efficacy of agonistmediated inhibition of adenylylcyclase, protein kinase, and lipolysis in adipocytes from the two phenotypes, and by comparing GTP analog-mediated inhibition of membrane adenylylcyclase. This strategy assumes that if either of these G proteinscan couple to adenylylcyclase inhibition, the observed decrease in expression could be translated into diminished function. It should be noted that the identity of the Gprotein which mediates inhibitory regulation of cyclase by specific receptors on adipocytes hasnot been established. Further, recent studies have suggested that pertussis toxinsensitive G-proteins can be promiscuous in the sense that they will interact with more than one signaling pathway (21). Whether a similar principal applies in the adipocyte remains to be shown. We detected no difference in the sensitivity of adenylylcyclase to inhibition by R-PIA or Gpp(NH)p, but it was noted that the magnitude of inhibition of cyclase by Gpp(NH)p was smaller in adipocytes from obese mice. Using low magnesium concentrations, Begin-Heick (9, 10) was unable to show GTP- or R-PIA-mediated inhibition of adenylylcyclase in adipocyte membranes from obesemice. The author also found substantially lower levels of “Gi” immunoreactivity in adipocytes from obese mice (10). In contrast, Greenberg et al. (8)reported more potent inhibition of cyclase by R-PIA in purified adipocyte membranes from obese mice and at least a 2-fold increase in pertussis toxin substrates compared with membranes from lean mice. It seems possible that subtle differences in assay conditions ( i e . magnesium concentration and protease inhibitors) and methods used in membrane preparation may account for some of the differences among these findings. The present findings suggest several interpretations including the possibility that Gi,.z is the mediator of cyclase inhibition in this system. Another possibility is that Gi,.l and Gin-xare not coupled to cyclase inhibition or thatthe decline in theseG-proteins is not sufficient to limit signaling through this pathway. Work from Houslay’s group (36) has suggested that defective inhibitory G-protein function seen in obese Zucker rats may be due to an inactivating phosphorylation of Gi,.z which prevents normal interaction with adenylylcyclase. Clarification of these possibilities awaits further study. It was assumed that observed changes in G-protein mRNA in adipose tissue from the two phenotypes would be reflected The abbreviations used are: ACTH, adrenocorticotropic hormone; KRH, Krebs Ringer Hepes; DATD, N,N”diallyltartardiamide; BSA, in expression of the encoded proteins. Inthe case of G~,.Ithat bovine serum albumin; TES, N-tris(hydroxylmethyl)methyl-2-ami- expectation was fully realized since both message levels and noethanesulfonic acid TCA, trichloroacetic acid; MOPS, 3-(N-mor- expression were -50% lower in adipose tissue fromobese pho1ino)propanesulfonicacid; Gpp(NH)p, guanyl-5”yl imidodiphos- mice. The findings are quitedifferent for Gin.’ and Gin.3where phate; DTT, dithiothreitol; NAD, nicotinic adenine dinucleotide; RPIA, phenylisopropyladenosine;SDS-PAGE, sodium dodecyl sulfate- expression appears not to reflect the relative mRNA levels in polyacrylamide gel electrophoresis; ADA, adenosine deaminase; the two phenotypes. Messenger RNAlevels for Gin-z were increased %fold in adipose tissue from obese mice despite no HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.
!s of Obese ~ob/ob) Mice G-proteins in Ad~pocyt~
15951
16. Ros. M.. NorthuD. J. K.. and Malbon. C. C. 11989) . . Biochem J. 2 6 7 , 737-744 17. Malbon, C. C., Rapiejko, P. J., and Mangano, R. J. (1985) J. Biol. Chem. 260.2558-2564 18. Rapiejko, P. J., and Malbon, C. C . (1987) Biochem. J. 241,765771 19. Levine, M.A., Feldman, A. M., Robishaw, J. D., Ladenson, P. W., Ahn, T. G., Moroney, J. F., and Smallwood, P. M.(1990) J. Bwl. Chem. 265,3553-3560 20. Milligan, G., and Saggerson, E. D. (1990) Biochem. J. 2 7 0 , 765769 21. Yatani, A., Mattera, R., Codina, J., Graf, R., Okabe, K., Padrell, E., Iyengar, R., Brown, A. M., and Birnhaumer, L. (1988) Nature 336,680-682 22. Begin-Heick, N., and Welsh, J. (1988)Mol. Cell. Endocrinol. 5 9 , 187-194 23. Bushfield, M., Griffiths, S. L., Murphy, G. J., Pyne, N. J., Knowler, J. T., Milligan, G., Parker, P. J., Mollner, S., and Houslay, M. D. (1990) Bwchem. J. 271,365-372 24. Dehaye, J. P., Winand, J., and Christophe, J. (1978) D i ~ e ~ o ~ g i a 15,45-51 25. Begin-Heick, N.(1980) Can. J. Biochem. 