Organ culture of rat white adipose tissue

BIOCHIMICA ET BIOPHYSICA ACTA 579

BBA 56137

ORGAN CULTURE OF RAT WHITE ADIPOSE TISSUE

JIRf FROHLICH, ALAN VOST AND CHARLES H. HOLLENBERG*

McGill University Medical Clinic, The Montreal General Hospital, Montreal, Quebec (Canada)

(Received June 19th. 1972)

SUMMARY

I. Intact rat epididymal fat pads were incubated for up to 6 days in Trowell’s medium with zoo/O fetal calf serum. Fat pad DNA content fell by 30% after 6 days of incubation. The lipid content of the incubated fat pads increased by 11% after 3 days of incubation and then remained constant. Insulin when present throughout the incubation increased the lipid content and reduced the fall in fat pad DNA content.

2. [SH]Thymidine incorporation into DNA and [U-14C]glucose incorporation into lipid were not impaired after 3 days of incubation but were reduced by 6 days of incubation.

3. The fat pad lipolytic response to norepinephrine and active and passive uptake of 8sRb were reduced by 50% after 3 days of incubation.

4. Fat pads were pulse-labeled by incubating with [aH]thymidine for 2 h and were then digested with collagenase immediately or after several days incubation. There was a significant increase in fat cell DNA radioactivity after 3 days of incubation. This finding suggests that during the period of incubation primordial fat cells matured into lipid containing adipocytes.

5. The presence of insulin during the period of incubation did not enhance the formation or maturation of primordial fat cells.

INTRODUCTION

The finding that obesity is not only constantly associated with an increase in lipid content per fat cell but also in some instances with an increase in fat cell numberlpa has stimulated studies of the factors controlling fat cell proliferation and maturation. We had previously studied adipocyte proliferation by a technique involving intra- peritoneal injection of [3H]thymidine into rats, followed by separation of the adipose tissue into fat cells and stromal vascular cells by collagenase digestiona. Prior to the second day after injection of [aH]thymidine almost all label was found in stromal vascular DNA. Between 2 and 5 days after isotope injection, radioactivity in fat cell DNA increased markedly and then remained relatively constant for at least the ensuing

* Present address: Department of Medicine, University of Toronto, Toronto, Canada.

Biochim. Biophys. Acta, 280 (1972) 579-587

580 J. FROHLICH et al.

IO days. These results were interpreted as being compatible with a process in which labeled primordial fat cells, initially collected in the stromal pool, matured and ac- cumulated sufficient lipid within 5 days to be harvested in the fat cell fraction.

While this in viva system provided useful information concerning major factors controlling fat cell proliferation, the detailed study of nutritional and hormonal regulation of fat cell multiplication and maturation requires an in vitro system for prolonged incubation of adipose tissue. The present study describes organ culture of white adipose tissue and attempts to determine whether primordial fat cells continue to synthesize DNA and mature into fat laden adipocytes in this system.

MATERIAL AND METHODS

Wistar rats, weighing 70-90 g maintained on Purina Chow were purchased from Carworth Breeding Laboratories, N.Y.. Allcomponentsof tissue culture medium were obtained from Grand Island Biological Company, N.Y.. Crystalline zinc insulin was purchased from Connaught Laboratories, Toronto, ouabain from Sigma Chemical Company, St. Louis, MO. and collagenase (crude extract) from Worthington Bio- medical Company, Freehold, N. J.. [3H]Thymidine (Schwarz Bioresearch Incorporated, Orangeburg, N.Y. spec. act. 6.0 Ci/mmole, stated radiochemical purity > 99%) was adjusted to a final spec. act. of zoo &i/PM by addition of unlabeled thymidine (Nutri- tional Biochemicals Corporation, Cleveland, Ohio); in incubation studies the final medium concentration was 5.0 PM. [U-X]Glucose (New England Nuclear Corp., Boston, Mass., spec. act. 6.65 mCi/mmole, stated radiochemical purity > 99%) was added to the tissue culture medium to obtain a final spec. act. of 11.2 ,uCi/mole and a glucose concentration of 19 mM. saRb (New England Nuclear Corp., Boston, Mass.) was added to the medium to achieve a final concentration of 3.9 mg/ml and a spec. act. of 4.7 &i/ml.

