Comp. Biochem. Physiol. Vol. 101B,No. 1/2, pp. 147-151, 1992
0305-0491/92 $5.00+ 0.00 Pergamon Presspie
Printed in Great Britain
IDENTIFICATION OF CHICKEN (GALLUS DOMESTICUS) ADIPOCYTE PLASMA MEMBRANE A N D DIFFERENTIATION SPECIFIC PROTEINS USING SDS-PAGE A N D WESTERN BLOTTING S. C. BUTTERWITH,* S. KESTIN,t H. D. GRIFHN,* J. BEATTm and D. J. FLINT:~ *Agricultural and Food Research Council, Institute of Animal Physiology and Genetics Research, Edinburgh Research Station, Department of Cellular and Molecular Biology, Roslin, Midlothian EH25 9PS, UK (Tel: 031 440 2726; Fax: 031 440 0434) tUniversity of Bristol, Department of Meat Animal Science, Langford, Bristol BS18 7DY, UK; and :~Hannah Research Institute, Ayr, KA6 5HL, UK (Received 17 June 1991)
Abstract--1. Affinity-purified adipocyte membrane proteins were used to raise antisera in two sheep. 2. Using one of the antisera 15 proteins were identified as being adipocyte specific by comparison on Western blots of plasma membrane proteins from various tissues. 3. Of these 15 proteins eight appeared to be present only in mature adipocytes and not in the adipocyte precursor. 4. In the presence of guinea pig complement the two antisera raised were cytotoxic to adipocytes and their precursors. 5. Characterization and further study of these adipocyte differentiation specific proteins will provide valuable information about the process of adipocyte differentiation.
INTRODUCTION
The study of adipose tissue growth has been greatly aided by the isolation and development of culture techniques for adipocyte precursor cells from a number of species and the development of stable cell lines. Cell culture systems have proved invaluable for studies designed to identify possible regulators of adipocyte proliferation and differentiation. A number of enzyme activities increase during adipocyte differentiation (see Cryer, 1985) and some of these have been used to quantitate the degree of differentiation in vitro. The most commonly used are lipoprotein lipase and glycerol-3-phosphate dehydrogenase. A morphological measure of differentiation involves the direct measurement of lipid accretion by staining with Oil Red O. Very little is known about the cell surface changes which take place during the differentiation of adipocyte precursors into the mature cell type. Thompson and Abraham (1979) reported the presence of antigens on the surface of adipocytes which could be used to distinguish mammary adipose cells from mammary epithelial cells and fibroblasts using an antiserum raised against whole adipocytes. Plaas et al. (1981) and Cryer et al. (1984) have used antisera raised against adipocyte plasma membranes to follow the differentiation of 3T3-L1 cells and of bovine adipocyte precursors in vitro. However the polypeptide composition of the adipocyte plasma membrane appears to differ between species (Tume et al., 1985). The identification of specific adipocyte membrane proteins has recently taken on new importance with attempts being made to manipulate adiposity in a
number of species by the use of anti-fat cell antibodies. Although this approach has worked well in the rat (Flint et al., 1986), it has been less successful in the chicken. Injection of crude chicken fat cell membranes into sheep produced an antibody response to the adipocyte but the antiserum produced had considerable reactivity towards other cell types including liver and red blood cells (Butterwith et al., 1989). This has necessitated the identification of adipoctye specific antigens in the chicken. In this paper we describe the use of a specific anti-adipocyte plasma membrane antiserum to identify adipocyte plasma membrane specific proteins. Some of these also appear to be differentiation specific. MATERIALS AND METHODS
Preparation of chicken adipocyte specific antigens and antisera
The following strategy was used to prepare antisera that were specific to chicken adipocytes. Firstly, an antiserum was raised in sheep to purified chicken adipocyte plasma membranes, as described by Butterwith et al. (1989). IgG from these antisera were passed in succession through three affinity columns containing bound liver, red blood cell and kidney plasma membrane proteins. Antibodies not bound to these columns were used to prepare an affinity column for purification of adipocyte specific antigens. Preparation and use of affinity columns
Membranes were prepared from tissue or cell homogenates by centrifugation on sucrose gradients as described before (Butterwith et al., 1989). Membranes were resuspended in 0.1 M sodium bicarbonate buffer, pH 8.0, and sonicated. Triton X-100 was added to a final concentration of 2% (w/v) and the suspension sonicated again. After 147
148
S.C. BUTTERWITnet al.