5 8 , 1033-1038 26. Kahn, C . R.,Neville, D.M., Jr., and Roth, J. (1973) J. Biol. Chem. 248,244-250 27. Begin-Heick, N. (1984) Can. J. Bwchem. 62,819-826 28. Rapiejko, P. J., Northup, J. K., Evans, T., Brown, J. E., and Malbon, C. C. (1986) Biochem. J. 240,35-40 29. Hinsch, K. D., Rosenthal, W., Spicher, K., Binder, T., Gausepohl, H., Frank, R., Schultz, G., and Joost, H. G. (1988) FEBS Lett. 238,191-196 30. Green, A., Johnson, J. L., and Milligan, G. (1990) J. Biol. Chem. 265,5206-5210 31. Green, A., and Johnson,J. L. (1991) Diabetes 40,88-94 32. Birnhaumer, L., Codina, J., Mattera, R., Yatani, A., Graf, R., Olate, J., Sanford, J., and Brown, A. M. (1988) Cold Spring Harbor Symp. Qwmt. BWL 53,229-239 33. Goldsmith, P., Rossiter, K., Carter, A., Simonds, W., Unson, C . G., Vinitsky, R., and Spiegel, A. M. (1988) J. Biol. Chem. 263, 6476-6479 34. Strassheim, D., Milligan, G., and Houslay, M. D. (1990) Biochem. J. 266, 521-526 Acknowledgments-We acknowledge Dr. Christopher J. Lynch 35. Ramkumar, V., and Stiles, G. L. (1990) Endocrinology 126,1295(Molecular Physiology Department, Hershey Medical Center, Her1304 shey, PA) for the gift of antiserum to the cy subunits of Gi+21,Gi.3, 36. Bushfield, M., Pyne, N. J., and Houslay, M. D. (1990) Eur. J. and G,, and Dr. Janet Robishaw (Weis Center for Research, Geisinger Biochem. 192,537-542 Clinic, Danville, PA) for the gift of antiserum to thecy subunit of Go. 37. Longabaugh, J. P., Didsbury, J., Spiegel, A., and Stiles, G. L. (1989) Mol. Phrmacol. 36,681-688 REFERENCES 38. Griffiths, S. L., Knowler, J. T., and Houslay, M. D. (1990) Eur. 1. Thurlby, P. L., and Trayhurn, P. (1978) Br. J. Nutr. 3 9 , 397J. Biochem. 193,367-374 39. Morre, D. J., and Morre, D. M. (1989) B w t e c h n i q ~ s7,946-958 402 2. Trayhurn, P. (1984) CEin. Endocrinol. Metab. 13,451-474 40. Spydevold, S. O., Greenbaum, A. L., Baquer, N. Z., and McLean, P. (1978) Eur. J. Biochem. 89,329-339 3. Marshall, N. B., and Engel, F. L. (1960) J. Lipid Res. 1,339-342 4. Begin-Heick, N.,and Heick, H. M. (1977) Can. J. Physiol. Phar- 41. Rodhell, M. (1964) J. Biol. Chem. 239,375-380 42. Kono, T. (19841 in Methods in Diabetes Research (Larner, J., and m a ~ O l .55,1320-1329 Pehl, S. L., eds) pp. 83-91, John Wiley & Sons, New York 5. Dehaye, J. P., Winand, J., and Christophe, J. (1977) Diabetologia 43. Uhing, R. J., Polakis, P. G., and Snyderman, R. (1987) J. Biol. 13,553-561 Chem. 262,15575-15579 6. Gawler, D., Milligan, G., Spiegel, A. M., Unson, C.G., and 44. Mumby, S., Pang, 1.-K., Gilman, A.G., and Sternweiss, P. C. Wouslay, M. D. (1987) Nature 327,229-232 (1988) J. Biol. Chem. 263,2020-2026 7. Lynch, C. J., Blackmore, P. F., Johnson, E. H., Wange, R. L., 45. Smith, C. J., and Manganiello, V. C. (1989) Mol. Pharmacol. 36, Krone, P. K., and Exton, J. H. (1989) J. Clin. Inuest. 83,2050381-386 2062 8. Greenberg, A. S., Taylor, S. I., and Londos, C. (1987) J. Biol. 46. Gettys, T. W., Blackmore, P. F., Redmon, J. B., Beebe, S. J,, and Corbin, J. D. (1987) J. Biol. Chem. 2 6 2 , 333-339 Chem. 262,4564-4568 47. Beebe, S. J., Holloway, R., Rannels, S. R., and Corbin, J. D. 9. Begin-Heick, N. (1985) J. BioL Chem. 269,6187-6193 (1984) J. Riol. Chem. 269,3359-3547 10. Begin-Heick, N. (1990) Biochem. J. 268,83-89 48. Gettys, T. W., Okonogi, K., Tarry, W. C., Johnston, J., Horton, 11. Vannucci, S, J., Klim, C.M., Martin, L. F., and LaNoue, K. F. C., and Taylor, I. L. (1990) Second Messengers Phosphoproteins (1990) Am. J. Physiol. 257, E871-E878 13,37-50 12. Begin-Heick, N. (1987) Mol. CeU. ~ ~ o c r i53, ~ l1-8 . 49. Harper, J. F., and Brooker, G. (1975) J. Cyclic ~ ~ ~ e o tRes. i d 1e , 13. Bray, G. A., and York, D. A. (1979) Physiol. Reu. 51,598-646 207-218 14. de Mazancourt, P., Giot, J., and Giudicelli, Y, (1989) Biochem. 50. Gettys, T. W., Burrows, P. M., and Henricks, D. M. (1986) Am. Int. 1 9 , 505-512 J. Physiol. 251, E357-E361 15, Mazancourt, P. D., Lacasa, D., Giot, J., and Giudicelli, Y. (1989) 51. Gettys, T. W., Vine, A. J., Simonds, M. F., and Corbin, J. D. Endocrinology 124, 1131-1139 (1988) J. Biol. Chem. 263,10359-10363
