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Excessive proliferation in culture of reverted adipocytes from massively obese persons
Metabolism Clinical and Experimental JANUARY 1986
VOL XXXV, NO 1
Excessive
Proliferation in Culture of Reverted Massively Obese Persons
Adipocytes
From
Daniel A.K. Roncari, Sarah Kindler, and Charles H. Hollenberg Differentiated omental adipocytes from lean and massively obese persons lost their spherical shape in culture and regained the ability to replicate. In propagating culture, reverted cells from each group multiplied to a greater extent than corresponding stromal adipocyte precursors. Reverted adipocytes from the massively obese proliferated at significantly higher rates than those from lean subjects. Reverted cells derived from l-2 adipocytes also revealed these differences. Q 1988 by Grune & Stratton. Inc.
XCESSIVE REPLICATION in culture of omental adipocyte precursors from persons with massive obesity may at least partly explain the adipocyte hyperplasia characteristic of that state.“* Indeed, induction of nutritional obesity in rats is associated with exaggerated replication of stromal precursors in vivo.3 We now report that when mature fat cells (which cannot replicate) revert in culture to a delipidated state, those from the massively obese proliferate to a significantly greater extent than cells from lean subjects. E
MATERIALS
AND
METHODS
Omental adipose tissue was obtained from 13 massively obese and 6 lean adult persons by the reported criteria and methods.‘.’ Fat tissue was digested with collagenase as reported.4 Of the 3 layers formed by gravity, the upper was transferred to a plastic centrifuge tube, and after dilution with Hanks’ solution, spun at 400 x g for 5 min. The floating layer was removed, centrifuged 3 more times, and aspirated. While holding the syringe upright, the pink suspension was discarded, with retention of the cells. At this stage, phase-contrast light microscopy revealed only mature adipocytes. For morphologic studies of delipidation, as well as for some comparisons in terms of replication, the cells were suspended in medium consisting of (complete) MEM Alpha Medium (Gibco) buffered to pH 7.4 with I5 mmol/L HEPES (N-2-hydroxyethylpiperazine-N’-2-ethane sulfonic acid) supplemented with antibiotics and 10% fetal bovine serum (Gibco). After dilution to 1-2 cells per low-power field prior to plating, the base of the 25-cm2 Corning flasks was marked to follow single cells. For systematic studies of replication, about 150,000 cells were plated on 75-cm* Corning flasks using the described medium except for a higher serum concentration (15%). Twenty-four hours after inoculation, the cells were washed 3 times with Hanks’ solution, and the growth medium was changed every third day, up to about 2 weeks when monolayer confluence was usually reached. For subculMetabolism,
Vol 35,
No
1 (January)
1986:
pp l-4
ture, the cells were detached with 0.5 mg/mL trypsin-0.2 mg/mL Na,EDTA in Na,Citrate-KCI, pH 7.4. When replication was assessed by incorporation of [methyl-‘HI-thymidine (sp act 80 Ci/mmol, New England Nuclear) into DNA, Medium 199 containing Hanks’ solution (Gibco) was substituted for Alpha Medium to allow greater incorporation of radioactivity.’ For quantitative studies of cloned cells, mature adipocytes were isolated from one lean and one (borderline) massively obese subject, counted with a hemocytometer, and diluted with MEM Alpha Medium supplemented with antibiotics and 15%fetal bovine serum. The estimated 10 cells were seeded on 24-well Falcon 3847 Primaria plates (Becton Dickinson). After three weeks, subculture, growth, cell counting, and incorporation of [‘HI-thymidine into DNA, were carried out as described for the uncloned cells except that MEM Alpha Medium devoid of ribo- and deoxyribo-nucleosides, instead of Medium 199, was used for the studies of incorporation. Stromal adipocyte precursors were recovered from the intermediate layer formed after collagenase digestion of omental fat tissue, and cultured simultaneously with the delipidated cells and by the same methods. Aliquots of detached cells were counted with a Coulter Counter Model ZF (Coulter electronics), as reproted.‘,’ Replication was also assessed with [‘HI-thymidine using glass-microfibre filters (GF/C,
From the Institute of Medical Science, the Banting and Best Diabetes Centre. and the Department of Medicine, University of Toronto, Toronto, and the Julia McFarlane Diabetes Research Unit, and the Departments of Medicine and Medical Biochemistry, the University of Calgary, Calgary, Alberta, Canada. Supported in part by grants from the Medical Research Council of Canada (MT-5827 and MA-7679), the Alberta Heritage Foundation for Medical Research, and the Canadian (Ontario and Alberta) Heart Foundation. Address reprint requests to Dr D.A.K. Roncari, Faculty of Medicine, Health Sciences Centre, The University of Calgary, Calgary, Alberta, Canada T2N 4NI 0 1986 by Grune & Stratton, Inc. 0026-0495/86/3501-0001$03.00/0/
2
RONCARI ET AL
Whatman), as previously described’ except for the omission of trichloroacetic acid and the use of 10 mL Econofluor (New England Nuclear) for counting of radioactivity.
