Adiposity increases as humans' age. Similar phenotypic changes are also observed in FBN hybrid rats as they become old (>26 mo) 
. This increase in age-related fat accumulation in humans 
and rodents is mainly attributed to altered insulin sensitivity, which is a major risk factor for obesity, diabetes and atherosclerosis. Understanding the role of RNA regulatory mechanisms influenced by aging and its impact on age-related diseases is an area of intense investigation. Despite growing evidence in the literature on the role of miRNAs on RNA regulatory mechanisms, its potential role in age-related decline in function has not been well investigated. Alterations in miRNAs have been shown in rodents and humans with senescence or increasing age. MiRNAs such as hsa-let-7f, hsa-miR-499, hsa-miR-373, hsa-miR-372, hsa-miR-371, hsa-miR-369-5p, hsa-miR-34c, hsa-miR-34b, hsa-miR-34a, hsa-miR-29c, hsa-miR-217, and hsa-miR-20a might influence senescence or aging 
. Similarly, miRNAs that influence adipogenesis or osteogenesis pathways have been studied 
. However, no studies have correlated miRNA regulation on adipose tissue function with age.
The aging process independently influences adipose tissue morphology, distribution and function 
. The higher ratio of central versus peripheral fat is attributed to weight gain 
with aging. We recently showed that the gene expression of various adipose derived factors was dramatically altered with age and was dependent on both the source of the fat depot (visceral versus epicardial fat) and gender 
. These fat depot changes observed with age are highly correlated to the dysfunction of the SVF derived stem cells 
. The gene regulation and secretion profiles of SVF derived stem cells also dramatically influence fat tissue function 
The ability to undergo adipogenic, chondrogenic and osteogenic differentiations constitute the elementary capacity of multi-lineage potential of mesenchymal stem cells, including adipose SVF derived stem cells 
. The reciprocal differentiation pathway to adipocyte or osteocyte is switched through activation of either PPARg 
or Runx2 
. These regulators exist in the mesenchymal stem cells and other progenitor cells 
. Moreover, these two differentiation pathways are found to reciprocally inhibit each other 
. Studies have shown that preadipocytes retain their aging phenotype in culture, but are altered in their capacity to undergo adipogenesis and osteogenesis 
. The senescent preadipocytes with lower adipogenic capacity express decreasing levels of C/EBPa, C/EBPd, and PPARg expression 
. Any alterations in preadipocyte gene regulation result in fat redistribution and dysfunction during aging 
. Perturbation in lipid metabolism in preadipocytes enhances lipotoxicity and impairs adipogenesis and lipid oxidation with age 
. These inherent differences in the nature of adipose SVF derived stem cells determine the differences in fat depot function due to overfeeding 
. Regulators involved in phenotypic or functional changes in preadipocytes are not known. Since miRNAs regulate adipogenic and osteogenic pathways, our findings support their role in the aging mediated switch in differentiation capacities of preadipocytes.
Reciprocal induction of miR-143 and its target gene ERK5 plays an important role in adipocyte differentiation 
. The activation of ERK5 enhances and activates PPARg through the interaction of the hinge-helix 1 region of PPARg and ERK5 
. In our study, there was a down-regulation of miR-143 levels post adipogenesis, in adipose SVF derived cells isolated from older rats compared to those from younger rats. In addition, the gain or knockdown of miR-143 only altered ERK5 mRNA levels in adipose SVF derived cells from younger rats, but not from 30 mo old rats, with very little change in ERK5 protein expression in adipose SVF derived cells from both groups of rats, suggesting a dysregulation of miR-143 with age. ERK5 is responsible for insulin induced adipogenesis 
. The observed increase in ERK5, PPARg, ap2 and adiponectin levels in 6 mo old rat adipose SVF derived cells post-adipogenesis, but not in older rats supports the active role of ERK5 during adipogenesis in young cells. However, although miR-143 inhibited ERK5, the levels of the adipogenic factors were increased after adipogenesis in adipose SVF derived cells from young rats. This may possibly be due to a negative feedback regulation by the adipogenic factors to maintain differentiation. Increases in miR-143 levels by premir transfection enhanced the adipogenic differentiation capacity of young adipose SVF derived cells with a concomitant increase in PPARg and miR-204.
Our data also showed that adipose SVF derived cells from 30 mo rats expressed significantly higher Runx2 mRNA level, which may indicate the phenotypic switch during aging in adipose SVF derived cells. Moreover, miR-204 was activated post-adipogenesis in 6 mo SVF derived cells to inhibit Runx2; however, this process was not seen in older rats, resulting in increased levels of Runx2 in these cells. Similarly, a failure of PPARg induction was also observed in the 30 mo cells after adipogenic induction. This supports the hypothesis that miRNA mediated adipogenic differentiation was impaired in aging preadipocytes which was accompanied by an inability of adipogenic miR-204 to suppress RunX2 and other factors (PPARg).
Interestingly, it seems like aging utilizes opposing mechanisms to influence progenitor cell function in adipose tissue and bone marrow. Contrary to what was observed in this study, the mesenchymal stem cells in bone marrow were found to exhibit activated adipogenic but suppressed osteogenic capacity, thus contributing to osteoporosis in seniors 
. In addition, it is possible that the differences in CD markers (switch in number of CD90+versus CD34+cells) in young versus old SVF derived cells might play a role in the phenotypic switch observed in our studies with aging.
Our findings suggest a new regulatory mechanism in adipose function during the aging process. An impairment of adipogenic program accompanied with an unregulated osteogenic program, influences preadipocyte perturbation in the aging process. This phenomenon correlated with the impairment of the miRNA regulatory pathway. Moreover, unresponsiveness to miRNA intervention by the adipose SVF derived cells from old rats (30 mo) suggested that miRNA dysregulation contributed to the pathological processes observed in certain tissues during aging. This dysregulation might be attributed to epigenetic alterations in both miRNA and its target messenger RNA (mRNA), alterations in the proteins involved in miRNA biogenesis or due to alternate regulatory phenomenon such as miRNA editing.