Adipocytes

Chapter 18 Adipocytes Shinobu Gamou, Yoshiko Shim&u, and Nobuyoshi Shim&u 1. Introduction There are several preadipocyte cell lines reported, but in this chapter, we will describe mainly the 3T3-Ll cells, which are best characterized and widely used for molecular biological studies. The 3T3-Ll cells were clonally isolated by Green and Kehinde from 3T3-Swiss albino. When these cells are growing exponentially, they appear indistinguishable from their parental Swiss/3T3 cells. 3T3-Ll cells, however, undergo a differentiation to adipocytes when they enter a confluent and contact-inhibited resting stage (2). Many clones isolated from the original 3T3 stock are able to convert to adipocytes, in most cases, with a much lower frequency than that of Ll cells. Subclones can be generated by serial cloning, which differentiate to adipocytes at a high frequency. 3T3-F422A is such a clone isolated from the same 3T3-Swiss albino stock culture as Ll cells (2). A number of morphological, biochemical, and genetic changes occur during the differentiation of 3T3-Ll cells to adipocytes. Ll cells start to accumulate triglyceride in their cytoplasm, which can be seen as Oil Red 0 staining droplets as they change from a spindle-like fibroblastic cell to a spherical cell (3). Thus, the differentiation can be easily identified under the microscope. The differentiation is also accompanied by a large increase in the enzyme activities involved in de ~OVOfatty acid and triglyceride synthesis

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such as glycerol-3-phosphate dehydrogenase (4). The differentiated cells acquire a sensitivity to physiological concentrations of insulin via an increased number of insulin receptors that have a higher binding capacity than those in preadipocyte cells (5). Although the differentiation of Ll cells can occur spontaneously over a period of 2-4 wk following confluency, the differentiation can be synchronously induced at a high frequency by treating growth-arrested Ll cells with dexamethasone (DEX) and l-methyl-3-isobutylxanthine (MIX) for 2 d, followed by incubation in normal medium for 2-5 d (6). The combinations of DEX and MIX with insulin and biotin can accelerate the differentiation. Growth arrest at a specific stage of the cell cycle plays an important role in the induction of the adipocyte differentiation, and various factors appear to be involved in this process (7). The relationship between cell division in growth-arrested Ll cells and their initiation of differentiation can be closely examined under serum-free, hormone-supplemented conditions (8). We can observe the complex differentiation phenomenon under the microscope within a week by rather simple induction procedures. Thus, Ll cells’ differentiation is useful as a model of in vivo differentiation. The differentiated adipocytes are also useful as a model hormone-responsive cell sys tern.

2. Materials 2.1. Culture

Medium

1. Medium: DMEM/FCSlO. Dulbecco’s Modified Eagle’s Medium with a high concentration of glucose supplemented with fetal calf serum (FCS) at 10% and kanamycin (100 pg/mL). FCS concentration can be reduced to 5% by replacing with heat-inactivated calf serum. 2. PBS(-): Ca2+-and Mg2+-free Dulbecco’s phosphate buffered saline (PBS; 2.7 mM KCl, 1.5 mM KH.J?O, 8.1 mA4 Na,HPO,, 137 mM NaCl). 3. Trypsin/EDTA solution: 0.1% trypsin and 0.01% EDTA in PBS(-).

2.2. Inducers

for Adipocyte

Differentiation

1. Dexamethasone (DEX) 1,000 x stock solution: 0.25 mM (98 pg/mL). Dissolve in DMSO and store at -2OOC in small aliquots. 2. l-methyl-3-isobutylxanthine (MIX) 1,000 x stock solution: 0.5M (111 mg/mL). Dissolve in DMSO and store at -2OOC in small aliquots.

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3. Insulin 1,000x stock solution: 10 mg/mL. Dissolve in O.OlNHCl, sterilize by filtration through a sterile Millipore filter (0.22 pm> and store at -2OOC. 4. Biotin 1,000 x stock solution: 100 pg/mL. Dissolve in distilled water, sterilize by filtration through a sterile Millipore filter (0.22 pm) and store at -2OOC.