RESULTS AND DISCUSSION
Figure 1 (A, B, C) illustrates morphologic changes during reversion of mature omental adipocytes to earlier forms. For comparison, adipocyte precursors from the stromal fraction of omental adipose tissue, grown at a comparable stage, are shown in Fig 1D. These bear a greater resemblance to skin fibroblasts. In fact, cells with the morphologic features displayed in Fig lB, C are not recovered from adipose tissue by our isolation techniques. This point and the fact that single cells were carefully followed, suggest that reverting mature adipocytes were indeed studied. In the
Delipidation in vitro of mature adipocytes isolated from Fig 1. omental adipose tissue of e lean 52-year-old man undergoing elective repair of an abdominal eortic aneurysm. IA) One mature adipocyte diluted in the growth medium described in the text. (B) The cell in A, 24 hours later. (Cl Cells derived by multiplication after delipidetion of the ndipocyte shown in (A) and (B), at 14 days in first subculture. (01 Adipocyte precursors obtained from the stromal fraction of omentel adipose tissue from a lean 69-year-old man undergoing elective repair of an abdominal aortic aneurysm. The precursors were grown concurrently with the cells shown in (C), and are at a comparable stage in culture (original magnificetion for (A) and (Cl x 200; for (B) end (D) x 400).
current investigations, similar morphologic changes were observed whether mature adipocytes were obtained from lean or from massively obese subjects. In previous reports, we4 and other&’ have presented some evidence for reversion of mature fat cells; we studied cells from lean adults,4 in another study’ the body weights of the adults were not provided, while adipocytes from a 6-day-old infant were used in an earlier investigation.6 In the present study, despite sequential observation of apparently single mature adipocytes, the remote possibility exists that the “reverted” cells did not originate from adipocytes, but were actually fibroblast-like cells all along, because of incomplete separation of mature fat cells from very closely associated fibroblast-like cells, as has been the experience of others.8 In vitro reversion of differentiated adipocytes to less mature, fibroblast-like forms probably does not simply reflect loss of lipid. Changes in cell shape are dependent not on triglyceride accumulation, but on alterations in gene expression for cytoskeletal elements.’ Further, the reverted cells regain replicative capability. Chondrocytes also revert or “dedifferentiate” in culture.” Should such reversion also occur in vivo, it would signify that at least certain mesenchymal cells are not committed to terminal differentiation. Thus, the number of mature adipocytes might flexibly and reversibly increase or decrease in adaption to (opposite) sustained stimuli. Since necrosis of fat cells is very limited, reversion would also account for the proposed adipocyte turnover.4 Figure 2 illustrates the point that the extent of t’H]-thymidine incorporation into DNA of reverted mature omental fat cells in third subculture is appreciably greater than that of corresponding stromal adipocyte precursors both in the case of tissue derived from lean and that from massively obese subjects. In addition to confirming differences between stromal precursors, a major new finding is the significantly greater proliferation of reverted adipocytes from massively obese as compared to reverted cells from lean subjects (Fig 2). For all comparisons, cell counts indicated similar trends. Basically the same findings were obtained through the first three subcultures. When cultures were derived from l-2 cells, corresponding differences were observed for each comparison. However, quantification, in duplicate, throughout 12 hours was only carried out in the case of reverted adipocytes from one lean and from one obese subject (in first subculture). CPM of [3H]-thymidine incorporated were too low for meaningful comparisons prior to 24 hours, at which time mean CPM were 2726. During the ensuing 24 hours (24 to 48 hours of growth in first subculture), CPM incorporated rose 5.0-fold in
REVERTED ADIPOCYTES IN OBESE PERSONS
3
Obese 0
n
Fig 2. Incorporation of [“HIthymidine for omental adipose cells from lean and massively obese subjects. Data are shown for 2 lean and 2 obese because each pair (obese-lean) was not only identical with respect to experimental conditions, but all steps, including tissue resection, were also conducted almost simultaneously. Means + SEM are shown for 72 hr in third subculture. By the paired t-test, “lean” (reverted) adipocytes Y precursors. P < 0.01; “obese” precursors v “lean” precursors, P < 0.05; “obese” adipocytes v “lean” adipocytes, P < 0.0125. For the other comparisons involving all lean and all massively obese subjects, the level of significance was at P < 0.025.