Rats were killed by a blow on the head. Epididymal fat pads, each weighing 60-80 mg, were transferred under sterile conditions to plastic tissue-culture T-flasks (Falcon Plastics, Los Angeles) containing 5 ml of medium per IOO mg of the tissue. The medium was composed of 80 parts of Trowell’s medium4 without insulin or chloramphenicol and 20 parts of fetal calf serum. Penicillin (IOO units/ml) and strep- tomycin (IOO pg/ml) were added.

The pH of the medium was brough to 7.4 by flushing the flask with an air-CO, mixture (95 : 5, v/v) and the fat pads were incubated at 37 “C, without agitation. The medium was changed at least every 24 h and this prevented a fall in pH below 7.3 during the first three days of incubation. After three days of incubation, the medium had to be changed 2 or 3 times daily to maintain the pH above 7.2. The sterility of tissue and medium was checked repeatedly for viral, bacterial or fungal growth.

The first series of experiments was designed to assess viability of fat pads maintained in tissue culture medium for 3 or 6 days. As indices of viability the following were studied; incorporation of [3H]thymidine into DNA, incorporation of [14C]glucose into tissue lipid, the lipolytic response to norepinephrine and tissue uptake of 86Rb. In each study the right and left pads of 2 rats were placed alternately in one of two pools; one pool was processed immediately, the other after 3 or 6 days of incubation in the tissue culture medium. Thymidine incorporation into total tissue DNA and glucose conversion to triglyceride were assessed simultaneously by incubation of fat

Biochim. Biophys. Ada, 280 (1972) 579-587

ORGAN CULTURE OF RAT WHITE ADIPOSE TISSUE 581

pads for 2 h under air-CO, (95 : 5, v/v) in 2 ml of medium containing 5 PM [SH]thymidine and rg mM [U-l’C]glucose. The fat pads were then extracted with acetone and ether, the extract evaporated, dissolved in heptane and washed 3 times with water. The DNA content of the tissue was measured in the tissue residue after acetone-ether extraction3.

The effect of incubation for I and 3 days on basal lipolysis and norepinephrine- stimulated lipolysis was measured by incubating single pads for I h in 1.0 ml of phosphate buffered saline6 containing 4% albumin at pH 7.4 with or without I ,ug/ml of norepinephrine (Levophed, Winthrop); free fatty acids were extracted and titrated according to the method of Dolee. Lipolysis in alternate right or left pads was examined on excision and the corresponding pads in each animal cultured for I or 3 days before lipolysis was measured.

In studies in which s6Rb uptake was measured fat pads were bisected into pieces of approximately 30 mg wet wt. 8eRb uptake was measured in the two halves of each pad with or without ouabain andthe halves of the alternate fat pad were incubated for 3 days before measuring 8BRb uptake. Fat pieces were preincubated for I h with and without 5. IO-~ M ouabain in 2.9 ml of phosphate-buffered saline with 4% albumin; 0.1 ml of phosphate-buffered saline containing 86Rb (3.9 mg/ml) was then added and after IO min of incubation the fat pieces were dried, washed with saccharose and placed intact in vials for counting radioactivity by liquid scintillation spectrophoto- metry. a6Rb uptake was calculated as cpm per mg of wet weight of adipose tissue. Preliminary studies established that uptake of 8BRb was linearly related to tissue weight and to the duration of incubation.

In a second series of experiments an assessment was made of the effects of insulin on the DNA and lipid content of tissue incubated for 3 or 6 days. In these studies, paired pools of 2 epididymal fat pads were incubated for 3 or 6 days in the tissue culture medium, with and without insulin (IO munits/ml). The insulin-enriched medium was renewed at the usual intervals.

