- Email: [email protected]
[60] Growth and cytodifferentiation of 3T3-L1 preadipocytes into Adipocytes
720
CELL AND TISSUE TECHNIQUES
[60]
Acknowledgment This work was supported, in part, by grant GM-18278from the National Institutes of Health.
[60] G r o w t h a n d C y t o d i f f e r e n t i a t i o n o f 3 T 3 - L 1 P r e a d i p o c y t e s into Adipocytes
By THOMAS R. RUSSELL The 3T3-L1 preadipose cell line was isolated from the original uncloned stock (3T3M) of Swiss mouse fibroblasts.1 This preadipose cell line exhibits fibroblast-like properties during growth. However, when confluent cultures are maintained in a resting state for 2-3 weeks, adipose-like cells develop as clusters throughout the monolayer. The cultured adipocytes have many properties that are similar to those of normal adipocytes, z-4 Thus, this cell line is a convenient model system for studying both the development and the biochemical properties of adipocytes. Growth and Maintenance of Stock Cultures 3T3-L1 preadipocytes can be obtained from American Type Culture Collection, Rockville, Maryland (catalog No. ATCC-CCL-92.1). Stock cultures are grown in Dulbecco-Vogt's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). In our laboratory the medium is also supplemented with penicillin (30/zg/ml) and streptomycin (50 tzg/ml). Cultures are fed three times per week (10 ml DMEM-10% FCS per 100-mm culture dish) and maintained in a humidified 5% CO2 atmosphere at 37°. All culture operations are performed under sterile conditions. Stock cultures serve as a source of cells for experimental use and are maintained in a growing state. If stock cultures are allowed to grow to confluence and cease dividing before they are subcultured, the ability of the cells to convert into adipose cells is decreased. To subculture the cells, the medium is removed from the culture dish and the stock culture is 1 H. Green and O. Kehinde, Cell 1, 113 (1974). 2 H. Green, in "Differentiation and Development" (F. Ahmad, T. R. Russell, J. Schultz, and R. Werner, eds.), p. 13. Academic Press, N e w York, 1978. 3 j. Mackall, A. Student, S. Polakis, and M, D. Lane, J. Biol. Chem. 251, 6462 (1976). 4 C. S. Rubin, E. Lai, and O. Rosen, J. Biol. Chem. 252, 3554 (1977).
METHODS IN ENZYMOLOGY,VOL. 72
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181972-8
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incubated with 3 ml of a t r y p s i n - E D T A solution (0.25% trypsin, 0.6 m M EDTA in phosphate-buffered saline) for 3-5 min at 37°. The trypsinization is stopped by the addition of 3 ml of D M E M - 1 0 % FCS. The resulting cell suspension is transferred into a sterile conical centrifuge tube and pelleted in a clinical centrifuge at room temperature for 5 min (Model C L International Clinical Centrifuge at a setting of 4). The pellet is resuspended in 6 ml of D M E M - 1 0 % FCS and diluted to the appropriate cell density to establish new stock cultures (10,000 cells per 100-ram dish) and experimental plates (20,000 cells per 60-mm dish). Cell number is determined with a hemacytometer. Stock cultures are subcultured before the cell population reaches 30% confluence (700,000 cells per 100-mm dish). 3T3-L1 is subcultured 4-6 times and then a new stock culture is established from a frozen stock o f cells. To prepare frozen stocks, a stock culture is trypsinized, pelleted, and resuspended (800,000 cells/ml) in D M E M - 1 0 % FCS containing 10% sterilized glycerol. (Glycerol is sterilized by autoclaving.) Aliquots of 1 ml are distributed into N U N C freezing vials (Vanguard International, Inc. catalog No. 1076). The vials are placed in a 0 ° freezer for 2 hr, transferred to a - 7 0 ° freezer overnight, and then stored in liquid nitrogen. To establish stock cultures from cells stored in liquid nitrogen, the frozen cells are rapidly thawed by vigorous hand shaking in a 37° water bath (thawing should be complete within 90-120 sec). The vial is then soaked in a 70% ethanol solution and wiped dry. The thawed cells are transferred to a culture dish that contains 10 ml of prewarmed (37 °) D M E M - 1 0 % FCS and placed in the incubator. The medium is changed the following day to further dilute the glycerol. Growth and Cytodifferentiation of 3T3-L1 Preadipocytes into Adipocytes 3T3-L1 cells are plated at 20,000 cells per 60-mm culture dish (1000 cells/cm 2) and fed three times per week with D M E M - 1 0 % FCS (5 ml per 60-mm dish). When plated in this manner, the cells grow to confluence in 5-7 days, reaching a cell number of 50 to 55 × 103 cells/cm 2. The adipose conversion does not occur until after the cells reach confluence and enter a resting state. Kuri-Harcuch and Green 5 have also shown that the adipose conversion requires a nondialyzable factor present in FCS. The cells appear to grow equally well in FCS or calf serum, but the adipose conversion is almost completely absent when the cultures are grown and maintained in calf serum. Green and Kehinde 6 have shown that the adipose conversion is accelW. K u r i - H a r c u c h and H. Green, Proc. Natl. Acad. Sci. U . S . A . 75, 6107 (1979). 6 H. Green and O. Kehinde, Cell 7, 105 (1976).