T
Precursors Delipidated Adipocytes
-_I
lean -I-
reverted adipocytes from the lean and 7.1-fold in reverted cells from the obese subject, ie, the incorporation was 42% higher for the obese. Further, cells from this subject had 3 1% higher incorporation during 48 to 72 hours of growth. The higher replicative rates of reverted as compared to stromal cells might reflect the smaller number of divisions (until reaching the nondividing differentiated stage) undergone by the average clone of reverted adipocytes. As previously discussed,” an inverse relation may exist between number of cell divisions and replicative rate of preadipose cells. Indeed, some precursor clones from the massively obese might undergo the least number of divisions because they have been shown to have an unusual propensity to differentiation.2 We postulate that such sequence may explain why the reverted cells from these subjects have the highest replicative indices. These are observed consistently in culture under growth medium conditions favoring proliferation. As examples, two in vivo situations will now be described. During successful treatment of obesity, reversion of mature adipocytes would be promoted. Once reversion has occurred, the replica-
tion of these cells would be suppressed because of the negative energy state. This conjecture is an extrapolation of early studies indicating virtual abolition of replication of rat adipose tissue stromal cells during starvation.12 Thus, potential reduction in the excessive number of lipid-containing adipocytes characteristic of massive obesity is conceivable. The second example relates to the commonly observed cycles of compliance and excessive nutrient energy intake by obese persons. The cells that had reverted during periods of relative caloric deprivation would proliferate inordinately during times of excess. Coupling of exaggerated replication with promotion of maturation would lead, over a number of cycles, to a progressively higher complement of supernumerary adipocytes. These proposals require confirmation in vivo.
ACKNOWLEDGMENT The authors are grateful to Drs M.M. Cohen, L.E. Rotstein, P.M. Walker, and the late Dr J.A. Palmer, Department of Surgery, University of Toronto, for their interest and valuable help.
REFERENCES I. Roncari DAK, Lau DCW, Kindler S: Exaggerated
replication obese persons.
in culture of adipocyte precursors from massively Metabolism 30:425-427, 198 1 2. Roncari DAK, Lau DCW, Djian P, et al: Culture and cloning of adipocyte precursors from lean and obese subjects: Effects of growth factors, in Angel A, Hollenberg CH. Roncari DAK feds): The Adipocyte and Obesity: Cellular and Molecular Mechanisms. New York, Raven Press. 1983, pp 65-73 3. Klyde BJ, Hirsch J: Increased cellular proliferation in adipose
tissue of adult 1979
rats fed a high-fat
diet. J Lipid Res 20:705-715,
4. Van RLR, Bayliss CE, Roncari DAK: Cytological and enzymological characterization of adult human adipocyte precursors in culture. J Clin Invest 58:699-704, 1976 5. Roncari DAK, precursor replication 62:503-508, 1978
Van RLR: Promotion of human adipocyte by 17/3-estradiol in culture. J Clin Invest
4
RONCARI ET AL
6. Adenobojo FO: Synthesis and storage adipocytes of a human neonate. Biol Neonate
of lipids by cultured 23:366-370, 1973
7. Dixon-Shanies D, Rudick J, Knittle JL: Observations on the growth and metabolic functions of cultured cells derived from human adipose tissue. Proc Sot Exp Biol Med 149:541-545, 1975 8. Klyde BJ, Hirsch J: Isotopic labeling of DNA in rat adiopose tissue: evidence for proliferating cells associated with mature adipocytes. J Lipid Res 20:69 l-704, 1979 9. Spiegelman
BM, Farmer
SR: Decreases
in tubulin
and actin
gene expression prior to morphological differentiation of 3T3 adipocytes. Cell 29:53-60, 1982 10. Schiltz JR, Mayne R, Holtzer H: The synthesis of collagen and glycosaminoglycans by dedifferentiated chondroblasts in culture. Differentiation 1:97-108, 1973 11. Djian P, Roncari DAK, Hollenberg CH: Influence of anatomic site and age on the replication and differentiation of rat adipocyte precursors in culture. J Clin Invest 72:120&1208, 1983 12. Hollenberg CH, Vost A: Regulation of DNA synthesis in fat cells and stromal elements from rat adipose tissue. J Clin Invest 47:2485-2498, 1968
VOL XXXV, NO 1
Excessive
Proliferation in Culture of Reverted Massively Obese Persons
Adipocytes
From
Daniel A.K. Roncari, Sarah Kindler, and Charles H. Hollenberg Differentiated omental adipocytes from lean and massively obese persons lost their spherical shape in culture and regained the ability to replicate. In propagating culture, reverted cells from each group multiplied to a greater extent than corresponding stromal adipocyte precursors. Reverted adipocytes from the massively obese proliferated at significantly higher rates than those from lean subjects. Reverted cells derived from l-2 adipocytes also revealed these differences. Q 1988 by Grune & Stratton. Inc.