In the third series of experiments fat cell maturation in vitro was studied by pulse-labeling DNA of fat pads with [3H]thymidine immediately after excision; the pads were then incubated in the tissue culture medium. After varying periods of organ culture the labeled fat pads were digested with collagenase and the mature free fat cells were separated from all other cells which were designated as stromal cells7. It was anticipated that labeled primordial adipocytes, initially recovered with stromal elements, might mature after several days incubation resulting in increased radioactivity of DNA in the free fat cell fraction; analogous results have been previously observed in vivo3. Epididymal fat pads of 15 rats were divided into two pools of alternate right and left pads; both pools were pulse-labeled by incubation for 2 h in tissue- culture medium with [3H]thymidine. Paired pools had identical weights and the mean wet weights were 1070 & 206 mg. After washing in phosphate-buffered saline one pool was digested with collagenase immediately while the other pool (containing the contralateral fat pads) was processed after I, 2,3 or 6 days of incubation in the culture medium. The further purification of the fat cell fraction after collagenase digestion and the determination of DNA content and radioactivity of both fractions have been previously describeda.

In all experiments, except where otherwise described, the results derived from pools of 15 fat pads incubated for varying time intervals were expressed as a percent

Biochim. Bio$hys. Acta, 280 (1972) 579-587

582 J. FROHLICH et al.

of values obtained from corresponding pools of contralateral fat pads analysed immediately after removal from the animals.

To follow the histological changes associated with prolonged incubation, intact tissue was fixed in 3% glutaraldehyde for 20 min, delipidated with ethanol and em- bedded in paraffin. The histology of fat cells and stromal elements, was followed by preparing smears of these elements, fixing in glutaraldehyde and staining with he- matoxylin and eosin.

Radioactivity of 3H, 14C and s6Rb was counted in a Mark I Nuclear Chicago scintillation spectrophotometer and quenching of counts was calculated by a channel ratios method using an external standard. Bray’s scintillation fluidlo, containing dioxane as a solvent, was used. In studies where tissues were exposed simultaneously to :sH]thymidine and [Xjglucose both radioisotopes were counted simultaneousl! as described by Vostrl.

All statistics were tested by Student’s t-test for probability differences.

RESULTS

The viability of intact fat pads in vitro

The first series of experiments were carried out in order to assess the viability of the intact fat pads incubated for a maximum of 6 days. Histological preparations of fat pads incubated for 6 days showed no evidence of cell necrosis despite the reduction in tissue DhTA content described below.

The changes in fat pad DhTA and lipid content during 6 days of incubation are shown in Fig. I. DNA content declined slightly during the first 3 days of incubation

LIPID

DNA

0 D&S

6

Fig. I. Effect of incubation on DNA and lipid content of epididymal fat pads. One of a pair of fat pad pools was processed immediately after excision, the other after 3 or 6 days of incubation. Results derived from the incubated pads are expressed as a percentage of values derived from fresh tissue. Means and the S.E. of I I experiments are shown at 3 days, of 6 experiments at 6 days. The difference in lipid content between o time and 3 days is significant (P < 0.05). The difference between DNA content between o time and 6 days is significant (P < 0.01).

but more markedly thereafter. Lipid content increased slightly but significantly (P < 0.05) during the first 3 days of incubation. When insulin was present throughout incubation, fat pads had a significantly higher lipid content (P < 0.05) and DNA content (P < 0.01) than in the absence of this hormone (Figs 2 and 3). The higher recovery of DNA in the insulin-exposed pads was observed in both fat cell and stromal DNA.