apparent difference in expression ofGi,.z between the phenotypes. Our crude attempts to resolve and detect Gia-*,coupled with its lower overall expression, may have hampered our ability to detect differences between lean and obese mice. In contrast, levels of Gin.ain adipose tissue from obese mice were subs~ntiallylower than inlean mice without a concomitant change in Gia-3mRNA. The lack of concordancebetween protein expression and mRNAlevelsfor the various Gi, subunits suggests regulation at a level other than transcription, butbased on the current unders~ndingof their regulation, the reasons for these differences are unclear. We have encountered similar situations inadipocytes from rats treated with pertussis toxin (35) or R-PIA (37)where changes in expression of Gi,., and Gi,.z were noted without a concomi~nt change in message levels for these proteins (35). Disparities have also been reported in adipocytes of diabetic rats (38) where mRNA forGi,.l increased 4-fold without a corresponding change in Gia.,expression. Previous reports using the ob/ ob model have providedconflicting reports with regard to the levels of plasma membrane-associated G-proteins (8-10). The use of a highly purified membrane preparation (39) has allowed the characterization of G-protein levels with minimal contribution from other organelles whose levels may differ between Iean and obese mice (40).Our results demonstrate disparities between mRNA levels and plasma membrane protein expression for Gia+ and perhaps Gia.a,suggesting that G-protein expression in this genetic model of diabetes involves regulation at levels other than transcription. Further studies will berequired to determine at which levelexpression is altered in adipocytes from obese mice. In addition, studies in other tissues will be required to determine whether there is a genetic basis for the alterations in G-proteins noted in the adipocyte.
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'
G-proteins in Adipocytes of Obese (oblob) Mice
15952
supplemental Material To: ALTERATIONS IN mRNA LEVELS, EXPRESSION AND FUNCTION OF GTP-BINDING REGUIATORY PROTEINS IN ADIPOCYTES FROM OBESE MICE (C57BL/6J-Ob/Ob)
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Waterials and Wethods
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mterials Obese mice (C578L/J6-0b/ob) and their lean siblinqs (OB/OB) of Certified genotype were Obtained one week after weaninq from Jackson Labs (Bar Harbor, ME) and used at 9-14 vk of aqe. Recombinant human insulin Was a gift from D r . Jack Wagner at Eli Lilly (Indianapolis. IN). collaqenase vas purchased from Worthinqton Biochemlcals (Freehold, NJ). Guanylyliaidodiphosphate. B-phenylIsopropyladenoslne, qlyserokinase, and qlycer'ol-3-phosphate dehydrogenase were from Boehrinqer-Mannheim (Indianapolls, IN). Kemptide and rat ACTH were from Penninsula Labs (Fremont, CAI. Pertussis and cholera toxin Were from List BiOlogical Labs (Campbell, CA). Dextran T-500 was obtained from Pharmacla (Nutley. NJ) and polyethylene qlycol 1350 vas from Fischer Scientific (Atlanta, GA). Labeled ('zP]NAD, y - [ 'PlATP, aind ("'1)qoat anti-rabbit IqG Were from New England Nuclear (Boston. MA). All other Chemicals vere from S l v a (St. Louis. MO).
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wads of Response variables from experiments in the present study were characterlred usinq relationship functions appropriate to the shape of the response surface ( 4 6 . 5 0 . 5 1 ) . The activation of adenylyl cyclase and protein kinase, and qlycerol release in response to lnsreasinq concentrations of isoproterenol, or the activation of adenylyl cyclase by forskolin were Characterized by the risinq loqistic ogive qiven as (1)
y x y
Adipocytes were isolated from epididpal fat pads of mala mice by collaqenase treatment scsordinq to Rodbell ( 4 1 ) . The cells were washed and resuspended in Krebs-Rinqer-Hepes (KRH') buffer contalninq 1 mU CaC1. and 21 BSA. In cells desiqnated for measurement of CAMP-dependent prot'ein kinase and lipolysis, the adipocytes were resuspended ( 5 0 mq cells/ml) in m buffer containing 4% BSA. 200 nu adenosine. and 5 mn qlucose and preinoubated for 15 mi".
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Cells were preincubated for 5 Preuaration of Membrues mi", washed tvice with 0.25 M sucrose in 10 M TES buffer (pH 7.0). and S N d e microsomal fractions (P-2) yare prepared accordinq to the method of K O ~ O (42). The microsomal pellets Yere reSU5pEndEd in 10 mB TES (pH 7.5) containlnq 0.25 M sucrose and purlfied plasma membranes Were obtained by ammous two-nhase Dartition as described oreviouslv 1 3 9 1 . In brief. 500 "1
6
response variable (i.e. cyclase activation) concentration of isoproterenol or forskolin, asymptotic value of y at infinite x , asymptotic value Of y at x-0, concentration of hormone producing 1/2( Y-6)+6. Standardized elope parameter where - B / W is the rate of chanqe In the response at X-#.