XCESSIVE REPLICATION in culture of omental adipocyte precursors from persons with massive obesity may at least partly explain the adipocyte hyperplasia characteristic of that state.“* Indeed, induction of nutritional obesity in rats is associated with exaggerated replication of stromal precursors in vivo.3 We now report that when mature fat cells (which cannot replicate) revert in culture to a delipidated state, those from the massively obese proliferate to a significantly greater extent than cells from lean subjects. E
MATERIALS
AND
METHODS
Omental adipose tissue was obtained from 13 massively obese and 6 lean adult persons by the reported criteria and methods.‘.’ Fat tissue was digested with collagenase as reported.4 Of the 3 layers formed by gravity, the upper was transferred to a plastic centrifuge tube, and after dilution with Hanks’ solution, spun at 400 x g for 5 min. The floating layer was removed, centrifuged 3 more times, and aspirated. While holding the syringe upright, the pink suspension was discarded, with retention of the cells. At this stage, phase-contrast light microscopy revealed only mature adipocytes. For morphologic studies of delipidation, as well as for some comparisons in terms of replication, the cells were suspended in medium consisting of (complete) MEM Alpha Medium (Gibco) buffered to pH 7.4 with I5 mmol/L HEPES (N-2-hydroxyethylpiperazine-N’-2-ethane sulfonic acid) supplemented with antibiotics and 10% fetal bovine serum (Gibco). After dilution to 1-2 cells per low-power field prior to plating, the base of the 25-cm2 Corning flasks was marked to follow single cells. For systematic studies of replication, about 150,000 cells were plated on 75-cm* Corning flasks using the described medium except for a higher serum concentration (15%). Twenty-four hours after inoculation, the cells were washed 3 times with Hanks’ solution, and the growth medium was changed every third day, up to about 2 weeks when monolayer confluence was usually reached. For subculMetabolism,
Vol 35,
No
1 (January)
1986:
pp l-4
ture, the cells were detached with 0.5 mg/mL trypsin-0.2 mg/mL Na,EDTA in Na,Citrate-KCI, pH 7.4. When replication was assessed by incorporation of [methyl-‘HI-thymidine (sp act 80 Ci/mmol, New England Nuclear) into DNA, Medium 199 containing Hanks’ solution (Gibco) was substituted for Alpha Medium to allow greater incorporation of radioactivity.’ For quantitative studies of cloned cells, mature adipocytes were isolated from one lean and one (borderline) massively obese subject, counted with a hemocytometer, and diluted with MEM Alpha Medium supplemented with antibiotics and 15%fetal bovine serum. The estimated 10 cells were seeded on 24-well Falcon 3847 Primaria plates (Becton Dickinson). After three weeks, subculture, growth, cell counting, and incorporation of [‘HI-thymidine into DNA, were carried out as described for the uncloned cells except that MEM Alpha Medium devoid of ribo- and deoxyribo-nucleosides, instead of Medium 199, was used for the studies of incorporation. Stromal adipocyte precursors were recovered from the intermediate layer formed after collagenase digestion of omental fat tissue, and cultured simultaneously with the delipidated cells and by the same methods. Aliquots of detached cells were counted with a Coulter Counter Model ZF (Coulter electronics), as reproted.‘,’ Replication was also assessed with [‘HI-thymidine using glass-microfibre filters (GF/C,
From the Institute of Medical Science, the Banting and Best Diabetes Centre. and the Department of Medicine, University of Toronto, Toronto, and the Julia McFarlane Diabetes Research Unit, and the Departments of Medicine and Medical Biochemistry, the University of Calgary, Calgary, Alberta, Canada. Supported in part by grants from the Medical Research Council of Canada (MT-5827 and MA-7679), the Alberta Heritage Foundation for Medical Research, and the Canadian (Ontario and Alberta) Heart Foundation. Address reprint requests to Dr D.A.K. Roncari, Faculty of Medicine, Health Sciences Centre, The University of Calgary, Calgary, Alberta, Canada T2N 4NI 0 1986 by Grune & Stratton, Inc. 0026-0495/86/3501-0001$03.00/0/
2
RONCARI ET AL
Whatman), as previously described’ except for the omission of trichloroacetic acid and the use of 10 mL Econofluor (New England Nuclear) for counting of radioactivity.