stirring for I hr, the membrane solution was centrifuged at 75,000 g for I hr at 4°C and the sediment discarded. The membrane solution was then incubated overnight with cyanogen bromide activated Sepharose 4B in the proportions two parts membrane solution to one part gel. Any remaining active groups were blocked by treatment with 1 M ethanolamine, pH 9.0, containing 0.1% Triton X-100 for 1 hr. Affinity Sepharose was then washed with borate buffer containing 0.1 M disodium tetraborate, pH 8.0, 0.5 M NaCI and 0.1% Triton X-100, and glycine buffer containing 0.1 M glycine, pH 2.5, and 0.1% Triton X-100. Using this method, application of 5 mg protein per ml of Sepharose gave a binding efficiency of 50-70%. Antibody was coupled to Sepharose 4B in the same way except that detergent was absent. Preparation o f chicken adipocyte specific antigens
Antiserum against crude chicken adipocyte plasma membranes was produced as described by Butterwith et al. (1989). A 7-globulin preparation from this antiserum containing 0.1% Triton X-100 was in turn passed down liver, red blood cell and kidney plasma membrane affinity columns ( l m g globulin protein per ml of column). Antibodies were left on the columns for 4-16 hr at 4°C. Unbound materials were washed from the column with 10 column volumes of borate buffer containing 0.1% Triton X-100. This affinity purified antibody preparation was linked to Sepharose 4B and used as an at~nity column to prepare adipocyte specific proteins. Immunization o f animals
Sheep were immunized with adipocyte specific antigens by the method of Butterwith et al. (1989). Two sheep were immunized resulting in antisera identified in the text by the numbers 460 and 484. Preparation o f membrane proteins for S D S - P A G E
Plasma membranes were prepared from tissue homogenates of brain, muscle, kidney, liver and adipose tissue on a sucrose gradient as described by Butterwith et al. (1989). Red blood cell membranes were prepared by centrifuging homogenates of red blood cells at 20,000 g and collecting the pellet. For the preparation of adipocyte precursor membranes, cells were prepared and grown in 25 cm 2 flasks by the method of Butterwith et al. (1989). After 5 days in culture (when the cells had not yet reached confluence and had not therefore started to differentiate) the cells from six flasks were scraped into 1 ml of phosphate buffered saline (PBS), pH 7.4 containing 0.2 mM phenylmethylsulphonylfluoride. The cells were homogenized using a Polytron homogeniser (Kinematica, Lucerne, Switzerland) before centrifuging at 100,000g at 4°C. The membrane pellet was washed three times with PBS. Membrane proteins were extracted with 2% (w/v) sodium deoxycholate and dialysed overnight against 10mM Tris-HC1, pH 7.4, containing 0.02% sodium deoxycholate. Protein concentration was determined by the method of Bradford (1976). S D S - P A G E and Western blotting SDS-PAGE was performed by the method of Laemmli (1970). Gels were blotted onto 0.2/~m nitrocellulose sheets using a semi-dry blotting system (LKB-Produkter, Bromma, Sweden). A continuous buffer system was used for the transfer which contained 39mM glycine, 48 mM Tris, 0.0375% sodium dodecyl sulphate and 20% methanol. Transfer time was I hr at 200 mA. Unreacted binding sites on the nitrocellulose were blocked by incubation overnight in washing buffer (100 mM Tris-HC1, pH 7.4, containing 0.9% NaC1, 0.05% Tween-20) containing 4% (v/v) horse serum. Blots were then washed three times with washing buffer and incubated for 1 hr in a 200-fold dilution of antiserum 460 in the same buffer. Bound antibodies were
detected using a biotinylated anti-sheep second antibody and an avidin-alkaline phosphatase conjugate and stained with an alkaline phosphatase detection kit (Vector Laboratories, Peterborough, UK). Blots were scanned using a Shimadzu CS 9000 dual-wavelength flying spot scanner and mol. wts calculated by comparison of unknown bands to biotinylated standard proteins (Sigma Chemical Company, Poole, Dorset, UK) run on the same blot. Cytotoxicity against adipocytes and their precursors Adipocyte precursors were grown and differentiated in culture by the method of Butterwith et aL (1989). For this study adipocyte precursors were plated at 32,000 per well in 24-well plates, and grown for 5 days for studies on precursors and for 12 days post-confluency for studies on mature adipocytes. Cells were washed once with 200 #1 of phosphate buffered saline per well, and incubated in 200 #1 of medium 199 containing 10 mM HEPES, pH 7.4, 2 mM glutamine, 1 mM pyruvate, 50gg/ml gentamycin, 8.9% (v/v) sheep complement and 28% (v/v) of the serum to be tested which had been beat-inactivated at 56°C for 1 hr to remove endogenous complement activity. Incubations were for 2 hr at 37°C in an atmosphere of 95% 02/5% CO2. Cytotoxicity was determined by measurement of release of lactate dehydrogenase which was assayed by the method of Saggerson and Greenbaum (1969) using a microplate reader. Total cellular lactate dehydrogenase was determined by disruption of a sample of cells with 0.2% (v/v) Triton-X100.