Responses such as the inhibition of sdenylyl cyclase. protein kinase. and qlycer'ol release in response to increasinq concentrations of B-PIA, or the inhlbition of adenylyl cyclase by Gpp(NH)p were characterized by the fallinq loqlstic ogive qiven as
y The ippir phasi coneainlnq blnost exclusi&ly plasma membranes was removed and conblned wlth 5 volumes of cold 10 mn TES 1oH . 7.51. buffer containing 0.25 M SUE~OSB, and centrifuqed at 48,000 x q for 10 man. The supernatant was aspirated and the pellet was resuspended ln buffer containinq 25 w Hepes (pH 7.4). 150 w NaC1, 1 lall EDTA, 40 pM leupeptin, and I Irgl.1 soybean trypsin inhibitor, and stored at -80' c. q.
----
x 6 Y p
---
--
response variable (i.e. ~ y c l a s einhibition) concentration of B-PIA or Gpp(NH1p. asymptotic value of y at infinite x, asymptotic value of y at x-0, concentration of hormone producing 1/2( I - K ) + 6 . Standardized slope parameter where -#/4!A is the rate Of chanqe 1" the response at X-".
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Purified O-f P and ADP--On adipocyte plasma membranes (50-100 pq membrane protein) were solubilized on ice for I h in 20 mn Tris. 1 rN EDTA, 1 mu DIT, 100 mn NaCl and 0.91 Na The su9DenSion vas then centrifuqed at 11,000 X 9 for 10 cholate (DH 8 . 0 1 . min and protein was assayed in the supernatant. Five pq of selubilized m o t e i n from each treatment vas Lncubated vith 1.5 wq of activated pertussis
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Antisera raised against the C-terminal for western EL%s decapeptides of cia-1, Gie-3, and Gs(1 (7) yere obtained from D r . Christopher J. Lynch (Dept. of Molecular Physioloqy, Hershey Medical Center, Herehey, PA) and antisera SpecIfic for G0-z yere obtained from Dr. Janet D. Robishav (Wais Ctr. Res., Geiainqer Clinic, Danville. PA). Antisera aqeinst Gie-1 also resogqnlred cia-2 smce both share the same E-terminal decapeptide. This antisera was desiqnated anti-Glo(1-2) and w e d at a final dilution of 1:200. The anti-Gio-I also was used at a final dilution Of 1:200. while antisera aqainst Gso and Goa were used at 1:lOO and 1:500, respectively.
- Solubilized
adipocyte plasma
membranes yere resolved on SDS-polyacrylamide qel electrophoresis (12.5% acrylamide, 0.051% DATD), and then electrophorectically transferred tO nitrocellulose. The gels were loaded with 20 pq solubilized membrane protein per l a n e for incubation with antisera aqalnst Gia(1-2) and Cia-I, and 30 eq per lane for incubation with GO= and Gsa antisera. Follovinq the procedure of Mumby ( 4 4 ) . nitrocellulose membranes were blocked with 5 % dried skim milk in buffer (pH8 . 0 ) containing 5 0 DM Tris. 2 mU CaCI2, 80 lall NZICI, 0.028 Na azide. and 0.21 nonidet P-40 for 1.5 h at r w m temperature with gentle shskinq, followed by two washes with the same low-deterqent blotto buffer. The primary antisera were diluted as specified in low-deterqent blotto and incubated with the mmbranes for 3 h at room temperature with gentle shakinq. After extensive vashinq in low-deterwnt blotto, "'I-labeled qoat antl-rabbit
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n and NO The epididymal fat pads from 2 lean were %!%er m 1 of Cold quanidinium thiocyanate (GIT) buffer and homoqenized for 20 8 with a polytron, folloved by addition of 2 m l of GIT bufferand immediate freerinq with llquid nitrogen. Total RNA vas isolated as previously described ( 3 5 ) . after whlih purity and concentration were estimated by UV spectrophotometry. Samples from each phenotype containinq 12 119 total RNA were denatured at 90. C for 10 min and resolved on 11 aqar-ose gels at 8 5 V for 2 h. RNA was transferred to nylon membranes aind baked for 2 h at 80' C in a vacuum oven. followed by incubation with buffer containinq 0.1$ SDS before hybridization with labeled Drobes. The membranes were probed with OliqodeOY~nUCleotide5 complementary~to s p e c l f ~ cmRNAS of the alpha Subunits of Gi-I. Gi-2, Gi-I, and G 5 118 described by Ramkumar ( 3 5 ) . The amount Of RNA loaded per lane Was normalized to e-tubulin mRNA. as detected bv an 0-tubulin =DNA. The relative chanqes in G-protein trakcripts were determined by densitometric scanning of the autoradiographs. (or I%%%%
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Plasma nmkrmea Preliminary studies were undertaken to assure that the same amount of membrane protein Was beinq applied to qela for each phenotype. These Studies employed purified Plasma membranes and the crude microsomal membrane preparatlons (P-2) from YhlCh plasma membranes were prepared. Similar amounts of membrane protein from each type Of membrane preparation yere subjected to solubilization under the conditions described in the Materials and Methods. The extracts from each type of membrane preparatlon and phenotype were cantrlfuqed and protein was assayed in the supernatant and equal amounts of protein were loaded on SDS-poly(lCrylamideq e l s . Coomassie staining of the resolved proteins revealed the presence of several more prorament bands in lanes loaded with extracts of Crude microsomal membranes from o b p b adipocytes compared to lean adipocytes. However, lanes containing extracts of purified plasma membranes from each phenotype were essentlally Identical in their Staining Patterns. This difference may be due to the enrichment of mitochondrial markers noted in C N d e membrane preparations Of adipocytes from some rodent models Of Obesity (40). Purlfled plasma membranes Were used henceforth Unless Othemvlse indicated.