RESULTS AND DISCUSSION
Figure 1 (A, B, C) illustrates morphologic changes during reversion of mature omental adipocytes to earlier forms. For comparison, adipocyte precursors from the stromal fraction of omental adipose tissue, grown at a comparable stage, are shown in Fig 1D. These bear a greater resemblance to skin fibroblasts. In fact, cells with the morphologic features displayed in Fig lB, C are not recovered from adipose tissue by our isolation techniques. This point and the fact that single cells were carefully followed, suggest that reverting mature adipocytes were indeed studied. In the
Delipidation in vitro of mature adipocytes isolated from Fig 1. omental adipose tissue of e lean 52-year-old man undergoing elective repair of an abdominal eortic aneurysm. IA) One mature adipocyte diluted in the growth medium described in the text. (B) The cell in A, 24 hours later. (Cl Cells derived by multiplication after delipidetion of the ndipocyte shown in (A) and (B), at 14 days in first subculture. (01 Adipocyte precursors obtained from the stromal fraction of omentel adipose tissue from a lean 69-year-old man undergoing elective repair of an abdominal aortic aneurysm. The precursors were grown concurrently with the cells shown in (C), and are at a comparable stage in culture (original magnificetion for (A) and (Cl x 200; for (B) end (D) x 400).
current investigations, similar morphologic changes were observed whether mature adipocytes were obtained from lean or from massively obese subjects. In previous reports, we4 and other&’ have presented some evidence for reversion of mature fat cells; we studied cells from lean adults,4 in another study’ the body weights of the adults were not provided, while adipocytes from a 6-day-old infant were used in an earlier investigation.6 In the present study, despite sequential observation of apparently single mature adipocytes, the remote possibility exists that the “reverted” cells did not originate from adipocytes, but were actually fibroblast-like cells all along, because of incomplete separation of mature fat cells from very closely associated fibroblast-like cells, as has been the experience of others.8 In vitro reversion of differentiated adipocytes to less mature, fibroblast-like forms probably does not simply reflect loss of lipid. Changes in cell shape are dependent not on triglyceride accumulation, but on alterations in gene expression for cytoskeletal elements.’ Further, the reverted cells regain replicative capability. Chondrocytes also revert or “dedifferentiate” in culture.” Should such reversion also occur in vivo, it would signify that at least certain mesenchymal cells are not committed to terminal differentiation. Thus, the number of mature adipocytes might flexibly and reversibly increase or decrease in adaption to (opposite) sustained stimuli. Since necrosis of fat cells is very limited, reversion would also account for the proposed adipocyte turnover.4 Figure 2 illustrates the point that the extent of t’H]-thymidine incorporation into DNA of reverted mature omental fat cells in third subculture is appreciably greater than that of corresponding stromal adipocyte precursors both in the case of tissue derived from lean and that from massively obese subjects. In addition to confirming differences between stromal precursors, a major new finding is the significantly greater proliferation of reverted adipocytes from massively obese as compared to reverted cells from lean subjects (Fig 2). For all comparisons, cell counts indicated similar trends. Basically the same findings were obtained through the first three subcultures. When cultures were derived from l-2 cells, corresponding differences were observed for each comparison. However, quantification, in duplicate, throughout 12 hours was only carried out in the case of reverted adipocytes from one lean and from one obese subject (in first subculture). CPM of [3H]-thymidine incorporated were too low for meaningful comparisons prior to 24 hours, at which time mean CPM were 2726. During the ensuing 24 hours (24 to 48 hours of growth in first subculture), CPM incorporated rose 5.0-fold in
REVERTED ADIPOCYTES IN OBESE PERSONS
3
Obese 0
n
Fig 2. Incorporation of [“HIthymidine for omental adipose cells from lean and massively obese subjects. Data are shown for 2 lean and 2 obese because each pair (obese-lean) was not only identical with respect to experimental conditions, but all steps, including tissue resection, were also conducted almost simultaneously. Means + SEM are shown for 72 hr in third subculture. By the paired t-test, “lean” (reverted) adipocytes Y precursors. P < 0.01; “obese” precursors v “lean” precursors, P < 0.05; “obese” adipocytes v “lean” adipocytes, P < 0.0125. For the other comparisons involving all lean and all massively obese subjects, the level of significance was at P < 0.025.
