A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirement for the degree of Doctor of Philosophy.
Johannesburg, 2004Alkaline phosphatase (ALP) is a ubiquitously expressed enzyme, that has been shown to play a role in cell differentiation and organogenesis. One study has also demonstrated ALP activity in rat adipocytes. The purpose of the present study was therefore to determine whether ALP is expressed in preadipocytes and what role it may have in adipogenesis. ALP activity was detected in the murine preadipocyte cell line, 3T3-L1, and in human preadipocytes isolated from mammary tissue, and from subcutaneous abdominal fat depots. In all the cell types studied ALP activity increased in parallel with adipogenesis. In the 3T3 -L1 cell line the tissue- non -specific ALP inhibitors, levamisole and histidine inhibited ALP activity, and adipogenesis, whereas the tissue specific ALP inhibitor Phe- Gly-Gly did not inhibit ALP or adipogenesis.
In human preadipocytes, histidine inhibited adipogenesis and ALP activity,
whereas levamisole inhibited adipogenesis, but did not block ALP
activity in intact cells. However, levamisole did inhibit ALP activity by 50% in cell
extracts. Levamisole was able to inhibit adipogenesis in human
preadipocytes. The tissue specific ALP inhibitor, Phe Gly Gly, did not
inhibit ALP activity or adipogenesis in human preadipocytes.
ALP activity and adipogenesis, were compared in preadipocytes isolated from mammary tissue taken from black (13) and white (15) female subjects. Both ALP activity and adipogenesis, were lower in white compared to black female subjects.
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Immunocytochemical, analysis of the 3T3-L1 cell line and human preadipocytes demonstrated that ALP activity was restricted to the lipid droplets of these cells.
ALP activity was also measured in serum samples obtained from 100 African subjects (74 females and 26 males) of varying BMI. ALP activity was found to be higher in obese than lean subjects, whereas, the other liver enzymes or products measured in serum were not. In fact these variables correlated to varying degrees with waist-hip ratio, whereas ALP levels did not. This suggest that liver function is predominantly influenced by abdominal obesity whereas serum ALP levels are more influenced by overall body adiposity.
In conclusion, ALP, may be involved in the control of adipogenesis, in the 3T3- L1 preadipocyte cell line and in human preadipocytes isolated from mammary adipose tissue and subcutaneous abdominal adipose tisssue. The presence of ALP activity in lipid droplets in 3T3-L1 cells and human preadipocytes, and the ability of ALP inhibitors to block adipogenesis strongly suggest that ALP plays a role in the control of adipogenesis.IT201