toxin. The membra"; proteins were resolved by SDS-polyacrylamide qel electrophoresis and vlsuallration of the labeled protelns by autoradiography revealed bands lpiqratinq at 40 and 4 1 kd (Fig. 1 ) . comparlson of the intensity of the bands by densitometry indlcated that the band mlqratinq at 41 kd was present in membranes from obese mice at 1 9 ? 4t the levels Seen in membranes from lean mice (Fiq. 1). In Contrast, the lower band ( 4 0 kdl was much le58 prominent and occurred at levels vhrsh appeared not to dlffer between the phenotypes ("-61.
97.400-
66.200-
-
Kinase and G v Duplicate 1600 el Cell suspensions (50 m=/ml) were prelncubated for 15 ain, and hormones or effectors were added in a Cocktail containinq adenosine deaminase (ADA) toproduce LI final concentration o f 0.5 U l m l . The sells were incubated for 10 min and 800 c 1 vas removed from each tube into tubes containinq 200 P I of homogenizing buffer containing 50 mU TriS/EDTA and 2.5 rW RO-20-1724. These cella Yere dounced and centrifuqed at 42,000 x q for 20 mi", after which CAMP-dependent protein klnans activity ratios were determined as described previously (45). In brief, 25 p1 of crude extract vas added to 50 p l of kinase test m 1 x containinq 25 aW MOPS (pH 7.01. 16 mI4 Mqso,, 125 pH ATP, 4.2 OII MT, 100 pll kenptide, and 800.000 dprn of Y-["Pl-ATP (3000 ci/nnole). and incubated for 10 min at 30' C. Flfty p 1 of reaction mixture was spotted onto Uhstman P-81 phosphocellulose papers and the papers were washed aind counted as previously dessrlbed ( 4 6 ) . The 850 p1 aliquot of cell suspension renaininq in the oriqinal tubes after removing the aliquot for assay of protein kinase continued incubating at 37' c in the shakinq water bath for a total of 10 mi", after which extracts were prepared and qlyCerOl assayed as previously described (47). Glycerol release from the cellsvas expressed as "mol glycerol/h/q tissue.
-
lase assay Adenylyl cyclase activity was determined in purified adipocyte plasma membranes from the two phenotypes accordinq to In brief, 5 pq of purified plasma methods described previously ( 4 8 ) . mCmbranES were incubated for 10 mi" at 10' c in a buffer containing 5 0 W4 TES 150 OM NaCl. 0.5 U / m l of ADA. 2 mH creatine (pH 7.6). 4.0 mU MqCl phosphokinase, 50 pH ATP and 10 p M GTP. The phosphate, 25 U/al reaction was conducted I" a final volume of 100 p l and initiated bv addino
creatine
50
42,700-
41.000. 4OxK4'
1
2
3
4
5
G-proteins in Adipoq Solubilized adipocyte plasma membranes resolved by SDS-polyacrylamide gel elsctrophoreais were transferred to nitrofelluloss membranes alectrophoretlcally for further Characterization using antibodies Specific for the alpha subunits of various C-proteins. This approach was a160 taken to test whether the differencesin pertuesis toxin substrates between the phenotypes noted in Fig. 1 could be verified by independent methods. Reference to Fig. 2 illustrates that antisera raised against the C-terminal desapeptide shared by Gie-1 and Gie-2 reccqnired primarily a band migrating at 4 1 kd and a fainter band at 40 kd. The band at 40 kd was not completely resolved from the upper band, but appeared to be present at sinllar levels in both phenotypes ("-61. In contrast the upper band was present in membranes from obese mice at 59 t 4% the 1;vele seen in membrane(l from lean mice (Fig. 2 ) . The antisera raised against the C-terminal decapeptide of Gio-3 recognized one band migrating at 4 1 kd, and this band was present in membranes from obese mice at 4 1 t 9% the levels seen in membranes from lean mice (Fiq. 2). The possibility that the lower band mlqrating at 40 kd vas GO was evaluated with antisera specific for the alpha subunit of GO. Using 30 pg of solubilized membrane protein, the GO specific antlbody detected no protein at or near 40 kd (Fig. 3 ) . Using the same methods, GO was readily identified in solubilized brain membranes (data not Shownl. Using antisera specific for the alpha subunits Of 68 two bands mlqrating at 42 and 48 kd were detected in adipocyte membrane; from both PhenOtypeS (Fig. 3 1 . Reference to Fig. 3 also indicates that membranes from obese mice contained substantially lesa (42% of Control level-) of both bands than membranes from lean mise ("-2).
