Effects of fat depot site on differentiation-dependent gene expression in rat preadipocytes.

Variability in physiological characteristics among adipose depots could be caused, in part, by mechanisms intrinsic to cells comprising the depots. To evaluate the contribution of intrinsic mechanisms to depot variability, we studied levels of differentiation-dependent mRNAs in differentiating cultured rat epididymal and perirenal preadipocytes and in fat cells isolated from these depots. The magnitude of the change in levels of adipsin and glycerol-3 phosphate dehydrogenase mRNAs, which increase late during differentiation, was greater in perirenal than epididymal preadipocytes. The magnitude of the change in beta-actin mRNA, which decreases early during differentiation, was not site-dependent. Effects of anatomic site on changes in differentiation-dependent mRNAs observed in differentiating preadipocytes in vitro were similar to effects of site on these mRNAs in freshly isolated fat cells: those mRNA species whose levels increase late during preadipocyte differentiation were present in greater abundance in perirenal than epididymal fat cells. Hence, mechanisms which underlie site-dependent variability in adipose function may be intrinsic and could become evident midway through the differentiation process.

Increasing vimentin expression associated with differentiation of human and rat preadipocytes.

Alterations in the cytoskeletal apparatus constitute some of the earliest changes during assumption of an adipogenic phenotype. We examined three major cytoskeletal elements, beta-actin, alpha-tubulin and vimentin, during adipogenesis in euploid cells from human and rat adipose tissue. As reported with 3T3 sub-lines, mRNA level for beta-actin and alpha-tubulin were decreased upon differentiation. However, in contrast to reports with 3T3 cells, levels of vimentin were increased during differentiation. Furthermore, immunological analyses confirmed that there was no decrease in vimentin protein levels during adipogenic development. As well as highlighting a difference between 3T3 cell lines and preadipocytes isolated from fat depots, these studies indicate that the pattern of cytoskeletal gene expression undergoes complex changes early during preadipocyte differentiation.

Effects of age and anatomic site on preadipocyte number in rat fat depots.

Preadipocytes are cells which are capable of either replication or of differentiation into fat cells. As with other stem or progenitor cell types, the replicative capacity of preadipocytes declines with increasing age; however, little information about effects of age on preadipocyte cell number in vivo is available. We determined preadipocyte number in the perirenal and epididymal fat depots of 3-, 17-, and 27-month-old Fischer 344 rats in 23 experiments. Increasing age was not associated with a decrease in preadipocyte number; indeed, the number of preadipocytes increased in epididymal depots throughout maturation and senescence. Hence, the tenet that aging causes a decline in the size of stem cell or progenitor pools is not generalizable to all tissues, even if the cells exhibit reduced replicative capacity in culture.

Ageing, differentiation, and gene expression in rat epididymal preadipocytes.

Ageing results in decreased replicative potential of preadipocytes, as well as reduced capacities for the lipid accumulation and increases in lipogenic enzyme activities during differentiation of preadipocytes into fat cells. To determine whether decreased differentiation is associated with decreased levels of mRNA for differentiation-dependent genes and whether early as well as late components of the differentiation programme are affected by ageing, we measured beta-actin, alpha-tubulin, lipoprotein lipase, and glycerol-3-phosphate dehydrogenase mRNA levels in undifferentiated and differentiated epididymal preadipocytes from 3-, 17-, and 24-month-old Fischer 344 rats. During ageing, diminished differentiation-related changes occurred in mRNAs affected early (actin, tubulin), midway through (lipoprotein lipase), and late (glycerol-3-phosphate dehydrogenase) in the preadipocyte differentiation process. Hence, early as well as late phases of the differentiation programme were affected by ageing. The effects involved changes in gene transcription or mRNA processing. Our results were not consistent with the hypothesis that age-related decreases in replication are caused by an increased tendency for cell differentiation.

Effects of aging on ribosomal protein L7 messenger RNA levels in cultured rat preadipocytes.

Ribosomal protein L7 mRNA is a cell cycle-independent message whose levels are lower in late passage "senescent" fibroblasts than early passage cultures. To determine whether decreases in L7 mRNA levels also occur during aging in tissues in vivo and whether reduced L7 mRNA is caused by terminal differentiation, we measured L7 and adipsin (a differentiation-dependent serine protease) mRNA levels in undifferentiated and differentiated preadipocytes and glyceraldehyde-3-phosphate dehydrogenase mRNA in differentiated preadipocytes cultured from perirenal fat depots of 3-, 17-, and 24-month-old male rats. L7 mRNA levels decreased with increasing age and were not affected by differentiation. In the same cultures, adipsin mRNA levels did not increase with age but did increase with differentiation, confirming that the preadipocytes exposed to enriched medium had, in fact, differentiated. Glyceraldehyde-3-phosphate dehydrogenase mRNA levels did not change with age indicating that the decrease in L7 mRNA was not a result of a general decrease in mRNA with age. These observations are consistent with the hypotheses that decreasing L7 mRNA levels are associated with aging and that late passage fibroblasts have features in common with senescence. The observations are not consistent with the hypothesis that senescent changes in cellular function are caused by terminal differentiation.

Two preadipocyte subtypes cloned from human omental fat.

To determine whether the characteristics of preadipocytes derived from human fat are uniform or variable, we developed methods for culturing and differentiating cloned human preadipocytes. Individual human omental preadipocytes were cultured for six weeks. The number of cells varied considerably among clones derived from the same subject, implying that human preadipocytes vary in replicative capacity. Indeed, two cell subtypes were found in human omental fat; one type replicated slowly and the other was capable of extensive replication. Cells of both subtypes were capable of differentiation into adipocytes, confirming that both subtypes were preadipocytes. When rat perirenal and epididymal preadipocytes were cloned, a slowly replicating and an extensively replicating preadipocyte subtype were also found. It is proposed that preadipocytes of the rapidly and the slowly replicating subtypes may be at different stages along the pathway between uncommitted precursor cells and differentiated adipocytes.

Age, anatomic site, and the replication and differentiation of adipocyte precursors.

The effects of donor age and anatomic site on cellular replication and differentiation were studied in adipocyte precursors cloned from epididymal and perirenal depots of young, middle-aged, and senescent rats. As animals aged from 3 to 29 mo, there was a progressive reduction in the proportion of cells capable of extensive replication in both depots. An inverse relation between clonal capacity for replication and differentiation was found. This relation was affected by donor site but not age. Aging was, however, associated with a reduction in the frequency of clones capable of full differentiation into cells with single, large, central lipid inclusions. Hence, age and donor site may affect adipocyte precursor replication and differentiation by different mechanisms.