Preadipocytes, which give rise to new fat cells throughout life, are among the most abundant type of progenitor in the body. Their numbers can exceed those of other cell types in fat tissue, including fat cells (9
). The fat tissue that develops from them is, in turn, at the nexus of processes involved in longevity and the genesis of age-related metabolic disease. Preadipocyte gene expression profiles are distinct from those of fat cells, as is their function (17
). For example, preadipocytes are a greater source of inflammatory cytokines than fat cells and produce chemokines that attract lymphocytes and macrophages (27
). They also process and release paracrine factors and hormones in a fashion distinct from fat cells. Thus, given their numbers and distinct function, they are an important cell type to study in their own right (30
). Unlike many other progenitor types, fat cell progenitors are generally resident in fat tissue (17
), with circulating progenitors making a minor contribution to new fat cell development under certain conditions (31
). Preadipocytes from different fat depots vary in capacities for replication and differentiation. Distinct characteristics of preadipocytes from different depots affect the fat tissue that develops from them, as we showed previously (18
), with different fat depots being effectively distinct mini-organs. Thus, trajectories of age-related changes in preadipocyte function with aging could vary among fat depots, potentially contributing to the fat redistribution and metabolic dysfunction that are common in old age (1
To our surprise, we found that although changes in preadipocyte gene expression patterns do occur with aging, they are considerably less prominent than differences among depots. This finding is consistent with parabiosis experiments, which suggested that tissue microenvironmental changes with aging might have a bigger effect on progenitor function than inherent effects of aging on progenitors themselves (34
). Transcriptional measures of 921 probe sets showed statistically significant differential expression across depots, with developmental genes being prominent among those that vary, consistent with studies in mice and humans (17
). Although more than 8% of genes differed among depots in our array analyses using stringent criteria, only two genes varied with aging using less stringent criteria. Unlike differences among fat depots, aging did not involve substantial changes in developmental gene expression.
Because (a) overall expression measures of preadipocytes from different depots were distinct in the array analyses with respect to 921 probe sets, (b) regional variation in extent of age-related changes in preadipocyte replication and capacity for adipogenesis have been reported (11
), and (c) trajectories of age-related changes in fat depot size, preadipocyte number, and fat cell size and number are depot dependent (6
), we tested the hypothesis that age-related changes in preadipocyte gene expression are depot dependent. We conducted further studies by real-time PCR of genes that appeared to have age-dependent expression changes in the microarray studies, but did not meet our criteria for identification as age-dependent genes in the microarray study. The PCR analyses were done in cells from additional animals and included cells from middle-aged in addition to young and old animals. With the increase in numbers of animals and ages and the greatly reduced multiple comparison correction, the PCR studies uncovered further genes that changed significantly with aging.
The genes whose expression changed with aging in the PCR analyses included cellular stress response–, injury-, and differentiation-related genes. These age-related changes in preadipocyte gene expression were evident, despite culturing cells from the same animals under identical conditions in parallel for a week. This suggests that these changes in gene expression are at least partly inherent, consistent with our previous finding that age-related declines in preadipocyte replicative potential and adipogenesis remain evident in colonies derived from single preadipocytes isolated from rats of different ages, even after a month in culture (13
). Furthermore, the larger changes in perirenal than epididymal preadipocyte gene expression with aging in the current study are consistent with the larger declines in adipogenesis and replicative potential with aging in the perirenal than epididymal clones. The increases in preadipocyte stress response– and injury-related genes and declines in differentiation-related gene expression changes with aging in our study are similar to age-related changes in these gene categories reported in whole fat tissue from old compared with younger mice (35
). Thus, the potentially inherent age-related changes in expression of these gene categories that occur in preadipocytes appear to carry through to the fat tissue that develops from them.
In the cases of genes that changed with aging, expression in middle-aged animals appeared to be intermediate between that of young and old animals, consistent with changes in fat cell progenitor function occurring steadily with chronological aging. Expression of these genes did not correlate with changes in body weight and fat, which increase during maturation (between 3 and 17 months of age) and decline in old age. The progressive nature of the changes in gene expression is consistent with the contention chronological processes, such as progressive accumulation of senescent progenitors or cellular damage, make a major contribution to age-related changes in progenitor function. Indeed, the gene categories involved (increased inflammation decreased differentiation genes with aging) and individual genes (eg, IL-6, Mmp-3) are related to cellular senescence and stress responses.
