Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Major/Program

Biochemistry

First Advisor's Name

Alejandro Barbieri

First Advisor's Committee Title

Committee chair

Second Advisor's Name

Fernando Noriega

Second Advisor's Committee Title

Committee member

Third Advisor's Name

Lou W. Kim

Third Advisor's Committee Title

Committee member

Fourth Advisor's Name

Xiaotang Wang

Fourth Advisor's Committee Title

Committee member

Keywords

Rab5, Adipogenesis, GEF, GAP, Insulin signaling pathwhay

Date of Defense

6-25-2018

Abstract

The formation of adipocytes is a complicated process in which insulin and IGF-1 signaling pathways and numerous transcription factors control the conversion of precursor cells to mature fat cells. The Rab5 protein acts as a rate-limiting protein during receptor-mediated endocytosis by switching between a GDP-bound inactive form and a GTP-bound active form. The inactivation and activation of Rab5 are regulated by several Rab5 GTPase activating proteins (GAPs) and Rab5 guanine nucleotide exchange factors (GEFs), respectively.

This dissertation demonstrated that the activity of the small GTPase Rab5 and its regulators are essential for the differentiation of 3T3-L1 pre-adipocytes. Specifically, it showed that Rab5 activation is detrimental to the differentiation process. The overexpression of a dominant-negative Rab5:S34N mutant, but not an active counterpart (Rab5:Q79L), stimulated the differentiation of 3T3-L1 pre-adipocytes. Consequently, the expression of Rab5:S34N increased the expression of two adipogenic-specific transcriptional factors, PPARγ and C/EBPα. siRNA-mediated depletion of Rab5 inhibited the differentiation of 3T3-L1 pre-adipocytes, providing further evidence for the requirement of Rab5 in the process of adipogenesis.

A dramatic decrease of the Rab5-GTP level is also observed during the differentiation of 3T3-L1 pre-adipocytes. Consistent with these observations, I found that the expression of Rab5 GEFs (i.e., RIN1, Rabex-5, and RAP6), which increased the GTP-bound form of Rab5, blocked the differentiation process. In contrast, the expression of Rab5 GAPs (i.e., RN-tre and RabGAP-5), which decreased the GTP-bound form of Rab5, stimulated differentiation of 3T3-L1 pre-adipocytes.

I also found a novel interaction between the VPS9 domain of the Rab5 GEFs and the activated insulin receptor. This interaction is specific since the VPS9 domain did not interact with the catalytic inactive mutant of the insulin receptor and the Rab5 GAPs (no VPS9 domain) did not bind to the activated insulin receptor.

The data point out that a reduction on the GTP-bound form of Rab5 is required for the rapid differentiation of 3T3-L1 pre-adipocytes, identifying Rab5 inactivation as an important contributor of adipogenesis. Also, these observations suggest a novel cellular mechanism of Rab5 activity in the adipogenesis process in connection with the insulin receptor, the Rab5 GAPs, and the Rab5 GEFs.

Identifier

FIDC006840

ORCID

https://orcid.org/0000-0002-8736-7002

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