722
CELL AND TISSUE TECHNIQUES
[60]
erated by the addition of insulin (1 /zg/ml) to DMEM-10% FCS. Under these conditions maximal adipose cluster formation in 3T3-LI occurred within 2-3 weeks after the cells reached confluence. We have demonstrated that treatment of confluent monolayers for 2 days with 1methyl-3-isobutylxanthine (MIX), an inhibitor of cyclic nucleotide phosphodiesterase, followed by feeding with medium containing insulin leads to maximal cluster formation by 7 d a y s / R u b i n e t al. s found that treatment of confluent cultures with a 2-day pulse of MIX (0.5 mM) plus dexamethasone (DEX) (0.25 /zM) results in maximal cluster development within 5 days and alleviates the need to add insulin to the culture medium. For further information on the role of MIX and DEX in the adipose conversion, see Russell and Ho, r Rubin e t a l . , 8 and Russell. ° We have found that a modification of the method of Rubin e t al. s which employs newborn calf serum (NBCS) and FCS yields a rapid and reproducible population of adipocytes. 9a Cells grown in DMEM-10% NBCS form a confluent monolayer but do not differentiate into adipocytes, presumably owing to lower levels of the adipogenic factor present in FCS. ~ However, if cells grown to confluence in DMEM-10% NBCS are promoted to differentiate with a 3-day pulse of DMEM-10% FCS, MIX, and DEX, the degree of differentiation is similar to that seen in cells maintained in DMEM-10% FCS. Since the price of NBCS is currently one-sixth that of FCS, there is a significant saving. To promote cytodifferentiation cells are grown to confluence in DMEM-10% NBCS. After the cells enter a resting state, cultures are fed for 3 days with DMEM-10% FCS containing 0.5 mM MIX and 0.25/zM DEX. At the end of 3 days the cultures are rinsed twice with warm, sterile phosphate-buffered saline and fed with 5 ml of DMEM-10% NBCS per 60 mm culture dish. Under these conditions maximum adipose conversion occurs within the following 3-5 days. Properties of 3T3-Adipocytes The most striking morphological feature of the adipose conversion is the appearance of triglyceride droplets in the cell cytoplasm. The altered metabolism that leads to the accumulation of triglyceride in the cultured adipocyte results from the development of hormonal responses and enzymatic activities characteristic of mature adipocytes. During the cytodifferentiation of preadipocytes into adipocytes the 7 T. Russell and R. J. Ho, Proc. Natl. Acad. Sci. U.S.A. 73, 4516 (1976). 8 C. Rubin, A. Hirsch, C. Fung, and O. Rosen, J. Biol. Chem. 253, 7570 (1978). a T. Russell, Proc. Natl. Acad. Sci. U.S.A. 76, 445• (1979). aa T. Murray and T. Russell, J. Supramolecular Structure (in press).