Importantly, the age-related changes in gene expression were fat depot specific. For example, expression of Stmn-2, Lbp, and Mmp-3 increased markedly in perirenal preadipocytes with aging, but underwent little if any change in epididymal cells. Genes upregulated with aging tended to change to a greater extent in perirenal preadipocytes, whereas those downregulated did so more in epididymal cells. These findings support the contentions that (a) cell-autonomous properties of preadipocytes may contribute to age- and fat depot–dependent changes in adipose tissue growth and function and (b) progenitors from different regions of the same tissue can undergo age-related changes that are distinct.
There were greater increases in stress response, proinflammatory, and matrix-remodeling genes in perirenal than epididymal preadipocytes with aging, as indicated in our PCR studies. This could be related to regional variation in extent of preadipocyte turnover. Larger increases in perirenal than epididymal fat cell numbers occur during maturation (18
). These are associated with greater capacity of perirenal than epididymal preadipocytes for replication and differentiation in young animals (11
). Over a lifetime, more extensive utilization of the perirenal than epididymal preadipocyte pools may lead to greater activation of stress, proinflammatory, and matrix-remodeling responses in older individuals. Alternatively, regional differences in the local microenvironment or abundance of other cell types, such as macrophages, could contribute. In support of this, the already high numbers of macrophages in rat epididymal depots of young animals do not increase further with aging, whereas the lower numbers of macrophages in the inguinal subcutaneous fat depots of young animals do increase (37
). As in subcutaneous fat, macrophage markers increase with aging in perirenal fat tissue fragments (38
). Despite lack of exposure to macrophages for a week in our culture system, possibly macrophages in the vicinity of preadipocytes in vivo impart a persisting effect. Thus, cell autonomous mechanisms and persistent microenvironmental influences could be responsible for depot-dependent age-related changes in preadipocyte function.
Little is known about preadipocyte or fat cell expression of stathmin-like 2 (Stmn-2
; also called Scg-11
). Of all 10,983 transcripts detected in the array study, Stmn-2
increased most with aging. This pattern was also found in the real-time PCR analysis. Stmn-2
is a stress-responsive microtubule-destabilizing protein that regulates neurite outgrowth and differentiation of oligodendrocytes (39
). It is upregulated in response to neural injury, but this becomes attenuated with aging (42
). Given its regionally specific role in regulation of nerve cell differentiation, a role of Stmn-2
in development of different fat depots is possible. Tetranectin, a 68-kDa cell surface protease that regulates mesenchymal development and cell migration, proliferation, and differentiation (43
), also varied with aging in both the array and PCR studies. It might also have a role in fat tissue development, particularly because preadipocytes arise from mesenchymal progenitors.
Both Mmp-3 and Mmp-12 mRNA and protein increased in perirenal preadipocytes with aging, but much less so (or not at all) in epididymal cells. Mmp-3 and Mmp-12 are involved in inflammation and tissue remodeling. Consistent with these increases in Mmp-3 with aging in perirenal preadipocytes, Mmp-3 increases in human skin fibroblasts and mouse subcutaneous fat cells with aging (46
). Mmp-3 is lower in preadipocytes from obese than lean subjects (48
). To date, little information about regional differences in fat tissue Mmp-3 has been available. Fat tissue Mmp-12 expression and activity are increased in obesity and after high fat feeding (49
), as well as in unstable atherosclerotic plaques and invasive cancers (51
), conditions associated with inflammation and tissue remodeling, consistent with the upregulation of preadipocyte proinflammatory genes we found with aging [ (27
Genes involved in early developmental segmentation and patterning were prominent among those that varied among fat depots. These genes, which regulate such fundamental cell dynamic processes as replication, differentiation, and susceptibility to apoptosis, may affect progenitor pool utilization and set the stage for regional variation in trajectories of age-related changes in gene expression. Differing nature and rates of age-related changes in progenitor gene expression profiles could themselves contribute to age-related changes in fat distribution and depot function. Altered fat distribution is a prominent feature of the aging phenotype in humans. Subcutaneous fat begins to decrease in the 70s, whereas intra-abdominal fat decreases later (53
), leading to an effective shift of fat from subcutaneous to intra-abdominal depots, with eventual deposition of fat ectopically in muscle, bone marrow, liver, and elsewhere. This is associated with increased prevalence of metabolic syndrome, particularly after age 70 (4
). It will be interesting to test if the depot-related differences in trajectories of age-related changes in preadipocyte gene expression in rats occur in humans and predispose to fat redistribution and metabolic disease.