Adipocyte Development. Measurement of the Size and Number of Adipocytes

The development of a region of adipose tissue starts with penetration of a capillary bud onto which an adipocyte lobule differentiates. Primitive fat cells may arise from pericapillary primordial cells, which are difficult to separate from endothelial cells by light microscopy. Such “adipogenic reticular cells” seem to sequester small sudanophilic (incorporating Sudan dyes) lipid droplets, which appear near the center of the cell and also near the endoplasmic reticulum and mitochondria. Presumably, the next step is the accumulation of more lipid droplets, which ultimately coalesce.

Staging of the cells into adipoblasts and preadipocytes may well take place with the former containing less lipid than the latter. The mature adipocyte contains the major single lipid droplet (unilocular) and assumes a spherical configuration surrounded by the thin layer of cytoplasm and a displaced nucleus that identifies the cell as a “signet ring” in appearance.

A more recent view of adipocyte origin involves the stem cell as being indistinguishable from a fibroblast. While the origin of adipocytes is under investigation, researchers apparently agree that capillaries and some form of precursor adipocyte appear together in developing adipose tissue.

Several techniques have been utilized to clarify adipocyte development. These include studies of DNA synthesis by presumed adipocyte precursor cells, adipocyte sizing and counting, lipid accumulation by cultured precursor cells including the mouse fibroblast cell line 3T3, and incorporation in vitro of metabolic substrates by cultured precursor cells.

The presence of adipogenic activity in human plasma has been established in both cell lines and in primary cultures of stromal-vascular cells from rat adipose tissue. Growth hormone and glucocorticoid hormones as well as insulin have been identified as promoting the differentiation of adipocyte precursor cells.

Measurement of the Size and Number of Adipocytes. A variety of methods have been utilized to determine the number and size of adipocytes and a universally accepted measurement regimen has not been established. Measurement of the quantity of DNA in adipose tissue overestimates the number of adipocytes simply because other cells contribute to the measurement. Conventional histologic sections are not acceptable because of cellular retraction during specimen preparation.

With thin sections, the plane of the section passes through several cells but only a few at their maximum dimension. Measurement of cell size on the periphery of a fat lobule underestimates cell size because the peripheral cells are smaller than those near the lobule’s center.

Samples of adipose tissue can be taken at surgery from almost any site, but only from subcutaneous sites with minor surgery in healthy people. Because the latter involves some risk, the needle transcutaneous biopsy technique has been employed under local anesthesia, as has the transcutaneous punch biopsy technique. A common site for taking these subcutaneous samples of adipose tissue is the superior quadrant of the gluteal region, although sites on the upper arm, thigh, abdomen, and upper back have been employed.

Fat cells are isolated from the small quantities of adipose tissue obtained by transcutaneous techniques using digestion with the enzyme collagenase, which breaks down the matrix connecting the cell. Cell separation can be done either before or after tissue fixation (i.e., treatment with substances that modify cellular molecules, holding them together and preventing their decay). Once the fat cells are isolated and appropriately fixed, the average diameter of the cells can be calculated by the direct measurement of a sufficiently large number under the microscope.

The assumption is made that the cells are spherical to calculate a mean cell volume. Mean cell weight can be calculated from mean cell volume assuming the density of the contained lipid to be that of triolein, or 0.915. Alternatively, a cell counter such as a Coulter Counter can be employed using a known volume of a cell suspension. Some investigators have used a thick section (100-200 /лm) of adipose tissue and then have repeatedly focused the microscope to measure individual cell sizes for those cells that remain intact.

The total number of adipocytes in the body can be calculated from the value of the total body fat mass and either the average cell weight or the number of adipocytes per milligram of adipose tissue. Average cell weight varies by subject, site, and measurement technique and ranges from about 0.020 to 0.300 fig of triglyceride per cell. Cell diameters range from about 40 to 160 fim. It should be emphasized that none of these techniques takes preadipocytes into account. Lipid must be present within the cell in identifiable amounts for the cell to be recognized as an adipocyte.