2.3. Oil Red 0 Staining 1. PBS(+): Dulbecco’s PBS with Ca2+(0.9 mM) and Mg2’ (0.5 mM). 2. 10% Formalin/PBS(+): Mixture of 10 mL formalin solution and 90 mL of PBS(+). 3. Oil Red 0 solution: Dissolve Oil Red 0 (700 mg) in isopropanol(200 mL), stir at 4OCovernight, and then filter through Whatman 3 MM paper. Mix 3 volumes of Oil Red 0 in isopropanol with 2 volumes of distilled water, allow to stand at 4°C overnight and filter through Whatman 3 MM. Store at 4OC. 4. 50% (v/v) glycerol solution. 5. Mayer’s Hematoxylin solution.

2.4. Detection of Glycerot3-Phosphate Dehydrogenase Activity 1. Extraction buffer: 0.5% lYiton X-100,10 mM Tris-HCl, pH 7.5,l mM MgCl,, 20 mM KCl, 0.2 mM Dithiothreitol, 10% Glycerol. 2. Reaction mixture: 125 mM Triethanolamine-HCl, pH 7.5, 2.5 mM EDTA, 0.16 mMNADH, 0.36 mM Dihydroxyacetonephosphate,0.125 mM P-mercaptoethanol. 3. Glycerol-3-phosphate dehydrogenase from rabbit muscle.

2.5. Insulin

Binding

Assay

1. Insulin binding buffer: 120 mM NaCl, 1.2 mM MgSO,, 5 mM KCl, 10 mM Glucose, 1 mM EDTA, 15 mM Sodium acetate, 50 mM HepesNaOH, pH 7.8,1% Bovine serum albumin. Store at -2OOC. 2. Cold insulin solution: Dilute the insulin stock solution described above with the insulin binding buffer at a final concentration of 1 pg/ mL and 10 pg/mL. Make fresh before use. 3. ?insulin: Insulin iodination can be carried out as described in Chapter 36, Vol. 1 of this series. Some technical skill is required to iodinate insulin at a high specific activity without compromising its biological activity. Thus, lZI-insulin obtained from commercial sources

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may be more convenient. Approximately 50,000 cpm/mL (-1 ng/ mL) of y251-insulin in insulin binding buffer is used for binding assay.

3. Methods 3.1. Maintenance

of 3T3-Ll

Cells

1. Ll cells can be purchased from American Type Culture Collection (12301 Parklawn Drive, Rockville, MD 20852-1776, USA). Ll cells are routinely maintained in DMEM/FCSlO at 37°C in 5% CO, and 100% humidity. Ll cells actively grow with a population doubling time of 20-30 h before becoming contact-inhibited at a saturation density of -5 x lo5 tells/3-cm dish. 2. The morphology of the parental 3T3-Swiss albino is quite uniform and shows typical 3T3 shape at confluency. Ll cells, however, show rather heterogeneous morphology, occasionally overlapping each other at confluency, but not piling up like transformed cells. 3. Contact-inhibited Ll cells will differentiate into the adipocytes spontaneously after 2-3 w when refed regularly. 4. Ll cells are easily detached after washing 2-3 times with PBS(-) and treating with trypsin/EDTA at 37°C. Transfer cells every4-5 d by 1:20 dilution to maintain the preadipocyte state.

3.2. Induction

of AcZipocyte Differentiation

1. The induction medium contains DEX (0.25 w), MIX (0.5 mM), insulin (10 pg/mL) and biotin (100 ng/mL) in DMEM/FCSlO. Add l/ 1000 vol of DEX and MIX stock solution into DMEM/FCSlO, mix immediately and vigorously, and then add insulin and biotin at the indicated concentration. Prepare the induction medium fresh before use. 2. Replace the medium with induction medium when Ll cells reach confluency, and incubate for 48 h at 37OC. 3. During the 48-h induction incubation, Ll cells start growing to approximately double in cell number, and the medium becomes slightly viscous because of triglyceride secretion. 4. Replace the induction medium with DMEM/FCSlO containing biotin (100 ng/mL) and insulin (10 pg/mL), and incubate for 5 d to allow differentiation into mature adipocytes.