-
The W A S e n c d i n g LLasaensnr RNA Levels Of G-vrotG-protein alpha subunits Were compared by Northern blot analysis Of total RNA from epididymal fat pads of lean and obese mice since expression of several Of the encoded protains were found to differ between the phenotypes. The G l m - 1 probe hybridized to a 3.4 kb mRNA. whlle the probe for Gim-3 hybridized to a message at 3.6 kb. The probe for Gio-2 hybridlred weakly to a 2.3 kb mRNA, while the CS probe hybridized to a 1.8 kb message that encodell the two forms (42 and 4 8 kd) of Gs alpha Subunit. The level Of RNA added per lane was normalized to a-tubulin mRNA. determined by reprobing the same blot with a-tubulin =DNA. Expressed (1s a percentage of the amount found in lean mice, transcripts for GSU (86 t 4%) and cia-1 (46 f 3%) Were lower in adipose tlSBUe from obese mice. and mRNA for Gim-3 did not dlffer between the phenotypes (Fig. 4). In contrast, adipose tissue from Obese mice contained a higher level of Cia-2 transcripts ( 1 9 4 t 12;) than adipose tissue from their lean littermates (Fig. 4 ) .
-
The functional significance of the E ~ G ~ Q of DGsa in decrease in G80 noted in adipocytes of obese mice was evaluated by incubating plasma membranes With various concentrations of isoproteranol and comparing
A
-
Fig. 4 Northern blot analysis Of total cellu1ar RNA isolated from adipose tissue of lean and Obese mice. Total cellular RNA was isolated from epididymal fat pads of lean and obese mice and subjected to Northern blot analysis as described in laterials and nethods using radiolabeled =DNA and oligonucleotide probes specific for mRNA encodinq a-tubulin (Ldne6 1 . 2 ) Gsm (lanes 3 , 4 l , cia-1 (lanes 5.6). cia-2 (lanes 7 . 8 ) . and cia-3 (lanes 9.101. Even numbered lanes Yere loaded with RNA from ob/ob mice and odd n u d r e d lanes were loaded with RNA from lean mice. The blots were scanned by densitometric methods and relative intensity was normalized relative to a-tubulin. The m R N A levels in Obese nice were expressed as a percentage of the corresponding mRNA occurring in l e a n mice and are summarized as follo~s: GSa, 86 t 4 % ; Cia-1, 4 6 t 3%: Gim-2, 1 9 4 t 12t: Cia-3, 104 t 10%. The blot 1s representative of 4 experiments.
the activation of adenylyl cyclase. This approach was used to evaluate the level of communication between 8-adrenergic receptors. Cso. and adenylyl Cvclase in thls tissue. Reference to Fia. 5 illUstrate6 that basal CYFlaSB artivity vas similar between the phenotypes, but maximal activatmn ~ 6 8 substantially higher In l e a n (223 t 3 pmol cAMP/mln/mq) than in obese mice (128 t 2 pmo1 cWP/min/mq). HOYBVB~,the concentration of isoproterenol nroducina half-maximal activation of adenvlvl cvclase vas similar between lean ( 5 . i t 015 pn) and Obese mice ( 5 . 3 t 1 . 0 m ) . The phenotyplc difference in response to 8-adrenergic stimulation in a broken cell preparation vas a l s o examined in a whole cell preparation by characterizing the ability of isoproterenol to activate cMP-dependent protein kinase and lipolysis in isolated adipocytes from each phenotype. Basal protein k m a s e activity was n e a r l y identical in the two phenotypes and the concentration Of isoproterenol producing half-mximal activaclon did not differ (Fig. 6). The differences in adenylyl cyclase activation in adlpOCyte membranes were mirrored by lower maximal protein kinase aictlvity ratios in adipocytes from ObeSe compared to lean mice (Lean, 0 . 8 1 f 0.04: Obese. 0.54 0.01; see Pig. 6). Basal glycerol release vas lower in adipocytes fro. obese mice (13.2 t 2.4 nmol/hr/gl compared to lean Dice (32.0 t 0.54 nmol/hr/g), and m(lx~ma1al~cerol release also was sUbstantiallY lower in cells from obese mise (Lean, 526 t 5 nmol/hr/q: Obese, 103 t 5 nmoljhr/q, see Fig. 71. In a d e m r t u r e from the results of F14. 6, the Concentration Of iSoDrotsrsno1
.~
~
~~
~
.~. I
.