RESULTS AND DISCUSSION Although many studies relating to adipocyte differentiation have been performed, little is known about the cell surface changes involved in this process. Most studies have preferred to focus on different responses to specific hormones during differentiation or changes in specific intracellular events such as changes in specific enzyme activities. We have therefore attempted to identify potential cell surface markers of the differentiation process using antibodies, as well as to identify which (if any) of these markers might be tissue specific. Separation by S D S - P A G E of plasma membrane proteins from adipose tissue, brain, muscle, liver and kidney and membrane proteins from red blood cells followed by Western blotting using our antiadipocyte plasma membrane antiserum produced the patterns shown in Fig. 1. A biotin-avidin-alkaline phosphatase system was used to detect bound antibodies in order to produce a highly sensitive response. As expected, very few proteins were detected in the liver, kidney and red blood Cell samples. However, a number of proteins appeared to cross-react in the brain and muscle samples. Comparison of the proteins detected in adipose tissue membranes with those from other tissues resulted in 15 proteins being identified as potential adipocyte specific antigens on the basis of their moi. wts. These were 110, 102.3, 96.3, 58.6, 38.4, 37, 31.3, 30.4, 28.4, 27.1, 24.3, 21.3, 17.2, 16.1 and 15.9kD. Two of these proteins of mol. wt 58.6 and 38.4 kD are similar to those of 56 and 37 kD reported by Lee et al. (1986) as being both adipocyte and species specific in the chicken. The same authors also reported that a 47 k D protein was adipocyte and species specific. We failed to detect a protein of this mol. wt as adipocyte specific. However, the criterion
149
Adipocyte membrane proteins
(a)
(b)
(d)
(c)
(e)
(f)
kD 1
110 + 1.8
2 3
102.3 + 3.6 96.3 ± 2.8
4
58.6 + 0.9
5 6
38.4 ± 2.1 37 ± 2.2
7 8
31.3 ± 2.1 30.4 ± 2.2 28.4 ± 1.3
9 10
|
27.1 ± 0.8
11
24.3 ± 1.3
12
21.3 ± 1.1
I
I
immmb ,
~i
,
.
13
17.2 ± 0.9
14
16.1
15
15.9 ± 1.0
± 0.9
Fig. 1. Western blot against proteins prepared from membranes of (a) liver, (b) red blood cell, (c) brain, (d) muscle, (e) kidney and (0 adipose tissue using an antiserum raised using a preparation of adipocyte specific antigens prepared as in Materials and Methods. The amount of protein loaded per lane was (a) 6.3, (b) 31.8, (c) 38.4, (d) 24, (e) 4.4 and (f) 9.3/tg. The location of 15 adipocyte specific proteins are indicated together with their respective mol. wts (mean __+SD of seven blots). Similar results were obtained with another antiserum raised against the same adipocyte antigen preparation. used for tissue specificity by Lee et al. (1986) was simply that the antiserum used in the experiments had been absorbed with red blood cells. This was because these authors were primarily concerned with identify-
ing species specific adipocyte membrane proteins rather than tissue specific components. They also relied on immunoprecipitation to identify antigens. Western blotting has the advantage that it does not