To determine the effect of prolonged incubation on incorporation over 2 h of [3H]thymidine into total tissue DNA.and [Xlglucose into lipid, fat pads were ex-

Biochim Biofihys. Ada, 280 (1972) 579-587


40

i_

30

-20

10

0 II 0 NSUN

3 DAIS 6 DAIS 3 DAYS 6 DAYS

Fig. 2. Effect of insulin on the lipid content of z incubated fat pads. Two paired fat pads were incubated for the time shown in the presence or absence of insulin. The results show the mean weight of 2 pads and the SE. of 5 experiments. The differences between control and insulin-treated fat pads were significant in each instance (P < 0.05).

Fig. 3, Effect of insulin on the DNA content of incubated fat pads. Paired fat pads were incubated for 3 and 6 days in the presence or absence of insulin. The results show the mean DNA content of a fat pads and the SE. of 5 experiments. The differences between control and insulin-treated fat pads were significant in each instance (P < o.or).

ONSIAN Hhu/ml

posed to the labeled substrates immediately or 3 to 6 days after excision. The results are shown in Fig. 4. Incorporation of [3H]thymidine into DNA was not affected by 3 days of incubation but decreased markedly thereafter. 3 days of incubation pro- duced no change in incorporation of ~aC]glucose into total fat pad lipid; radioactivity per mg of lipid fell slightly due to the increase in lipid content that occurred over the three day period. However by 6 days, lipogenesis from [14C]glucose had decreased significantly.

After I and 3 days of organ culture, fat pads showed changes in basal lipolysis and in norepinephrine stimulated lipolysis during a one hour incubation. Basal release of free fatty acids from the control pads incubated on removal from the rats was 1.8 rj, 0.5 PM/g wet wt per h (B = 12) and increased to 5.8 + 1.6 yM (n = 12) and 6.0 $I 2.3 PM (n = 12) after I and 3 days of incubation, respectively. The net lipolytic response to norepinephrine above basal values fell from 29 Ij, 4 PM/g per h at o time to 14 j, 4and 15 & ~~Mlgper h after I and3 daysincubation, respectively (P -c 0.005 for differences between control and both incubated groups).

I. 0 .A3 4

Fig. 4. Effect of incubation on incorporation of [SH]thymidine into fat pad DNA and [U-*VI- glucose into fat pad lipid. One of a pair of fat pad pools was exposed to the isotopes immediately after excision, the other after 3 or 6 days of incubation. Data are expressed as a percent of values that were obtained in fresh tissue. The means and S.E. of 7 experiments are shown. -, DNA specific radioactivity; ---, lipid specific radioactivity.


584 J. FROHLICH et d.

Another parameter of membrane activity, sGRb uptake, was examined before and after 3 days of organ culture. Both active (ouabain sensitive) and passive (ouabain insensitive) uptake of 8BRb decreased significantly (P < 0.01) after 3 days incubation (Fig. 5).

Fat cell maturation in vitro

The demonstration that lipogenesis and DNA synthesis were maintained after 3 days incubation encouraged an attempt to demonstrate formation and maturation of primordial fat cells in this in vitro system. This was approached by pulse labeling paired pools of 15 fat pads immediately after excision. One of the pair was then analysed and the other incubated for a period of up to 6 days. Fat pads were digested with collagenase and the content and radioactivity of DNA in the fat cell and stromal fractions determined.

The changes in fat pad adipocyte and stromal DNA content with incubation time are shown in Fig. 6. The recovery of DNA in both stromal and fat cell pools was

CONTROL

Fig. 5. Effect of incubation on active and passive transport of 8BRb. Fat pad halves were incubated in phosphate-buffered saline with 4% albumin with or without 5. IO-~ M ouabain for I h after which s6Rb was added and incubation continued for a further IO min. In each experiment all pieces were derived from a single rat and the results are expressed as a percent of values obtained per mg fresh tissue not exposed to ouabain. The means and S.E. of 12 experiments are shown.

