Insulin binding in differentiating rat preadipocytes in culture.

Binding, degradation, and antilipolytic effect of insulin were studied during the differentiation of preadipocytes into unilocular adipocytes. The precursor cells were isolated from the stromal-vascular fraction of adult rat epididymal fat pads and were cultured according to methods previously described. Under appropriate conditions the cells attained full morphological maturation after 6 days. A gradual increase in insulin binding was found concomitant with the morphological development of the preadipocytes into adipocytes. This increase was due to an enhanced number of binding sites whether expressed per cell or per unit cell surface area. The presence of a high insulin concentration (1.67 micrograms/ml or 278 nM) in the culture medium did not prevent this effect. The receptor density, expressed per unit surface area, was higher in the newly developed univacuolar cells than in mature fat cells from the same rat. The increased receptor density was also reflected by a leftward shift in the dose-response curve for the antilipolytic effect of insulin. In parallel with the increased binding, insulin degradation also increased. The lipolytic response to catecholamine also showed a gradual increase with development. When expressed per unit surface area, newly formed cells exhibited a considerably greater response (approximately 3.4 times) than mature cells from the same animals. The maximal antilipolytic effect of insulin in new cells was of the same order as in old cells when the data were expressed per unit cell surface area. Thus, the data show that developing adipocyte precursors gain membrane properties similar to those of mature fat cells. This cell system may serve as a useful model for studying receptor formation and factors that regulate hormone responsiveness.

The presence of epidermal growth factor receptors in cultured human adipocyte precursors.

The characteristics of 125I-epidermal growth factor urogastrone (125I-EGF) binding to human omental adipocyte precursors over a period of early differentiation in culture, is reported. The results show the presence of cell surface EGF receptor sites (38,000 per cell) that bind 125I-EGF with a high affinity (Ka = 2.7 X 10(9) l/mol). Their presence is not appreciably altered during a 28-day growth period in culture. The existence of such receptors suggests the possibility that growth factors might intervene in the regulatory processes involved in adipose tissue development.

Characterization of the collagen types synthesized by human and rat adipocyte precursors in vitro.

The types of collagen molecules synthesized by newly confluent rat and human adipocyte precursor cells were studied and compared with those synthesized by fibroblasts in culture. For this the cells were incubated in the presence of [2]3H]L-glycine and [5-3H]L-proline and the newly synthesized radiolabelled collagen produced was purified and analysed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This analysis revealed that the ratio of Types I and III collagen produced by four adipocyte precursor cell strains, each of distinct origin, was identical to that found with two different strains of lung fibroblasts. The mean ratio of Type I: Type III collagen in all cases was approximately 3.5:1. Because mature fat cells do not synthesize collagen and the adipocyte precursor cells lose their capacity for collagen synthesis during their post-confluent differentiation in vitro these data provide a biochemical basis to substantiate previous suggestions, based on morphology only, that the adipocyte stem cell is fibroblastic in nature.

Complete differentiation in vivo of implanted cultured adipocyte precursors from adult rats.

The formation of fully differentiated fat cells from adipocyte precursors, implanted into the same adult rats from which they were derived, is described. Precursor strains of rat epididymal adipocyte strains were grown through five subcultures, some in the presence of radioactive thymidine. While still at a relatively undifferentiated stage, the precursors were re-implanted into a superficial intramuscular location. At the time of resection six months later, "fat pads" were observed at the sites of implantation. These pads contained sheets of cells morphologically identical to mature epididymal adipocytes. The fat cells in pads developing from precursors grown in the presence of [3H]thymidine, were radiolabelled. Therefore, they represent fat cells that have differentiated in vivo from the implanted cultured precursors. Implanted skin fibroblasts did not lead to the formation of adipocytes. The finding that cultured adipocyte precursors from adult rats can differentiate fully not only in vitro, but also in adult animals, supports the probable physiological significance of these cells. The precursors probably participate in fat cell turnover, which likely persists throughout adulthood.

The contribution of a fat cell pool to adipose tissue cellularity.

The possible role of fat cell precursor differentiation and contribution to total adipose cellularity in the genetically obese Zucker fa/fa and non-obese Fa/-rat is reported. The study demonstrates that there is no difference in adipocyte precursor growth characteristics under normal culture conditions between genotypes. However, it is shown that there is a significantly larger fat cell pool maintained in the adult fa/fa epididymal adipose tissue, when compared to their lean littermates. The question as to the identity of the adipocyte precursor in the fat cell pool is addressed.