S. C. BUTTERWITHet al.
150
(a)
110 + 1.8
(b)
--~
102.3 _+ 3.6 96.3 + 2.8
93.8 _ 5.8
65.9 + 2.5
31.1 _+ 0.52 29.2 _+ 0.41 28.4 + 1.3 27.1 + 0.8
24.3 + 1.3
18.5 _+ 1.4 17.7 +_ 1.4 17.2 + 0.9
15.9 _ 1.0
Fig. 2. Western blot comparing membrane proteins from (a) mature adipose tissue with those prepared from (b) adipocyte precursors. The antiserum used was the same as in Fig. 1. The amount of protein loaded per lane was (a) 12.5 and (b) 9.3/~g. The mol. wts of eight putative adipocyte specific differentiation markers are indicated (mean + SD of three blots). rely on a precipitating antibody or the use of a second antibody to precipitate the antigen-antibody complex. In order to study the processes involved in adipocyte precursor differentiation we have attempted to identify any plasma membrane antigens which appeared to be present on the mature
adipocyte and not on the precursor. Previous antisera raised against adipocyte plasma membranes have always shown a significant a m o u n t of reactivity against the undifferentiated precursor cell type (Cryer et al., 1984; Lee et al., 1986). An exception to this was undifferentiated mouse 3T3-L1 cells which showed no reactivity to antiserum raised against mouse
Adipocyte membrane proteins
151
Table 1. Cytotoxicitytowards adipoeytesand their precursors of antisera 460 and 484 raised against adipocyte specificantigens Lactate dehydrogenase release (%) Precursors Adipoeytes Non-immune serum 19 +_.1.4(N = 3) 25 ___7.5(N = 5) Antiserum 460 64 + 8.8(N = 3) P < 0.005 68 + 11 (N = 4) P < 0.005 Antiserum 484 42 + 5.6(N = 3) P < 0.005 51 _ 5.7(N = 3) P < 0.005 Results are expressed as percentage release of lactate dehydrogenase relativeto total releaseof 0.2% Triton X-100 control (means+_SD). Significantdifferences from that of non-immune serum in an unpaired t-test are indicated. adipocytes (Plaas et al., 1981). This is perhaps partly explained by the fact that these cells are of embryonic origin, and therefore may resemble more closely a pluripotent stem cell. By comparing membrane proteins from adipocytes and their precursors using our specific antiserum, we have identified a number of proteins which appear only to be expressed after differentiation (Fig. 2). These proteins are identified by their mol. wts of 110, 102.3, 96.3, 28.4, 27.1, 24.3, 17.2 and 15.9kD. There are also some proteins of mol. wts 93.8, 65.9, 31.3, 29.2, 18.5 and 17.7kD which appear to decrease after differentiation. We also investigated whether the antisera we had raised were cytotoxic to adipocytes and their precursors in the presence of exogenously added complement. Only a low level o f cytotoxicity was seen (Table 1). This is a similar result to that found using a crude antiserum which had been adsorbed against liver cell membranes (Butterwith et al., 1989). The antibody response to specific antigens was much greater when they were used as immunogen so the lack of cytotoxicity in previous experiments with crude adsorbed antiserum was not due to a low concentration of specific antibodies. We are currently investigating the interaction of different complement sources with our specific antibodies in an attempt to increase the cytotoxic response.
SUMMARY
Affinity-purified adipocyte membrane proteins were used to raise antisera in sheep. These antisera were used on Western blots to compare membrane antigens from liver, kidney, muscle, red blood cell, brain and adipose tissue. Fifteen proteins were identified as being adipocyte specific on the basis of their mol. wt. O f these, eight appeared also to be differentiation-specific. Six proteins were also identified as appearing to decrease after differentiation but none of these were adipocyte specific. Characterization and further study of these adipocyte differentiation specific proteins will provide valuable information about the process of adipocyte differentiation. The antisera used to identify these proteins were cytotoxic to adipocytes and their precursors in the presence of complement.
Acknowledgements--This work was supported by a commission from the Ministry of Agriculture, Fisheries and Food. Financial support was also provided by the British Technology Group. REFERENCES
Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. Butterwith S. C., Kestin S., Griffin H. D. and Hint D. J. (1989) Cytotoxic antibodies to chicken adipocytes and their precursors: lack of tissue specificity. Br. Poult. Sci. 30, 371-378. Cryer A. (1985) New Perspectives in Adipose Tissue: Structure, Function and Development, pp. 383-405. Butterworths, London. Cryer A., Gray B. R. and Woodhead J. S. (1984) Studies on the characterisation of bovine adipocyte precursor cells and their differentiation in vitro, using an indirectlabelled-second-antibody cellular immunoassay. J. Devl. Physiol. 6, 159-176. Flint D. J., Coggrave H., Futter C. E., Gardner M. J. and Clarker T. J. (1986) Stimulatory and cytotoxic effects of an antiserum to adipoctye plasma membranes on adipose tissue metabolism in vitro and in vivo. Int. J. Obesity 10, 69-77. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680--685. Lee S. R., Tume R. K., Cryer J. and Cryer A. (1986) Studies on the expression of adipocyte-specific cell surface antigens during the differentiation of adipocyte precursor cells in vitro. J. Devl. Physiol. 8, 207-226. Plaas H. A. K., Woodhead J. S. and Cryer A. (1981) The use of antiserum with specific reactivity toward fat-cell surface antigen(s) to follow the progression of 3T3-L1 preadipoctye differentiation in vitro. Biosci. Rep. 1, 207-216. Saggerson E. D. and Greenbaum A. L. (1969) The effect of dietary and hormonal conditions on the activities of glycolytic enzymes in rat epididymal adipose tissue. Biochem. J. 115, 405-417. Thompson K. and Abraham S. (1979) Identification of mouse mammary adipose cells by membrane antigens. In Vitro 15, 441--445. Tume R. K., Lee S. R. and Cryer A. (1985) A comparison of the polypeptide composition of plasma membranes prepared from the white adipose tissue and adipocytes of the mouse, rat, rabbit and chicken by a percoll self-forming gradient procedure. Comp. Biochem. Physiol. 80B, 127-134.