Fig. 6. Effect of incubation on DNA content of fat cells and stroma-vascular cells. One of a pair of pools of 15 fat pads was processed immediately, the other after I, 2, 3 or 6 days of incubation. The pads were digested with collagenase and the DNA content of fat cell and stromal fractions determined. Paired pools had equal wet weights initially. The means and the S.E. of 7 experiments are shown. Results of pool DNA are expressed as a percent of pool DNA obtained in fresh tissue.

less than 60:/, of the zero time DNA after 3 days incubation. This DNA loss is much greater than that observed after 3 days incubation in intact tissue (Fig. I) and suggests that incubation with collagenase had increased adipose tissue cell destruction.

With increased incubation time there was a marked decline in DNA total radioactivity and specific radioactivity of stromal vascular cells per fat pad (Fig. 7).

In the free fat cell DNA there was some radioactivity at zero time but this amounted to only 3% of total fat pad DNA radioactivity; this probably reflected contamination of the fat cells by labeled stromal cells which had a specific radioactivity IO--20 times higher than fat cell DNA at zero time. The changes in fat cell radioactivity with incubation are shown in Fig. 8. Free fat cell DNA total radioactivity and specific activity increased markedly between 24 and 48 h of incubation and remained elevated. These results are compatible with labeling of primordial fat cells

Biochim. Biophys. Ada, 280 (1972) 579-587


DNA CONTENT 8 .

P 20.

Fig. 7. Effect of incubation of stroma-Vascular DNA content, specific radioactivity and total radioactivity. Paired fat pad pools were pulse-labeled with [aH]thymidine, one of each pair was used immediately, the other after I, 2, 3 or 6 days of incubation. The mean values per pool * S.E. of 5 (Days I, 2,6) and IO (Day 3) experiments are shown.

DNA YECIFK ACTIVIIY

0 1 2 D:YS

6

Fig. 8. Effect of incubation on fat cell DNA content, total radioactivity and specific radioactivity. The mean values per pool & S.E. of 5 (Days I. 2, 6) and IO (Day 3) experiments are shown. The difference in DNA radioactivity between o time and 3 days is significant (P < 0.01).

which are initially harvested with stromal cells but matured sufficiently during 48-h incubation to be recovered as free fat cells.

It was possible, however, that these changes in adipocytes were due to effects of incubation in increasing contamination of free fat cells by stromal cells. This pos- sibility was explored by comparing adipocyte DNA radioactivity in groups of paired fat pads, one of which was pulse-labeled with [3H]thymidine before 3 days of incubation and the other after 3 days of incubation. In tissues labeled before incubation both total adipocyte DNA radioactivity (6.2 .~oa & 1.4’10~ dpm) and the percentage of fat pad DNA radioactivity recovered in adipocytes (12.3 & 1.5%) were higher than in pads pulsed after incubation (4.0.10~ & 0.7.10~ dpm, 6.4 & 1.5 yo) (P < 0.01, n = 6). Obviously the increase in fat cell radioactivity associated with prolonged incubation was not related to enhanced contamination of the fat cell pool but was consistent with maturation of labeled primordial adipocytes initially harvested in the stromal fraction.

As previously shown (Fig. 3)) when insulin was added to the medium throughout 3 days of incubation, DNA content of intact fat pads was greater than in the absence of hormone. As shown in Table I, this insulin effect was observed in both fat cell and


$36 J. FROHLICH et al.

TABLE I

EFFECT OF INSULIN OK DNA CONTENT AND RADIOACTIVITY

Paired fat pad pools were pulse-labeled with [3H]thymidine and then incubated for 3 days in the tissue culture medium in the presence and absence of insulin (I o munits/ml), After incubation the pads were treated with collagenase and fat cell and stromal fractions prepared. Results of tissue incubated with insulin are expressed as percent difference from results in tissue not exposed to insulin (IOO~/~). Means and S.E. or 7 experiments are shown.

Cells 0/O change with insulin

DNA content DNA radioactivity

Fat +3I * 7.5* stromal +I8 + 3.0

* Insulin z)s control P < 0.01.