[60]
CONVERSION OF PREADIPOCYTES INTO ADIPOCYTES
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number of insulin receptors increase, s'l° the cells develop an ACTHsensitive adenylate cyclase 4 and an insulin-sensitive cyclic-AMP phosphodiesterase.Jl In the adipocyte, insulin increases glucose transport, 12'13 glucose oxidation 13 and the conversion of glucose, acetate, and palmitate into triglyceride. ~4 Epinephrine and dibutyrl cyclic-AMP, on the other hand, decrease the incorporation of glucose and palmitate into triglyceride. 14Thus, the reprogramming leads to hormonal responses characteristic of adipocytes. Along with the alterations in hormonal response, a number of enzymes involved in triglyceride metabolism increase in activity. These enzymes appear to fall into two groups. The first group of enzymes is primarily involved in lipid metabolism. These include fatty acid synthase3'~'~'~ acetyl-CoA carboxylase, 3 ATP-citrate lyase, 3'1~'17 glycerol phosphate acyltransferase, l~ fatty acyl-CoA synthatase, ~ and lipoprotein lipase. TM The increase in specific activity is usually 10- to 20-fold over that seen in the preadipocytes. However, glycerol phosphate acyltransferase 1~ and lipoprotein lipase TM increase at least 80-fold. In the case of two of these enzymes, acetyl-CoA carboxylase 3 and fatty acid synthase, TM the increase in specific activity is the result of new enzyme synthesis. The second group of enzymes, although involved in lipid metabolism, have other metabolic functions characteristic of many cell types. These include phosphofructokinase, aldolase, lactate dehydrogenase, and citrate synthaseJ ° The enzymes that fall into this group increase only 3- to 5-fold during the adipose conversion.
~0 B. C. Reed, S. K a u f m a n n , J. Mackall, A. Student, and M. D. Lane,Proc. Natl. Acad. Sci. U.S.A. 74, 4876 (1977). H T. Murray and T. R. Russell, Eur. J. Biochem. 107, 217 (1980). ~20. M. Rosen, C. J. Smith, C. Fung, and C. S. Rubin, J. Biol. Chem. 253, 7579 (1978). ~3 F. A. Karlsson, C. Grunfeld, C. R. K a h n , and J. Roth, Endocrinology 104, 1383 (1979). ~4 H. Green and O. Kehinde, Cell 5, 19 (1975). ~ P. Grimaldi, R. Negrel, and G. Ailhaud, Eur. J. Biochem. 84, 369 (1978). J~ P. A h m a d , T. R. Russell, and F. A h m a d , Biochem. J. 182, 509 (1979). ~7 I. Williams and S. Polakis, Biochem. Biophys. Res. Commun. 77, 175 (1977). ~8 W. K u r i - H a r c u c h and H. Green, J. Biol. Chem. 252, 2158 (1977). ~9 L. S. Wise and H. Green, Cell 13, 233 (1977). 20 j. Mackall and M. D. Lane, Biochem. Biophys. Res. Commun. 79, 720 (1977).
CELL AND TISSUE TECHNIQUES
[60]
Acknowledgment This work was supported, in part, by grant GM-18278from the National Institutes of Health.
[60] G r o w t h a n d C y t o d i f f e r e n t i a t i o n o f 3 T 3 - L 1 P r e a d i p o c y t e s into Adipocytes
By THOMAS R. RUSSELL The 3T3-L1 preadipose cell line was isolated from the original uncloned stock (3T3M) of Swiss mouse fibroblasts.1 This preadipose cell line exhibits fibroblast-like properties during growth. However, when confluent cultures are maintained in a resting state for 2-3 weeks, adipose-like cells develop as clusters throughout the monolayer. The cultured adipocytes have many properties that are similar to those of normal adipocytes, z-4 Thus, this cell line is a convenient model system for studying both the development and the biochemical properties of adipocytes. Growth and Maintenance of Stock Cultures 3T3-L1 preadipocytes can be obtained from American Type Culture Collection, Rockville, Maryland (catalog No. ATCC-CCL-92.1). Stock cultures are grown in Dulbecco-Vogt's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). In our laboratory the medium is also supplemented with penicillin (30/zg/ml) and streptomycin (50 tzg/ml). Cultures are fed three times per week (10 ml DMEM-10% FCS per 100-mm culture dish) and maintained in a humidified 5% CO2 atmosphere at 37°. All culture operations are performed under sterile conditions. Stock cultures serve as a source of cells for experimental use and are maintained in a growing state. If stock cultures are allowed to grow to confluence and cease dividing before they are subcultured, the ability of the cells to convert into adipose cells is decreased. To subculture the cells, the medium is removed from the culture dish and the stock culture is 1 H. Green and O. Kehinde, Cell 1, 113 (1974). 2 H. Green, in "Differentiation and Development" (F. Ahmad, T. R. Russell, J. Schultz, and R. Werner, eds.), p. 13. Academic Press, N e w York, 1978. 3 j. Mackall, A. Student, S. Polakis, and M, D. Lane, J. Biol. Chem. 251, 6462 (1976). 4 C. S. Rubin, E. Lai, and O. Rosen, J. Biol. Chem. 252, 3554 (1977).