0305-0491/92 $5.00+ 0.00 Pergamon Presspie
Printed in Great Britain
IDENTIFICATION OF CHICKEN (GALLUS DOMESTICUS) ADIPOCYTE PLASMA MEMBRANE A N D DIFFERENTIATION SPECIFIC PROTEINS USING SDS-PAGE A N D WESTERN BLOTTING S. C. BUTTERWITH,* S. KESTIN,t H. D. GRIFHN,* J. BEATTm and D. J. FLINT:~ *Agricultural and Food Research Council, Institute of Animal Physiology and Genetics Research, Edinburgh Research Station, Department of Cellular and Molecular Biology, Roslin, Midlothian EH25 9PS, UK (Tel: 031 440 2726; Fax: 031 440 0434) tUniversity of Bristol, Department of Meat Animal Science, Langford, Bristol BS18 7DY, UK; and :~Hannah Research Institute, Ayr, KA6 5HL, UK (Received 17 June 1991)
Abstract--1. Affinity-purified adipocyte membrane proteins were used to raise antisera in two sheep. 2. Using one of the antisera 15 proteins were identified as being adipocyte specific by comparison on Western blots of plasma membrane proteins from various tissues. 3. Of these 15 proteins eight appeared to be present only in mature adipocytes and not in the adipocyte precursor. 4. In the presence of guinea pig complement the two antisera raised were cytotoxic to adipocytes and their precursors. 5. Characterization and further study of these adipocyte differentiation specific proteins will provide valuable information about the process of adipocyte differentiation.
INTRODUCTION
The study of adipose tissue growth has been greatly aided by the isolation and development of culture techniques for adipocyte precursor cells from a number of species and the development of stable cell lines. Cell culture systems have proved invaluable for studies designed to identify possible regulators of adipocyte proliferation and differentiation. A number of enzyme activities increase during adipocyte differentiation (see Cryer, 1985) and some of these have been used to quantitate the degree of differentiation in vitro. The most commonly used are lipoprotein lipase and glycerol-3-phosphate dehydrogenase. A morphological measure of differentiation involves the direct measurement of lipid accretion by staining with Oil Red O. Very little is known about the cell surface changes which take place during the differentiation of adipocyte precursors into the mature cell type. Thompson and Abraham (1979) reported the presence of antigens on the surface of adipocytes which could be used to distinguish mammary adipose cells from mammary epithelial cells and fibroblasts using an antiserum raised against whole adipocytes. Plaas et al. (1981) and Cryer et al. (1984) have used antisera raised against adipocyte plasma membranes to follow the differentiation of 3T3-L1 cells and of bovine adipocyte precursors in vitro. However the polypeptide composition of the adipocyte plasma membrane appears to differ between species (Tume et al., 1985). The identification of specific adipocyte membrane proteins has recently taken on new importance with attempts being made to manipulate adiposity in a
number of species by the use of anti-fat cell antibodies. Although this approach has worked well in the rat (Flint et al., 1986), it has been less successful in the chicken. Injection of crude chicken fat cell membranes into sheep produced an antibody response to the adipocyte but the antiserum produced had considerable reactivity towards other cell types including liver and red blood cells (Butterwith et al., 1989). This has necessitated the identification of adipoctye specific antigens in the chicken. In this paper we describe the use of a specific anti-adipocyte plasma membrane antiserum to identify adipocyte plasma membrane specific proteins. Some of these also appear to be differentiation specific. MATERIALS AND METHODS
Preparation of chicken adipocyte specific antigens and antisera
The following strategy was used to prepare antisera that were specific to chicken adipocytes. Firstly, an antiserum was raised in sheep to purified chicken adipocyte plasma membranes, as described by Butterwith et al. (1989). IgG from these antisera were passed in succession through three affinity columns containing bound liver, red blood cell and kidney plasma membrane proteins. Antibodies not bound to these columns were used to prepare an affinity column for purification of adipocyte specific antigens. Preparation and use of affinity columns
Membranes were prepared from tissue or cell homogenates by centrifugation on sucrose gradients as described before (Butterwith et al., 1989). Membranes were resuspended in 0.1 M sodium bicarbonate buffer, pH 8.0, and sonicated. Triton X-100 was added to a final concentration of 2% (w/v) and the suspension sonicated again. After 147
148
S.C. BUTTERWITnet al.
