| Abstract |
Even though obesity is one of the major health care challenges of the 21st century, there is still a huge gap in our knowledge regarding the fundamental biology of mature adipocytes. The fact that mature adipocytes are categorized as post-mitotic cells is a deep-rooted notion in the scientific literature that has never been challenged directly but a handful of times. Thus, the aim of this study was to investigate the cell cycle in human mature adipocytes and the physiological and pathological effects it has in the biology of the tissue.
In the first part of the study we investigated whether mature human adipocytes can re-enter cell cycle in vivo and in vitro. Using immunocytochemistry and immunohistochemistry techniques, it was shown that human primary adipocytes were able to express several cell cycle markers spanning from G1 to G2 phase. These results were confirmed in vitro in a modified culture environment of isolated human mature adipocytes. In this model adipocytes were found to re-enter cell cycle, as suggested by the elevation of cyclin D1 nuclei levels. Moreover, the EdU nucleotide incorporation into cells’ DNA indicated that the adipocytes were able to pass through S-phase. These phenomena were augmented when insulin was added to culture medium which is proof for insulins mitogenic effects in mature adipocytes.
Despite cell cycle re-entry, however, there is no indication that adipocytes can go through cytokinesis. This might suggest that what is really happening is an alternative to cell cycle, called endoreplication which is responsible for development of polyploid cells. In order to investigate the possibility of adipocyte polyploidy we developed and improved qualitative techniques for staining of adipocytes’ nuclei and quantitative techniques, like the staining of Barr body, as a second part of the study. In addition to this, a new flow cytometry analysis method was proposed, that would allow for the simultaneous investigation of adipocyte hypertrophy (hallmark of polyploid cells) and other cell markers. If validated, these techniques can be applied in future studies of adipocyte polyploidy and hypertrophy.
In addition to possible polyploidy, the fact that mature adipocytes are able to re-enter cell cycle without finishing it has the implication that mature primary adipocytes might develop senescence. Senescence could potentially act as a unifying theory to explain the hypertrophic, pro-inflammatory and insulin resistant characteristics of “unhealthy adipocytes”. In the third part of the study we investigated the development and the factors leading to adipocyte senescence in vivo and in culture, as well as the role of cell cycle re-entry as a condition for senescence. When isolated human primary adipocytes were stained for senescence associated β-Galactosidase (SA-β-Gal), some of the adipocytes were found positive. Parallel to SA-β-Gal staining, two more known markers of senescence were used, the increased size of SA-β-Gal positive cells and their correlation with HMGB-1 nuclear absence, which established the presence of a senescent phenotype in the adipocytes. More importantly, the percentage of SA-β-Gal positive adipocytes was found to have a true physiological effect, as it was shown to correlate with the metabolic profile of adipose tissue donors in a cohort of 51 patients. In addition, in vitro experiments revealed that the presence of insulin, which previously led to cell cycle re-entry, increased senescence. The use of palbociclib that arrests cell cycle progression, also, augmented senescence, while metformin, which restricts cell cycle entry and inhibits the mTOR mitogenic signals, reduced senescence.
Finally, in order to investigate the heterogeneity between adipocytes, which might be responsible for leading some cells into senescence while protecting others from it, the insulin receptor expression was studied in human mature adipocytes, since insulin had such a pivotal role in cell cycle and senescence. Using flow cytometry the presence of an insulin receptor negative adipocyte population and its correlations to the metabolic profile of 14 patients was investigated, yielding however inconclusive results for the time being.
In conclusion, mature human adipocytes were found to be able to re-enter cell cycle which is an important condition for the development of senescence. Parallel to those, this study provides new methods for the investigation of adipocyte ploidy and hypertrophy. Finally, a new adipocyte population of insulin receptor negative cells was discovered and its correlation to patients’ metabolic profile was investigated. Future studies can address how these new biological processes affect adipose tissue and human health.
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