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3.3. Oil Red 0 Staining to Identifjt the Morphological Differentiation 1. After the five days of incubation, the medium becomes viscous and spherical adipocytes are seen under the microscope (Fig. 1). The adipocytes often appear in clusters of varying sizes. 2. Wash the differentiated cultures twice with PBS(+), and then fix with 10% formalin/PBS(+) for 30 min. Stain the fixed culture dishes with Oil Red 0 solution for 10 min, wash under tap water for a few minutes, and then cover with 50% glycerol. The adipocyte colonies can be readily identified. 3. For microscopic observation or photography, the cells can be stained with Mayer’s Hematoxylin for 2-3 min, washed for about 10 min under tap water, and covered with 50% glycerol. 4. To determine the differentiation rate, wash the adipocytes and undifferentiated cells 2-3 times with PBS(-) and detach them by trypsin/ EDTA treatment at 37OC. 5. Resuspend the cells with an appropriate volume of DMEM/FCSlO, and count the number of adipocyte and undifferentiated cells in the hemocytometer. The adipocytes are identified by the presence of large lipid droplets, 6. When the lipid droplets are not large enough to identify under the microscope, add half a volume of Oil Red 0 solution to the cell suspension and incubate for a minute at room temperature. The adipocytes can be easily identified by the presence of red droplets in their cytoplasm.

3.4. Detection of Glycerol-S-Phosphate Dehydrogenase Activity as a Biochemical Differentiation Marker 1. Ll cells grown in 6-cm dish are washed twice with ice-cold PBS(-) and scraped with a rubber policeman. Transfer the Ll cells to a 1.5-mL centrifuge tube, and wash the cells twice with ice-cold PBS(-) by brief centrifugation. 2. Add 100 PLof ice-cold extraction buffer, and incubate for 30 min at 4OC with gentle and continuous agitation. Centrifuge at 16,000 rpm for 60 min at 4°C in a Sorval SS34 rotor with a 1.5-mL tube adapter. 3. The resulting supernatant is kept frozen at -7OOC in small aliquots until use. The amount of protein is determined by Lowry’s method as described in Chapter 1, Vol. 1 of this series.

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Fig. 1. Morphology of 3T3-Ll cells. (A) 3T3-Ll cells at high density. 09 3T3-Ll cells after adipocyte differentiation. The cells were treated for 2 d with inducers (DEX, MDC, and insulin) and allowed to differentiate for 5 d.

4. Put 0.4 mL of reaction mixture into a narrow width cuvette and set it in a spectrophotometer. 5. Dilute the cell lysate to 100 PL (W-200 pg protein) with the extraction buffer. Add 100 PL of diluted cell extract into the cuvette and mix rapidly with the reaction mixture to initiate the reaction.

Adipocytes 6. Record the absorbance at 340 nm 30 s after initiation and at 30-s intervals. The absorbance will decrease. 7. The reaction is linear with time (for several minutes) and cell extract concentration. Calculate the difference in absorbance/min/pg protein. 8. To determine the absolute enzyme activity, purified glycerol-3-phosphate dehydrogenase from rabbit muscle (Sigma) can be used as a standard. 9. The enzyme activity in adipocytes increased lO-loo-fold over the activity in preadipocytes. The increase in enzyme activity can be detected just after the induction incubation.