stirring for I hr, the membrane solution was centrifuged at 75,000 g for I hr at 4°C and the sediment discarded. The membrane solution was then incubated overnight with cyanogen bromide activated Sepharose 4B in the proportions two parts membrane solution to one part gel. Any remaining active groups were blocked by treatment with 1 M ethanolamine, pH 9.0, containing 0.1% Triton X-100 for 1 hr. Affinity Sepharose was then washed with borate buffer containing 0.1 M disodium tetraborate, pH 8.0, 0.5 M NaCI and 0.1% Triton X-100, and glycine buffer containing 0.1 M glycine, pH 2.5, and 0.1% Triton X-100. Using this method, application of 5 mg protein per ml of Sepharose gave a binding efficiency of 50-70%. Antibody was coupled to Sepharose 4B in the same way except that detergent was absent. Preparation o f chicken adipocyte specific antigens
Antiserum against crude chicken adipocyte plasma membranes was produced as described by Butterwith et al. (1989). A 7-globulin preparation from this antiserum containing 0.1% Triton X-100 was in turn passed down liver, red blood cell and kidney plasma membrane affinity columns ( l m g globulin protein per ml of column). Antibodies were left on the columns for 4-16 hr at 4°C. Unbound materials were washed from the column with 10 column volumes of borate buffer containing 0.1% Triton X-100. This affinity purified antibody preparation was linked to Sepharose 4B and used as an at~nity column to prepare adipocyte specific proteins. Immunization o f animals
Sheep were immunized with adipocyte specific antigens by the method of Butterwith et al. (1989). Two sheep were immunized resulting in antisera identified in the text by the numbers 460 and 484. Preparation o f membrane proteins for S D S - P A G E
Plasma membranes were prepared from tissue homogenates of brain, muscle, kidney, liver and adipose tissue on a sucrose gradient as described by Butterwith et al. (1989). Red blood cell membranes were prepared by centrifuging homogenates of red blood cells at 20,000 g and collecting the pellet. For the preparation of adipocyte precursor membranes, cells were prepared and grown in 25 cm 2 flasks by the method of Butterwith et al. (1989). After 5 days in culture (when the cells had not yet reached confluence and had not therefore started to differentiate) the cells from six flasks were scraped into 1 ml of phosphate buffered saline (PBS), pH 7.4 containing 0.2 mM phenylmethylsulphonylfluoride. The cells were homogenized using a Polytron homogeniser (Kinematica, Lucerne, Switzerland) before centrifuging at 100,000g at 4°C. The membrane pellet was washed three times with PBS. Membrane proteins were extracted with 2% (w/v) sodium deoxycholate and dialysed overnight against 10mM Tris-HC1, pH 7.4, containing 0.02% sodium deoxycholate. Protein concentration was determined by the method of Bradford (1976). S D S - P A G E and Western blotting SDS-PAGE was performed by the method of Laemmli (1970). Gels were blotted onto 0.2/~m nitrocellulose sheets using a semi-dry blotting system (LKB-Produkter, Bromma, Sweden). A continuous buffer system was used for the transfer which contained 39mM glycine, 48 mM Tris, 0.0375% sodium dodecyl sulphate and 20% methanol. Transfer time was I hr at 200 mA. Unreacted binding sites on the nitrocellulose were blocked by incubation overnight in washing buffer (100 mM Tris-HC1, pH 7.4, containing 0.9% NaC1, 0.05% Tween-20) containing 4% (v/v) horse serum. Blots were then washed three times with washing buffer and incubated for 1 hr in a 200-fold dilution of antiserum 460 in the same buffer. Bound antibodies were
detected using a biotinylated anti-sheep second antibody and an avidin-alkaline phosphatase conjugate and stained with an alkaline phosphatase detection kit (Vector Laboratories, Peterborough, UK). Blots were scanned using a Shimadzu CS 9000 dual-wavelength flying spot scanner and mol. wts calculated by comparison of unknown bands to biotinylated standard proteins (Sigma Chemical Company, Poole, Dorset, UK) run on the same blot. Cytotoxicity against adipocytes and their precursors Adipocyte precursors were grown and differentiated in culture by the method of Butterwith et aL (1989). For this study adipocyte precursors were plated at 32,000 per well in 24-well plates, and grown for 5 days for studies on precursors and for 12 days post-confluency for studies on mature adipocytes. Cells were washed once with 200 #1 of phosphate buffered saline per well, and incubated in 200 #1 of medium 199 containing 10 mM HEPES, pH 7.4, 2 mM glutamine, 1 mM pyruvate, 50gg/ml gentamycin, 8.9% (v/v) sheep complement and 28% (v/v) of the serum to be tested which had been beat-inactivated at 56°C for 1 hr to remove endogenous complement activity. Incubations were for 2 hr at 37°C in an atmosphere of 95% 02/5% CO2. Cytotoxicity was determined by measurement of release of lactate dehydrogenase which was assayed by the method of Saggerson and Greenbaum (1969) using a microplate reader. Total cellular lactate dehydrogenase was determined by disruption of a sample of cells with 0.2% (v/v) Triton-X100.