Complete differentiation of adipocyte precursors. A culture system for studying the cellular nature of adipose tissue.

Evidence for the complete morphological maturation of precursor cells into adipocytes in vitro is presented. Cells were isolated from the stromal fraction of adipose tissue from adult humans and from rats and were grown in culture. Abdominal skin fibroblasts were used as controls. All cell strains were initially fusiform and replicated. On reaching monolayer confluency, they were transferred to an enriched growth medium in which the human and rat adipocyte precursors differentiated into a homogeneous population of cells, morphologically indistinguishable from mature adipocytes. In contrast, skin fibroblasts from the same person or animal, and grown under identical culture conditions, did not accumulate lipid and retained their fusiform contour. The same results were obtained in the first six subcultures that were studied. Thus, there is firm evidence that fat tissue of adult humans and rats contains adipocyte precursors that differentiate into mature fat cells. The culture system that has been described will facilitate the elucidation of the factors involved in replication and differentiation of adipocyte precursors.

Promotion of human adipocyte precursor replication by 17beta-estradiol in culture.

The influence of 17beta-estradiol and 17alpha-estradiol on adult human omental adipocyte precursors grown in a propagating culture system was studied. Cells were grown in subculture in the presence or absence of hormone. 17beta-estradiol resulted in significant promotion of adipocyte precursor replication, as determined by cell counting and incorporation of radioactive thymidine into DNA. The hormone stimulated cell multiplication in the concentration range 0.5--500 ng/ml growth medium. The highest level tested was 500 ng/ml. The maximal effects were obtained at 50 ng/ml (P less than 0.001 by paired t test, 48 h after hormone addition). All 10 cell strains (five were derived from men and five from women) that were tested responded similarly to the hormone. 17beta-estradiol did not affect cell size. 17alpha-estradiol did not promote the replication of adipocyte precursors, nor did it influence cell size. Thus, 17beta-estradiol, which is the active isomer in known target tissues, stimulates the multiplication of human adipocyte precursors in culture.

Isolation of fat cell precursors from adult rat adipose tissue.

The possible existence of adipocyte precursors in adult rat adipose tissue was investigated. Cells were isolated from the stromal fraction of adipose tissue and were grown in culture. Skin fibroblasts were used as controls. The stromal fraction cells were initially fusiform and proliferated; in culture, they accumulated lipid inclusions, became rounder and acquired an eccentric nucleus. In contrast, the skin fibroblasts from the same rat grown under identical culture conditions, did not exhibit any appreciable lipid accumulation. The doubling time for both the stromal fraction cells and skin fibroblasts was 40-60 h. At confluency, the stromal fraction cells contained 5-7 times more glyceride-glycerol than skin fibroblasts. Thus, adipose tissue of adult rats contains cells with the potential to proliferate and acquire morphological characteristics similar to those of adipocytes.

Cytological and enzymological characterization of adult human adipocyte precursors in culture.

Cell strains were derived from the stromal-vascular fraction of human omental adipose tissue and grown in culture. Since the purpose of this study was to isolate adipocyte precursors from adults, the cells were obtained from nonobese patients 40-60 yr of age. After treatment of adipose tissue with collagenase, mature adipocytes were separated from stromal-vascular fraction cells, and cell strains of the latter replicated in culture with a doubling time of 40-60 h. They were initially fusiform; upon reaching monolayer confluency, they accumulated lipid and became rounder. Skin fibroblasts from the same patients and grown under the same culture conditions remained fusiform and did not accumulate lipid. The stromal-vascular fraction cells of adipose tissue may be fibroblasts with the potential to become adipocyte precursors. Subcellular preparations of the cells grown from the stromal-vascular fraction revealed lipoprotein lipase activity (characterized by such properties as inhibition by 1 M NaCl) that was not detectable in skin fibroblasts. The overall specific activity of the enzymes that catalyze triglyceride synthesis was 15 times higher and that of fatty acid synthetase was 2 times higher in the cells cultured from the stromal-vascular fraction. The difference was significant in each case. Conversely, when isolated mature adipocytes were cultured, they lost considerable lipid and acquired morphological characteristics similar to those of stromal-vascular fraction cells. Thus, adipose tissue stromal-vascular fraction cells acquire in culture many of the morphological and enzymological characteristics of mature fat cells.