3.5. Insulin

Binding

Assay

I. Ll cells are grown in three 3-cm dishes. One of these is used to determine cell number and differentiation rate as described above. 2. Place the remaining two dishes on ice, and wash twice with ice-cold insulin-binding buffer. 3. Prepare 3 mL of *251-insulin solution, and save 0.5 mL to determine input radioactivity (cpm/mL) with a gamma counter (50,000 cpm/ mL, -1 ng/mL). 4. Add 1 mL of 1251-insulinsolution to one of the dishes, and incubate at 15OCfor 90 min with gentle agitation. 5. Wash the cells four times with ice-cold insulin binding buffer, and then dissolve in 1 mL of 1NNaOH by incubation at 37°C for 1 h. Cellasso-ciated radioactivity (TB) is determined with a gamma counter. 6. To determine nonspecific binding (NSB), the remaining dish is preincubated with cold-insulin solution containing 1 pg/mL insulin at 4°C for 30 min and then incubated with 1251-insulinsolution containing one-tenth the volume of the 10 pg/mL cold-insulin solution (final insulin cont. 1 pg/mL) at 15OCfor 90 min. Cell-associated radioactivity is determined as above. NSB is usually one-tenth to onefourth of TB, 7. Insulin specific binding (SB) is calculated as follow: SB = (TB-NSB)/ input/lo” cells x 100%. Specific binding in the undifferentiated Ll culture ranges from l-3% of input/l06 cells, whereas that in Ll adipocytes is 5-15% depending on the differentiation rate. Insulin binding capacity gradually increases after the induction incubation. 8. Duplicate or triplicate dishes of Ll cells are preferable for the TB and NSB determinations.

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4. Notes 1. Although the differentiation potential of Ll cells is heritable, they have a tendency to lose this capacity (2,2). The cells that have differentiated at 90% frequency may lose half of their differentiation ability after lo-20 transfers. Thus, a series of experiments must be carried out using cells with same differentiation potential. Numerous frozen stocks should be stored as early as possible. 2. If all of the frozen cell stocks with high differentiation potential have been depleted, subclones may be isolated using the same method by which the original Ll cells were established (1,2). Isolate lo-20 areas in which the cells have small lipid droplets in their cytoplasm at confluency by the standard cloning method. Measure the differentiation ability and subclone if necessary. 3. The timing for induction is an important factor in order to achieve a high differentiation rate. Sparse cultures can be induced, resulting in a high frequency of adipocyte differentiation. Induction during late confluency appears to be less effective in achieving the high differentiation rate than that induced during early confluency. Ll cells must traverse the cell cycle once before expressing the adipocyte phenotype

(8).

4. Incubation of more than five days for the expression of the adipocyte phenotype does not yield higher differentiation rates; however, the adipocytes become more fatted and anchorage independent with increased incubation time. 5. The differentiation rate may be measured quantitatively from the fixed, Oil Red 0 stained culture. Elute Oil Red 0 from the stained culture with isopropanol and measure the absorbance at 510 nm. Cultures containing no adipocytes occasionally bind some dye and give a high background. This, however, does not interfere the quantification of the differentiation rate. 6. When the cell lysates are analyzed by two-dimensional electrophoresis as described in Chapter 10, Vol. 1 of this series, an alteration in the biosynthesis of more than 60 species of differentiation-specific proteins is detected. This includes a decrease in the biosynthesic rates of p and y actins as well as a and p tubulins by 90% that reflect the changes in cell shape. These observations have been confirmed using cDNA probes (9). 7. The biochemical nature of the reaction of glycerol-3-phosphate dehydrogenase is: Dihydroxyaceton phosphate + NADI&&lycerol-3phosphate + NAD+. The decrease in the absorbance at 340 nm is the result of the conversion of NADH to NAD’.