I
--
l
l
4
1
1
1
4
Fiq. 2 ImmUnOblOts of purified plasma membranes from adipocytes of lean (Panel A Lanes 3 . 4 : Panel B Lanes 3 , 4 ) and obese mice (Panel A Lanes 1.2; Panel B Lanes 1.21. SDS-PAGE was performed as described in the naterials and Methods and transfers tonitrOCellulOse membranes were affected by 150 M for 3 h. Purified plasma membranes from adipocytes of lean and obese mice were solubilized as described in naterials and nethods and 2 0 pg Of membrane protein was loaded into each lane. The blots were treated With ['"I)-labeled goat anti-rabbit IgG as described by Humby ( 4 4 1 , following a 3 h incubation with specific antisera at the following dilutions: anti-Giell-21, 1:200: anti-Gin-3, 1:200. The lanes in Panel A were incubated with snti-Gi@(l-2) and the lanes of Panel B were incubated with anti-Gie-3. This autoradicqram is representative Of 6 experiments and each blot Was scanned by densitometric methods. Expressed as a percentage of the levels found in membranes from lean mice, Gio(1-2) and Cia-3 were present in Obese membranes at 59 t 4% and 4 1 t 9% of control levels.
-
-
-
.
the Obese mouse. Additional studies were conducted to rule out the WssibilitY that the poor adenylyl cyclase and protein kinase activation we& the resuit of deficient expression of adenylyl cyclase in adipocytes of obese mice. It was also of interest to determine whether the Observed difference in maximal lioolvtic rates could be overcome bv bvmssina the adenvlvl cvclase com~lex and a;tivating protein kinase diresi1y';ith a.cWP anal&: Ii was resabned that incubation Of adipocyte plasma membranes with various Concentrations of forskolin would bypass the hormone receptor-G protein complex and activate sdanylyl c y d a s e directly. thus testing the hypothesis that differences existed in the amount Of cyclase Catalytic subunit between the phenotypes. B a s a l adenylyl cyclase activity did not differ between the phenotypes (Fig. 8 ) : and forskolin produced m x m a l rates of activity that were s m i l a r in adlpOCyte membranes from lean 655 t 40 pmO1 cWP/minlmql and obese mice ( 6 1 5 t 36 PmOl sAnP/min/mq). In Diddltion, the concentration Of forakolin produclnq half-maximal cyc1ase activation was lndlstinguishable between the phenotypes (Lean. 2.63 t 1.14 #I+: Obese, 3.10 t 1 . 3 0 p U ) . These flndinga suggest that adlpocyte memhbranes contain essentially the same amount of adenylyl cyclase Catalytic subunit, and differences in the actlvatmn of
240 r
210
-
180
-
IS0
-
I20
-
90
-
18 Y
u td
60 ;
Fig. 5
I
-
2
3
4
Fig. 3 ImmYnOblots Of purified plasma membranes from adipocytes Of lean [lanes 1 . 3 ) and Obese mice (lanes 2.4). SDS-PAGE was performed as described in the nateriala and nethods and transfers tonitrocellulose membranes were for 3 h. Purified plasma membranes from adipocytes of affected by 150 lean and obese mice were solubilized as described in naterials and nethods and 30 pq of membrane protein was loaded into each lane. The blots Were treated with ["I)-labeled goat anti-rabbit IgG as described by Humby ( 4 4 ) . folloWinq a 3 h incubation with Specific antisera at the following dilution=: anti-Gs. 1:lOO: anti-Go. 1:500. Lanes 1-2 were Incubated with anti-Go and lanes 3-4 were incubated with anti-as. This autoradiogram is representative O f 2 experiments. The bands at 42 kd and 4 8 kd were present in membranes from Obese mice at 4 2 t of the levels seen in lean mice.
- The relation between isoproterenol concantration and adenylyl
cyclase activity in adipocyte plasma membranes Of lean ( a ) and obese 1 A I mice. The original observations are the mean of duplicate incubations Within each of four experiments, and the fitted curves were obtalned by least squares. The incubatlons were performed using 5 pg of membrane protein at 30' c for 10 min, and F M P was assayed as described in the Methods.
natermlsand
G-proteins in Adipocytes of Obese (oblob) Mice
15954 0.75
F-
0.65
.
0.55
-
0.45
-
0.35
-
0.25
J
through another reiiptoi &tern knbwn to be coupled to-adenylyl cyclise by GS(1 (ACTH) should a1110 be deficient if the decline in GB0 is Significant. Reference t o Table 1 illustrates that ALCTH and isoproterenol produced substantially higher adenylyl cyclase activity in adipooyte membranes from lean mice atall ConcentratiOnS of GTP tested. Direct activation of Cyclase by GTP in the absence of a stimulatory ligand did not differ between phenotypaa at lower GTP concentrations, but approached significance (P < 0.08) at 10 un GTP (see Table 11. Incubation Of idipocytas fkom lean and Obese mice with various concentrations of the CAMP analog. 8-bmmo-CAMP, activated lipolysis in aimilar fashion in both cell types (Pig. 9 ) . Easa1 lipolytic rates were lower in cells from Obese mice, but increaead at a11 exaept the highest concentration of 8-bromo-CAMP to rates not different from those seen in sells from lean mice (Fig. 9). These findinge a r v e that the defect in activation of lipOlySis in adipocytes from Obese lice by ieoproterenol (see Pig. 7) .1 UPBtream from Drotein kinase activation. and ma" well be due to a defect in
0.05
ooof
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'
'
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'
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I
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10
'
100
'
"'"* 1000
lroprolcrenol Concentralion (nM)
-
Fig. 6 The effects Of isoprotarenol on CAMP-dependent protein kinase activity in adipocytes ieolated from lean ( 0 ) and Obese (0) mice. After a 5 min preincubation, adipOCyteB were incubated with various concentrations of iaoproteranol and aliquota were removed at the 10sin tine point for assay of CAMP-dependent protein kinase. The remaining aliquot of cella was incubated for another 20 min and glycerol release was assayed in the extracts (Pig. 7). The protein kinase assay was perforred a8 described in the Materials and Methods and the fitted curves were obtained by least squares. Each point is the meanof duplicate incubations in each Of four experiments.