METHODS IN ENZYMOLOGY,VOL. 72
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181972-8
[60]
CONVERSION OF PREADIPOCYTES INTO ADIPOCYTES
721
incubated with 3 ml of a t r y p s i n - E D T A solution (0.25% trypsin, 0.6 m M EDTA in phosphate-buffered saline) for 3-5 min at 37°. The trypsinization is stopped by the addition of 3 ml of D M E M - 1 0 % FCS. The resulting cell suspension is transferred into a sterile conical centrifuge tube and pelleted in a clinical centrifuge at room temperature for 5 min (Model C L International Clinical Centrifuge at a setting of 4). The pellet is resuspended in 6 ml of D M E M - 1 0 % FCS and diluted to the appropriate cell density to establish new stock cultures (10,000 cells per 100-ram dish) and experimental plates (20,000 cells per 60-mm dish). Cell number is determined with a hemacytometer. Stock cultures are subcultured before the cell population reaches 30% confluence (700,000 cells per 100-mm dish). 3T3-L1 is subcultured 4-6 times and then a new stock culture is established from a frozen stock o f cells. To prepare frozen stocks, a stock culture is trypsinized, pelleted, and resuspended (800,000 cells/ml) in D M E M - 1 0 % FCS containing 10% sterilized glycerol. (Glycerol is sterilized by autoclaving.) Aliquots of 1 ml are distributed into N U N C freezing vials (Vanguard International, Inc. catalog No. 1076). The vials are placed in a 0 ° freezer for 2 hr, transferred to a - 7 0 ° freezer overnight, and then stored in liquid nitrogen. To establish stock cultures from cells stored in liquid nitrogen, the frozen cells are rapidly thawed by vigorous hand shaking in a 37° water bath (thawing should be complete within 90-120 sec). The vial is then soaked in a 70% ethanol solution and wiped dry. The thawed cells are transferred to a culture dish that contains 10 ml of prewarmed (37 °) D M E M - 1 0 % FCS and placed in the incubator. The medium is changed the following day to further dilute the glycerol. Growth and Cytodifferentiation of 3T3-L1 Preadipocytes into Adipocytes 3T3-L1 cells are plated at 20,000 cells per 60-mm culture dish (1000 cells/cm 2) and fed three times per week with D M E M - 1 0 % FCS (5 ml per 60-mm dish). When plated in this manner, the cells grow to confluence in 5-7 days, reaching a cell number of 50 to 55 × 103 cells/cm 2. The adipose conversion does not occur until after the cells reach confluence and enter a resting state. Kuri-Harcuch and Green 5 have also shown that the adipose conversion requires a nondialyzable factor present in FCS. The cells appear to grow equally well in FCS or calf serum, but the adipose conversion is almost completely absent when the cultures are grown and maintained in calf serum. Green and Kehinde 6 have shown that the adipose conversion is accelW. K u r i - H a r c u c h and H. Green, Proc. Natl. Acad. Sci. U . S . A . 75, 6107 (1979). 6 H. Green and O. Kehinde, Cell 7, 105 (1976).
722
CELL AND TISSUE TECHNIQUES
[60]
erated by the addition of insulin (1 /zg/ml) to DMEM-10% FCS. Under these conditions maximal adipose cluster formation in 3T3-LI occurred within 2-3 weeks after the cells reached confluence. We have demonstrated that treatment of confluent monolayers for 2 days with 1methyl-3-isobutylxanthine (MIX), an inhibitor of cyclic nucleotide phosphodiesterase, followed by feeding with medium containing insulin leads to maximal cluster formation by 7 d a y s / R u b i n e t al. s found that treatment of confluent cultures with a 2-day pulse of MIX (0.5 mM) plus dexamethasone (DEX) (0.25 /zM) results in maximal cluster development within 5 days and alleviates the need to add insulin to the culture medium. For further information on the role of MIX and DEX in the adipose conversion, see Russell and Ho, r Rubin e t a l . , 8 and Russell. ° We have found that a modification of the method of Rubin e t al. s which employs newborn calf serum (NBCS) and FCS yields a rapid and reproducible population of adipocytes. 