Adipocytes 8. Since glycerol-3-phosphate dehydrogenase activity is very low in predifferentiated Ll cells, the cell extract without dilution or the extract from more than two dishes may be required to measure the activity accurately. 9. An automated spectrophotometer with a recorder is preferable, but not necessary, for the measurement of the enzyme activity because the reaction will be linear 2-3 min after initiation. 10. cDNAs for glycerol-3-phosphate dehydrogenase and other major differentiation-related proteins have been cloned from cDNA libraries constructed from the differentiated 3T3-F422A cells (10) and Ll cells (11). 11. The increase in insulin binding capacity during adipocyte differentiation involves an increase in both receptor number and affinity. Thus, Scatchard plot analysis must be used to measure changes in insulin binding capacity during the differentiation process. Prepare 1251-insulin solutions with different specific activities (50,000 cpm/mL of *251-insulin with 1,2,4,10,20,40, and 100 ng/mL of cold-insulin, and 1,2,4,10,20,40, and 100 pg/mL of cold-insulin for nonspecific binding using duplicate or triplicate dishes for each specific activity). The 1251-insulinbinding assay is carried out as described above (3.5.). Calculate how much insulin is bound as follows: Sp. AC. = Input cpm/ [Input insulin concentration]. [Bound] = SB/Sp. AC. [Free] = [Input insulin concentration] - [Bound]. Plot [Bound]/ [Free] on the ordinate and [Bound] on the abscissa. The negative reciprocal of the slope indicates the affinity of the insulin receptor for insulin (Kd: dissociation constant). The intercept on the abscissa indicates the number for insulin receptor. Both predifferentiated Ll cells and adipocytes yield curvilinear plots, suggesting a heterogeneity in receptor affinity types. 12. Serum-free hormone-supplemented medium has been developed for 3T3-Ll cells (8,X?). The basal medium is a 3:l mixture of DMEM and Ham’s F12 supplemented with several amino acids, vitamins, and trace elements. Hormone-supplemented medium contains the following components in the basal medium: transferrin (2 pg/mL), insulin (10 pg/mL), epidermal growth factor (20 ng/mL), biotin (100 ng/mL), bovine serum albumin (O.l%, Fr. V), and ethanolamine (0.1 mM). Under these conditions, the cells’ commitment to adipocyte differentiation is separated from the expression of the adipocyte phenotype. 13. There are several drugs that can inhibit the differentiation through several distinct mechanisms. Nicotinamide possibly inhibits the differentiation through the inhibition of poly (ADP-ribose) synthetase (23). Dihydroteleocidin B (DHTB), a potent tumor promoter,

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almost completely inhibits the differentiation whether added before, during, or after the inducer treatment. Similar inhibition is observed by 12-0-tetradecanoylphorbol-13-acetate (TPA), but with over 90% less efficiency (14). Thus, these inhibitions are useful for dissecting the complex differentiation process. 14. Using insulin-diphtheria toxin A fragment conjugate, variant cells with altered insulin receptors have been isolated from Ll cells (25). Also, DHTB-nonresponsive variants have been isolated by mitotic shake-off method (16). Using these variants, the mechanism of mitogenic signal transduction is being investigated. 15. Proto-oncogene c-fos expression in Ll cells and their derived adipocyte have been studied in response to a variety of growth-promoting agents (17). The intracellular location of protein kinase C has been also studied in the Ll cell during growth and after exposure to phorbol esters (18). Vesicles containing insulin-responsive glucose transporters have been isolated from Ll cells (29). 16. 3T3-F442A cells isolated from 3T3-Swiss albino are able to differentiate into adipocytes with high frequency by growth hormone treatment (20).

References 1. Green, H. and Kehinde, 0. (1974) Sublines of mouse 3T3 cells

that accumulate lipid. Cell 1,113-l 16. 2. Green, H. and Kehinde, 0. (1976) Spontaneous heritable changes leading to increased adipose conversion in 3T3 cells. Cell 7,205-113. 3. Novikoff, A. B., Novikoff, I’. M., Rosen, 0. M., and Rubin, C. S. (1980) Organelle relationships in cultured 3T3-Ll preadipocytes. J. Cell Bid. 87,180-196. 4. Wise, L. S. and Green, H. (1979) Participation of one isozyme of cytosolic glycerophosphate dehydrogenase in the adipose conversion of 3T3 cells. J. Biol. Chem. 254,

273-275. 5. Rosen, 0. M., Smith, C. J.,Fung, C., and Rubin, C. S. (2978) Development

of hormone receptors and hormone responsiveness in vitro: Effect of prolonged insulin treatment on hexose uptake in 3T3-Ll adipocytes. J. Bid. Chem.253,7579-7583. 6. Rubin, C. S., Hirsch, A., Fung, C., and Rosen, 0. M. (1978) Development of hormone receptors and hormonal responsiveness in vitro: Insulin receptors and insulin sensitivity in the preadipocyte and adipocyte forms of 3T3-Ll cells. J. Biol Chem. 253,

