Investigation of the Functional Roles of Specific Protein Kinase C Isoforms in 3T3-F442A Adipocyte Development and Function

Millar, Iona M (1998) Investigation of the Functional Roles of Specific Protein Kinase C Isoforms in 3T3-F442A Adipocyte Development and Function. PhD thesis, University of Glasgow.

Abstract

It is clear, from a wide range of studies, that the protein kinase C (PKC) family has an important role in the regulation of cell growth, differentiation and function. However, as of yet, relatively little is known about the functions of the individual members of the PKC family in any system. The aim of this study was to investigate the roles of specific PKC isoforms in the regulation of adipocyte development and function using 3T3-F442A cells which differentiate in culture into cells with the charateristics of adipocytes. Initially, the PKC complement of 3T3-F442A cells was thoroughly characterised using a panel of isoform-specific antibodies with application of strict criteria to ensure the appropriate identification of PKC isoforms. The PKC complement of 3T3-F442A adipocytes was found to be identical to that of rat adipocytes. This supported the suitability of using 3T3-F442A cells as a model system of adipocyte development and function. Initially, in order to gain an indication of the functional roles of individual PKC isoforms in the process of preadipocyte differentiation, the temporal changes in cellular levels of PKC isoforms were examined throughout the time-course of differentiation of 3T3-F442A cells. The alpha, gamma and delta isoforms displayed similar temporal patterns of expression during the differentiation of 3T3-F442A cells; all increased rapidly, peaking at day two of differentiation. Subsequently, the expression of these isoforms decreased, resulting in markedly reduced levels in fully differentiated adipocytes as compared to preadipocytes. The expression of PKC s increased steadily during differentiation, resulting in markedly elevated levels in adipocytes. Although expression of PKCmu increased during differentiation, this was attributable to prolonged confluence rather than to the differentiation process per se. No change was observed in the expression of PKCzeta during adipocyte development. That selective changes in PKC isoform expression accompanied the differentiation process in 3T3-F442A cells implied distinct functional roles for PKC isoforms in the regulation of adipocyte development and function. To define the dependence of 3T3-F442A preadipocyte differentiation on PKC isoforms, antisense oligodeoxynucleotides (ODNs) were used to deplete the individual PKC subtypes selectively from 3T3-F442A preadipocytes prior to the induction of differentiation. Each of the ODNs depleted effectively the appropriate PKC isoform and did not affect the expression of other PKC subtypes. By this approach it was demonstrated that PKCzeta is not essential for the differentiation of 3T3-F442A preadipocytes. PKCalpha, delta and zeta each exert an inhibitory influence upon the early stages of adipocyte development and are therefore likely to modulate this stage of the differentiation process. PKCepsilon and PKCgamma are both essential for 3T3- F442A preadipocyte differentiation. Further antisense studies revealed that PKCgamma is necessary for the clonal expansion of differentiating cells. PKCepsilon is not required for clonal expansion but is essential for later stages of the differentiation process, when its expression is markedly elevated, for the attainment of the adipocyte phenotype. (Abstract shortened by ProQuest.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Miles D Houslay
• Keywords: Molecular biology, Developmental biology
• Date of Award: 1998
• Depositing User: Enlighten Team
• Unique ID: glathesis:1998-74703
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Sep 2019 17:04
• Last Modified: 27 Sep 2019 17:04
• URI: https://theses.gla.ac.uk/id/eprint/74703