9a Cells grown in DMEM-10% NBCS form a confluent monolayer but do not differentiate into adipocytes, presumably owing to lower levels of the adipogenic factor present in FCS. ~ However, if cells grown to confluence in DMEM-10% NBCS are promoted to differentiate with a 3-day pulse of DMEM-10% FCS, MIX, and DEX, the degree of differentiation is similar to that seen in cells maintained in DMEM-10% FCS. Since the price of NBCS is currently one-sixth that of FCS, there is a significant saving. To promote cytodifferentiation cells are grown to confluence in DMEM-10% NBCS. After the cells enter a resting state, cultures are fed for 3 days with DMEM-10% FCS containing 0.5 mM MIX and 0.25/zM DEX. At the end of 3 days the cultures are rinsed twice with warm, sterile phosphate-buffered saline and fed with 5 ml of DMEM-10% NBCS per 60 mm culture dish. Under these conditions maximum adipose conversion occurs within the following 3-5 days. Properties of 3T3-Adipocytes The most striking morphological feature of the adipose conversion is the appearance of triglyceride droplets in the cell cytoplasm. The altered metabolism that leads to the accumulation of triglyceride in the cultured adipocyte results from the development of hormonal responses and enzymatic activities characteristic of mature adipocytes. During the cytodifferentiation of preadipocytes into adipocytes the 7 T. Russell and R. J. Ho, Proc. Natl. Acad. Sci. U.S.A. 73, 4516 (1976). 8 C. Rubin, A. Hirsch, C. Fung, and O. Rosen, J. Biol. Chem. 253, 7570 (1978). a T. Russell, Proc. Natl. Acad. Sci. U.S.A. 76, 445• (1979). aa T. Murray and T. Russell, J. Supramolecular Structure (in press).
[60]
CONVERSION OF PREADIPOCYTES INTO ADIPOCYTES
723
number of insulin receptors increase, s'l° the cells develop an ACTHsensitive adenylate cyclase 4 and an insulin-sensitive cyclic-AMP phosphodiesterase.Jl In the adipocyte, insulin increases glucose transport, 12'13 glucose oxidation 13 and the conversion of glucose, acetate, and palmitate into triglyceride. ~4 Epinephrine and dibutyrl cyclic-AMP, on the other hand, decrease the incorporation of glucose and palmitate into triglyceride. 14Thus, the reprogramming leads to hormonal responses characteristic of adipocytes. Along with the alterations in hormonal response, a number of enzymes involved in triglyceride metabolism increase in activity. These enzymes appear to fall into two groups. The first group of enzymes is primarily involved in lipid metabolism. These include fatty acid synthase3'~'~'~ acetyl-CoA carboxylase, 3 ATP-citrate lyase, 3'1~'17 glycerol phosphate acyltransferase, l~ fatty acyl-CoA synthatase, ~ and lipoprotein lipase. TM The increase in specific activity is usually 10- to 20-fold over that seen in the preadipocytes. However, glycerol phosphate acyltransferase 1~ and lipoprotein lipase TM increase at least 80-fold. In the case of two of these enzymes, acetyl-CoA carboxylase 3 and fatty acid synthase, TM the increase in specific activity is the result of new enzyme synthesis. The second group of enzymes, although involved in lipid metabolism, have other metabolic functions characteristic of many cell types. These include phosphofructokinase, aldolase, lactate dehydrogenase, and citrate synthaseJ ° The enzymes that fall into this group increase only 3- to 5-fold during the adipose conversion.
~0 B. C. Reed, S. K a u f m a n n , J. Mackall, A. Student, and M. D. Lane,Proc. Natl. Acad. Sci. U.S.A. 74, 4876 (1977). H T. Murray and T. R. Russell, Eur. J. Biochem. 107, 217 (1980). ~20. M. Rosen, C. J. Smith, C. Fung, and C. S. Rubin, J. Biol. Chem. 253, 7579 (1978). ~3 F. A. Karlsson, C. Grunfeld, C. R. K a h n , and J. Roth, Endocrinology 104, 1383 (1979). ~4 H. Green and O. Kehinde, Cell 5, 19 (1975). ~ P. Grimaldi, R. Negrel, and G. Ailhaud, Eur. J. Biochem. 84, 369 (1978). J~ P. A h m a d , T. R. Russell, and F. A h m a d , Biochem. J. 182, 509 (1979). ~7 I. Williams and S. Polakis, Biochem. Biophys. Res. Commun. 77, 175 (1977). ~8 W. K u r i - H a r c u c h and H. Green, J. Biol. Chem. 252, 2158 (1977). ~9 L. S. Wise and H. Green, Cell 13, 233 (1977). 20 j. Mackall and M. D. Lane, Biochem. Biophys. Res. Commun. 79, 720 (1977).