-13 * 8.0

j-42 * 10.2*

stromal/DNA radioactivity over the 3 day interval but did not influence the appear-

ance of DNA radioactivity in the fat cell pool.

DISCUSSION

In earlier studies, Trowel14 demonstrated that fragments of white adipose

tissue could be maintained in vitro for 9 days without changes in histological appear-

ance. Herrera et aL8 incubated rat epididymal fat pads for 36 h without a significant

change in glucose metabolism. The results of the present study indicate that while

DNA and lipid synthesis were well maintained throughout 3 days of incubation, other

processes such as the lipolytic response to norepinephrine and uptake of 86Rb de-

creased. The latter changes probably reflected damage to fat cell membrane rather

than fat cell loss since total tissue DNA at that time had shown only a small reduction.

After 6 days in tissue culture medium the tissue was obviously abnormal as there was

a further definite decrease in all metabolic indices studied and an apparent reduction

in cell number.

In experiments in which the tissue was pulse-labeled with [3H]thymidine and

then incubated, total DNA radioactivity progressively decreased during incubation.

It is evident that this decrease affected only the stromal fraction and that total fat

cell DNA radioactivity actually increased after z days of incubation despite a reduc-

tion in fat cell DNA content. Since these results were evidently not due to an effect

of prolonged incubation or contamination of the fat cell fraction by stromal elements,

they may reasonably be attributed to a process of maturation of labeled primordial

fat cells. It is of interest and probably of significance that the time required for matura-

tion of these cells in vitro was the same as that required in Gvo3. The rise in fat cell DNA specific activity from Day 2 to Day 6 was due to a reduction in the DNA content

of this pool during a period in which fat cell DNA radioactivity remained constant. These data thus suggest that the cells that were lost from this pool during this interval were predominantly older cells with non-radioactive DNA.

Insulin, added to the medium after initial pulse-labeling of the fat pads with [sH]thymidine, partly prevented the decrease in DNA content in both the fat cell and stromal-vascular fractions and the decrease in DNA radioactivity in the stromal- vascular pool. However, the hormone had no effect on the amount of radioactivity found in fat cell DNA after 3 days of incubation. Thus, while insulin could have reduced destruction of stromal or fat cells, it had no evident effect on the maturation



of fat cells from stromal precursors. Previous in viva studies have also failed to show an unequivocal effect of insulin on production or maturation of fat cell precursors@.

The present study indicates the feasibility of maintaining adipose tissue in tissue culture medium with preservation of good function for 3 days and with preservation of some function for 6 days. It is apparent that under these in vitro conditions, DNA synthesis and maturation of fat cells can occur.

ACKNOWLEDGMENT

The authors wish to thank Drs B. Mosinger and R. L. Patten for their advice. Miss P. Walker (Mrs P. Mead) provided valuable technical assistance.

This work was supported by grants from the Medical Research Council of Canada and the Quebec Heart Foundation.

REFERENCES

I J. Hirsch, J. L. Knittle and L. B. Salans, J. C&z. Invest., 45 (1966) 1023. 2 J. L. Knittle and F. Ginsberg-Fellner, C&z. Res., XVIII (1969) 387. 3 C. H. Hollenberg and A. Vost, 1. Clin. Invest., 47 (1968) 2485. 4 0. A. Trowell, Exp. Cell Res., 16 (1959) 118. 5 R. Dulbecco and M. Vogt, J. Exp. Med., gg (1954) 167. 6 V. P. Dole, J. CL&. Invest., 35 (1956) 150. 7 M. Rodbell, J. Bid. Chem., 239 (1964) 375. 8 M. G. Herrera, G. R. Phillips and A. E. Renold, Biochim. Biophys. Acta, 106 (1965) 221. 9 A. Vost and C. H. Hollenberg, Endocrinology, 87 (1970) 606.

IO G. A. Bray, Anal. Biochem., I (1960) 279. I I A. Vost, J. Atheroscler. Res., g (1969) 221.

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Documents

Organ culture of rat white adipose tissue