7570-7578. 7. Scott, R. E., Florine, D. L., Wille, J. J., Jr., and Yun, K. (1982) Coupling

of growth arrest and differentiation at a distinct state in the G, phase of the cell cycle: GD. Proc. Natl. Ad. Sci. USA 79,845-849. of 3T3-Ll cells in 8. Gamou, S. and Shimizu, N. (1986) Adipocyte differentiation serum-free hormone-supplemented media: Effects of insulin and dihydroteleocidin B. Cell Struct. Fun& 11,21-30.

Adipocytes 9. Spiegelman, B. M. and Farmer, S. R. (1982) Decreases in tubulin and actin gene expression prior to morphological differentiation of 3T3 adipocytes. Cell 29,53-60. 10. Spiegelman, B. M., Frank, M., and Green, H. (1983) Molecular cloning of mRNA from 3T3 adipocytes: Regulation of mRNA content for glycerophosphate dehydrogenase and other differentiation-dependent proteins during adipocyte development. J. BioI. Chern.258,10083-10089. Il. Bernlohr, D. A., Angus, C. W., Lane, M. D., Bolanowski, M. A., and Kelly, T. J., Jr. (1984) Expression of specific mRNAs during adipose differentiation: Identification of an mRNA encoding a homologue of myelin P2 protein. Proc.N&Z. Acud. Sci. USA 81,5468-5472. 12. Shimizu, N. (1984) Use of hormone-toxin conjugates and serum-free media for the isolation and study of cell variants in hormone responses,inMethods for Serum-Free Culture ofEpitheliu1 and FibrobZusticCelZs.(Barnes, D. W., Sirubasku, D. A., and Sato, G. H., eds.), Alan R. Liss, New York, pp. 233-248. 13. Lewis, J. E., Shimizu, Y., and Shimizu, N. (1982) Nicotinamide inhibits adipocyte differentiation of 3T3-Ll cells. FEBS Lett. 146,37-41. 14. Shimizu, Y., Shimizu, N., Fujiki, H., and Sugimura, T. (1983) Distinct inhibitory effects of dihydroteleocidin B and the phorbol ester tumor promoters on the adipocyte differentiation of 3T3-Ll cells. Cancer Res. 43,49744979. 15. Shimizu, N., Miskimins, W. K., Gamou, S., and Shin&u, Y. (1982) A genetic approach to the mechanisms of insulin action: Use of the insulin-Diphtheria toxin fragment A conjugates for isolation of genetic variants. Cold Spring Harbor Conferences on Cell Proliferation 9,397-402. 16. Shimizu, Y., Fujiki, H., Sugimura, T., and Shimizu, N. (1986)Mouse 3T3-Ll cell var-

iants unable to respond to mitogenic stimulation of dihydroteleocidin B: Genetic evidence for the synergism of tumor promoters with growth factors. CancerRes. 46,

4027-4031. 17. Stumpo, D. J. and Blackshear, P. J. (1986) Insulin and growth factor effects on c-fos

expression in normal and protein kinase C-deficient 3T3-Ll fibroblasts and adipocytes. Proc. Nutl. Acud. Sci. USA 83,9453-9457. 18. Halsey, 0. L., Girard, I’. R., Kuo, J, F., and Blacksher, P. J. (1987) Protein kinase C in fibroblasts: Characteristics of its intracellular location during growth and after exposure to phorbol esters and other mitogens. J. Biol. Chem. 262,2234-2243. 19. Biber, J. W. and Lienhard, G. E. (1986) Isolation of vesicles containing insulin responsive, intracellular glucose transporters from 3T3-Ll adipocytes. 1. Bid. Chem. 261,16180-16184. 20. Morikawa, M., Nixon, T., and Green, H. (1982) Growth hormone and the adipose conversion of 3T3 cells. Cell 29,783-789.