T
Precursors Delipidated Adipocytes
-_I
lean -I-
reverted adipocytes from the lean and 7.1-fold in reverted cells from the obese subject, ie, the incorporation was 42% higher for the obese. Further, cells from this subject had 3 1% higher incorporation during 48 to 72 hours of growth. The higher replicative rates of reverted as compared to stromal cells might reflect the smaller number of divisions (until reaching the nondividing differentiated stage) undergone by the average clone of reverted adipocytes. As previously discussed,” an inverse relation may exist between number of cell divisions and replicative rate of preadipose cells. Indeed, some precursor clones from the massively obese might undergo the least number of divisions because they have been shown to have an unusual propensity to differentiation.2 We postulate that such sequence may explain why the reverted cells from these subjects have the highest replicative indices. These are observed consistently in culture under growth medium conditions favoring proliferation. As examples, two in vivo situations will now be described. During successful treatment of obesity, reversion of mature adipocytes would be promoted. Once reversion has occurred, the replica-
tion of these cells would be suppressed because of the negative energy state. This conjecture is an extrapolation of early studies indicating virtual abolition of replication of rat adipose tissue stromal cells during starvation.12 Thus, potential reduction in the excessive number of lipid-containing adipocytes characteristic of massive obesity is conceivable. The second example relates to the commonly observed cycles of compliance and excessive nutrient energy intake by obese persons. The cells that had reverted during periods of relative caloric deprivation would proliferate inordinately during times of excess. Coupling of exaggerated replication with promotion of maturation would lead, over a number of cycles, to a progressively higher complement of supernumerary adipocytes. These proposals require confirmation in vivo.
ACKNOWLEDGMENT The authors are grateful to Drs M.M. Cohen, L.E. Rotstein, P.M. Walker, and the late Dr J.A. Palmer, Department of Surgery, University of Toronto, for their interest and valuable help.
REFERENCES I. Roncari DAK, Lau DCW, Kindler S: Exaggerated
replication obese persons.
in culture of adipocyte precursors from massively Metabolism 30:425-427, 198 1 2. Roncari DAK, Lau DCW, Djian P, et al: Culture and cloning of adipocyte precursors from lean and obese subjects: Effects of growth factors, in Angel A, Hollenberg CH. Roncari DAK feds): The Adipocyte and Obesity: Cellular and Molecular Mechanisms. New York, Raven Press. 1983, pp 65-73 3. Klyde BJ, Hirsch J: Increased cellular proliferation in adipose
tissue of adult 1979
rats fed a high-fat
diet. J Lipid Res 20:705-715,
4. Van RLR, Bayliss CE, Roncari DAK: Cytological and enzymological characterization of adult human adipocyte precursors in culture. J Clin Invest 58:699-704, 1976 5. Roncari DAK, precursor replication 62:503-508, 1978
Van RLR: Promotion of human adipocyte by 17/3-estradiol in culture. J Clin Invest
4
RONCARI ET AL
6. Adenobojo FO: Synthesis and storage adipocytes of a human neonate. Biol Neonate
of lipids by cultured 23:366-370, 1973
7. Dixon-Shanies D, Rudick J, Knittle JL: Observations on the growth and metabolic functions of cultured cells derived from human adipose tissue. Proc Sot Exp Biol Med 149:541-545, 1975 8. Klyde BJ, Hirsch J: Isotopic labeling of DNA in rat adiopose tissue: evidence for proliferating cells associated with mature adipocytes. J Lipid Res 20:69 l-704, 1979 9. Spiegelman
BM, Farmer
SR: Decreases
in tubulin
and actin
gene expression prior to morphological differentiation of 3T3 adipocytes. Cell 29:53-60, 1982 10. Schiltz JR, Mayne R, Holtzer H: The synthesis of collagen and glycosaminoglycans by dedifferentiated chondroblasts in culture. Differentiation 1:97-108, 1973 11. Djian P, Roncari DAK, Hollenberg CH: Influence of anatomic site and age on the replication and differentiation of rat adipocyte precursors in culture. J Clin Invest 72:120&1208, 1983 12. Hollenberg CH, Vost A: Regulation of DNA synthesis in fat cells and stromal elements from rat adipose tissue. J Clin Invest 47:2485-2498, 1968