RESULTS AND DISCUSSION Although many studies relating to adipocyte differentiation have been performed, little is known about the cell surface changes involved in this process. Most studies have preferred to focus on different responses to specific hormones during differentiation or changes in specific intracellular events such as changes in specific enzyme activities. We have therefore attempted to identify potential cell surface markers of the differentiation process using antibodies, as well as to identify which (if any) of these markers might be tissue specific. Separation by S D S - P A G E of plasma membrane proteins from adipose tissue, brain, muscle, liver and kidney and membrane proteins from red blood cells followed by Western blotting using our antiadipocyte plasma membrane antiserum produced the patterns shown in Fig. 1. A biotin-avidin-alkaline phosphatase system was used to detect bound antibodies in order to produce a highly sensitive response. As expected, very few proteins were detected in the liver, kidney and red blood Cell samples. However, a number of proteins appeared to cross-react in the brain and muscle samples. Comparison of the proteins detected in adipose tissue membranes with those from other tissues resulted in 15 proteins being identified as potential adipocyte specific antigens on the basis of their moi. wts. These were 110, 102.3, 96.3, 58.6, 38.4, 37, 31.3, 30.4, 28.4, 27.1, 24.3, 21.3, 17.2, 16.1 and 15.9kD. Two of these proteins of mol. wt 58.6 and 38.4 kD are similar to those of 56 and 37 kD reported by Lee et al. (1986) as being both adipocyte and species specific in the chicken. The same authors also reported that a 47 k D protein was adipocyte and species specific. We failed to detect a protein of this mol. wt as adipocyte specific. However, the criterion
149
Adipocyte membrane proteins
(a)
(b)
(d)
(c)
(e)
(f)
kD 1
110 + 1.8
2 3
102.3 + 3.6 96.3 ± 2.8
4
58.6 + 0.9
5 6
38.4 ± 2.1 37 ± 2.2
7 8
31.3 ± 2.1 30.4 ± 2.2 28.4 ± 1.3
9 10
|
27.1 ± 0.8
11
24.3 ± 1.3
12
21.3 ± 1.1
I
I
immmb ,
~i
,
.
13
17.2 ± 0.9
14
16.1
15
15.9 ± 1.0
± 0.9
Fig. 1. Western blot against proteins prepared from membranes of (a) liver, (b) red blood cell, (c) brain, (d) muscle, (e) kidney and (0 adipose tissue using an antiserum raised using a preparation of adipocyte specific antigens prepared as in Materials and Methods. The amount of protein loaded per lane was (a) 6.3, (b) 31.8, (c) 38.4, (d) 24, (e) 4.4 and (f) 9.3/tg. The location of 15 adipocyte specific proteins are indicated together with their respective mol. wts (mean __+SD of seven blots). Similar results were obtained with another antiserum raised against the same adipocyte antigen preparation. used for tissue specificity by Lee et al. (1986) was simply that the antiserum used in the experiments had been absorbed with red blood cells. This was because these authors were primarily concerned with identify-
ing species specific adipocyte membrane proteins rather than tissue specific components. They also relied on immunoprecipitation to identify antigens. Western blotting has the advantage that it does not
S. C. BUTTERWITHet al.
150
(a)
110 + 1.8
(b)
--~
102.3 _+ 3.6 96.3 + 2.8
93.8 _ 5.8
65.9 + 2.5
31.1 _+ 0.52 29.2 _+ 0.41 28.4 + 1.3 27.1 + 0.8
24.3 + 1.3
18.5 _+ 1.4 17.7 +_ 1.4 17.2 + 0.9
15.9 _ 1.0
Fig. 2. Western blot comparing membrane proteins from (a) mature adipose tissue with those prepared from (b) adipocyte precursors. The antiserum used was the same as in Fig. 1. The amount of protein loaded per lane was (a) 12.5 and (b) 9.3/~g. The mol. wts of eight putative adipocyte specific differentiation markers are indicated (mean + SD of three blots). rely on a precipitating antibody or the use of a second antibody to precipitate the antigen-antibody complex. In order to study the processes involved in adipocyte precursor differentiation we have attempted to identify any plasma membrane antigens which appeared to be present on the mature
adipocyte and not on the precursor. Previous antisera raised against adipocyte plasma membranes have always shown a significant a m o u n t of reactivity against the undifferentiated precursor cell type (Cryer et al., 1984; Lee et al., 1986). An exception to this was undifferentiated mouse 3T3-L1 cells which showed no reactivity to antiserum raised against mouse
Adipocyte membrane proteins
151