r
240
180
-
I50
-
210
G-proteins noted in idipacytes from Obese mice.. The first approach involved comparing the inhibitory effects Of a ligand (E-PIA) known to function through pertussis toxin-sensitive G-proteins. Adipooyte plasma membrana. from each phenotype were stimulated with 10 PMisoproterenol in the premence of various concentrations of B-PIA. and reference to Pig. 10 illustrates that cyclase was activated to differing extents by isoproterenol [Lean, 1 0 0 f 1 Maximal inhibition or pmol cAMP/min/mg: Obese. 130 f 2 pmol cAnP/min/mg). adenylyl cyclase was reflective of the starting levels Of enzyme activity and also differed between the phenotypes (Lean, 114 f 2 pmol cAnP/min/mg; Obeme, 65 f 4 Dm01 EAnP/min/mal. It should be noted that, on a Dercentaae basim. the inhibition of cyclibe in membranes from Obese ;ice was slightiy high& than in lean membranes. However, the Concentration of E-PIA producing half-maximal effBCts did not differ ( e < 0.051 between the phenotypes (Lean, 4.3 N4; see Pig. 10). Experiments of identical 6.2 f 0.7 N4; Obese, 15.3 design were conducted in isolated adipocytas to determine the effectof E-PIA on CAMP-dependent protein kinase activity and glycerol release. A fixed Concentration of isoproterenol (loo n n ) in the absence of E-PIA produced higher protein kinase activity ratios in cella from lean (0.78 f 0.01) compared to Obese mice (0.50 f 0.01, see Pig. 11). The highest EDnCentrations of E-PIA reduced activity ratios in cells from Obes. mice to lower levels (Lean. 0.45 t 0 . 0 4 : Obese, 0.31 f 0.01). but the magnitude or
*
90 60 30 -
120
0' 0.01
GTP Concentration
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I
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10
100
Fig. 7 The effects ofisoproterenol on lipolysis in adipocytes isolated from lean (01 and Obese ( 0 ) mice. After a 5 min preincubation, adipocytes were incubated with various concentrations of isoproterenol and aliquots were removed at the10min time point for assay of CAMP-dependent protein kinase (Fig. 6). The remaining aliquot of cells was incubated for another 20 min and glycerol release was assayed in the extracts. The glycerol assay We.* perforred as described in the Materials and Methods and the fitted Curves were obtained by least squares. Each point is the mean of dupliaate incubations in each Of four experimentS.
700
r
600
-
500
-
400
-
300
-
200
-
100 -
100 nN
1 PM
10 PM
std err09
1000
Isoprolcrcnol Conrcnlralian (nM)
-
Treatment'
' " ' d
Lean
Control
31.4
39.3
45.3
3.04
Ob/Ob
Control
28.1
36.9
37.8
3.04
laan
Isoproterenol
70.5
152.1
174.4
3.04
Ob/Ob
Isoproterenol
30.5
47.2
58.0
3.04
Lean
ACTH
40.7
59.7
78.7
3.04
Ob/Ob
ACTH
31.9
47.2
52.9
3.04
'mrified adipocyte plasma membranes from each phenotype Yere incubated with buffer, 100 PM isoproterenol, or 10 PM ACTH in the presence Of increasing Adenylyl cyclase activity Was concentrations of GTP at 30' C for 20 mi". determined as described in the Materials and Methods except the testmix did not contain NaCl. Enzyme activity was expressed as pmol CAMP formed per min per mg protein. The mean cyclase activity in membranes treated with 100 PM forskolin was 496.3 pnol/nin/ng protein for lean membranes and 482.0 pnol/min/mg protein for ob/ob membranes. P n e means are from duplicate determinations in each of four experiments and the standard errDCe were obtained from the analyeis of variance
G-proteins in Adipocytesof Obese (oblob) Mice -
225
210
200
180
175
IS0
-
120
-
I50
I25
90 -
IO0 60
-
15
50 l0.f
. . ...
d
-l
0.01
0.I
100 I
10
1000
?-PIAConcentration (nM)
-
Fig. 10 The relation between 8-PIA concentration and adenylyl cyclase ( .I mice activity in adipocyte plasma membranes of lean ( r ) and ob" treated with 10 p l isoproterenol. The original observations are the mean of duplicate incubations within each of threeexperiments, and the fitted curves were obtained by least 8quare8. The incubations were performed using 5 Mg of membrane protein at 30' C for 10 min, a d UllP vas asbayed as described in the Materials and Methods.
0.80 0.70
-
0.60
-
0.50
-
0.40
-
-
P
550 500 -
d
450
g E
600
400
350
0.30 -