Table 1. Cytotoxicitytowards adipoeytesand their precursors of antisera 460 and 484 raised against adipocyte specificantigens Lactate dehydrogenase release (%) Precursors Adipoeytes Non-immune serum 19 +_.1.4(N = 3) 25 ___7.5(N = 5) Antiserum 460 64 + 8.8(N = 3) P < 0.005 68 + 11 (N = 4) P < 0.005 Antiserum 484 42 + 5.6(N = 3) P < 0.005 51 _ 5.7(N = 3) P < 0.005 Results are expressed as percentage release of lactate dehydrogenase relativeto total releaseof 0.2% Triton X-100 control (means+_SD). Significantdifferences from that of non-immune serum in an unpaired t-test are indicated. adipocytes (Plaas et al., 1981). This is perhaps partly explained by the fact that these cells are of embryonic origin, and therefore may resemble more closely a pluripotent stem cell. By comparing membrane proteins from adipocytes and their precursors using our specific antiserum, we have identified a number of proteins which appear only to be expressed after differentiation (Fig. 2). These proteins are identified by their mol. wts of 110, 102.3, 96.3, 28.4, 27.1, 24.3, 17.2 and 15.9kD. There are also some proteins of mol. wts 93.8, 65.9, 31.3, 29.2, 18.5 and 17.7kD which appear to decrease after differentiation. We also investigated whether the antisera we had raised were cytotoxic to adipocytes and their precursors in the presence of exogenously added complement. Only a low level o f cytotoxicity was seen (Table 1). This is a similar result to that found using a crude antiserum which had been adsorbed against liver cell membranes (Butterwith et al., 1989). The antibody response to specific antigens was much greater when they were used as immunogen so the lack of cytotoxicity in previous experiments with crude adsorbed antiserum was not due to a low concentration of specific antibodies. We are currently investigating the interaction of different complement sources with our specific antibodies in an attempt to increase the cytotoxic response.
SUMMARY
Affinity-purified adipocyte membrane proteins were used to raise antisera in sheep. These antisera were used on Western blots to compare membrane antigens from liver, kidney, muscle, red blood cell, brain and adipose tissue. Fifteen proteins were identified as being adipocyte specific on the basis of their mol. wt. O f these, eight appeared also to be differentiation-specific. Six proteins were also identified as appearing to decrease after differentiation but none of these were adipocyte specific. Characterization and further study of these adipocyte differentiation specific proteins will provide valuable information about the process of adipocyte differentiation. The antisera used to identify these proteins were cytotoxic to adipocytes and their precursors in the presence of complement.
Acknowledgements--This work was supported by a commission from the Ministry of Agriculture, Fisheries and Food. Financial support was also provided by the British Technology Group. REFERENCES
Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. Butterwith S. C., Kestin S., Griffin H. D. and Hint D. J. (1989) Cytotoxic antibodies to chicken adipocytes and their precursors: lack of tissue specificity. Br. Poult. Sci. 30, 371-378. Cryer A. (1985) New Perspectives in Adipose Tissue: Structure, Function and Development, pp. 383-405. Butterworths, London. Cryer A., Gray B. R. and Woodhead J. S. (1984) Studies on the characterisation of bovine adipocyte precursor cells and their differentiation in vitro, using an indirectlabelled-second-antibody cellular immunoassay. J. Devl. Physiol. 6, 159-176. Flint D. J., Coggrave H., Futter C. E., Gardner M. J. and Clarker T. J. (1986) Stimulatory and cytotoxic effects of an antiserum to adipoctye plasma membranes on adipose tissue metabolism in vitro and in vivo. Int. J. Obesity 10, 69-77. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680--685. Lee S. R., Tume R. K., Cryer J. and Cryer A. (1986) Studies on the expression of adipocyte-specific cell surface antigens during the differentiation of adipocyte precursor cells in vitro. J. Devl. Physiol. 8, 207-226. Plaas H. A. K., Woodhead J. S. and Cryer A. (1981) The use of antiserum with specific reactivity toward fat-cell surface antigen(s) to follow the progression of 3T3-L1 preadipoctye differentiation in vitro. Biosci. Rep. 1, 207-216. Saggerson E. D. and Greenbaum A. L. (1969) The effect of dietary and hormonal conditions on the activities of glycolytic enzymes in rat epididymal adipose tissue. Biochem. J. 115, 405-417. Thompson K. and Abraham S. (1979) Identification of mouse mammary adipose cells by membrane antigens. In Vitro 15, 441--445. Tume R. K., Lee S. R. and Cryer A. (1985) A comparison of the polypeptide composition of plasma membranes prepared from the white adipose tissue and adipocytes of the mouse, rat, rabbit and chicken by a percoll self-forming gradient procedure. Comp. Biochem. Physiol. 80B, 127-134.
