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GENETIC MANIPULATION OF RECEPTOR INTERACTING PROTEIN (RIP140)
UNCOVERS ITS CRITICAL ROLE IN THE REGULATION OF METABOLISM,
GENE EXPRESSION AND INSULIN SIGNALING IN SKELETAL MUSCLE CELLS
by
Silvana Constantinescu
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(INTEGRATIVE AND EVOLUTIONARY BIOLOGY)
May 2012
Copyright 2012 Silvana Constantinescu
Object Description
| Title | Genetic manipulation of receptor interacting protein (RIP140) uncovers its critical role in the regulation of metabolism, gene expression and insulin signaling in skeletal muscle cells |
| Author | Constantinescu, Silvana |
| Author email | sctinescu@gmail.com;sconstan@usc.edu |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Integrative and Evolutionary Biology |
| School | College of Letters, Arts And Sciences |
| Date defended/completed | 2012-01-10 |
| Date submitted | 2012-03-30 |
| Date approved | 2012-04-02 |
| Restricted until | 2014-03-30 |
| Date published | 2014-03-30 |
| Advisor (committee chair) | Turcotte, Lorraine |
| Advisor (committee member) |
McNitt-Gray, Jill Wong, Hung |
| Abstract | Oxidative capacity is commonly assessed by measuring the content and the activity of key mitochondrial enzymes and there is evidence suggesting that oxidative capacity is regulated by multiple regulatory factors which include among others, the actions of positive and negative nuclear factors on the transcriptional regulation of oxidative enzymes. Of specific interest is the role played by the nuclear co-repressor identified as Receptor Interacting Protein 140 (RIP140). RIP140 has been shown to be highly expressed in skeletal muscle and to inhibit mitochondrial biogenesis and oxidative capacity. In line with its role as a negative regulator of oxidative capacity, there is evidence suggesting that in adipose tissue RIP140 deletion increases cellular respiration and protein expression of cytochrome c. However, the role of RIP140 on fatty acid (FA) metabolism had not been fully delineated, especially as it relates to insulin sensitivity in skeletal muscle cells. ❧ Thus this dissertation project contains two experiments aimed at determining the role of low RIP140 expression in the regulation of basal and insulin-mediated FA metabolism in skeletal muscle cells under normal or short-term high FA treatment. Given the putative role of RIP140 as a negative regulator of oxidative capacity, the purpose of these studies was to determine in skeletal muscle cells 1) whether genetic down-regulation of RIP140 expression would increase oxidative capacity and improve insulin sensitivity in normal cells and cells exposed to short-term high FA treatment and 2) whether metabolic alterations would be associated with alterations in the mRNA and/or protein content of enzymes and proteins involved in metabolic regulation and signaling under both conditions. To accomplish our aims, we used L6 rat skeletal muscle cells and assessed insulin sensitivity by measuring the effects of insulin on glucose uptake and FA uptake and oxidation under normal and short-term high FA treatment. ❧ The findings in the first study provide novel information regarding the role of RIP140 in the regulation of FA metabolism and implicate the AKT-PKC zeta axis of the insulin signaling pathway in the high rates of insulin-mediated FA oxidation observed in L6 cells with low RIP140 expression. Multiple studies have shown that a rise in the activity of mitochondrial oxidative enzymes is often, though not always, accompanied by an increase in FA oxidation. However, our data showed that, under control conditions in skeletal muscle cells, while the protein or mRNA content of some oxidative enzymes (COX4) was increased, the content of other important FA oxidative enzymes (MCAD, CPT1) was reduced. Conversely, the activity of proximal insulin signaling intermediates was reduced. Given that the inhibitory action of insulin on FA oxidation was similarly reduced in RIP140-treated cells, our data suggest that proximal insulin signaling is critical for proper regulation of FA metabolism and that low RIP140 expression affects the activation of these signaling intermediates. ❧ Data from the second study provide further evidence for the involvement of RIP140 in the regulation of FA metabolism and metabolic signaling. In this study, L6 cells with or without low RIP140 expression were incubated with high FA for 36 h. Our results showed that high FA exposure alone affected the mRNA and/or protein expression of several proteins involved in the regulation of FA metabolism including FA transport proteins and oxidative enzymes. In muscle cells incubated with high FA, low RIP140 expression rescued basal FA uptake and basal and insulin-mediated glucose uptake, but it did not affect basal or insulin-mediated FA oxidation. In line with these metabolic alterations, low RIP140 expression was able to rescue some of the changes induced by high FA exposure via alterations in the activation state of signaling intermediates (e.g.: AKT) or changes in mRNA and/or protein expression of specific FA transporter proteins (e.g.: FAT/CD36) or oxidative enzymes (e.g.: CPT1). Taken together, our data suggest that low RIP140 expression may partially mitigate the negative impact of short-term FA exposure on metabolic regulation and cellular signaling. ❧ In summary, our results provide additional information regarding the role of RIP140 in the regulation of FA metabolism, gene expression and most importantly in metabolic signaling in skeletal muscle cells. More studies will be needed in order to decipher the cellular mechanisms that regulate the RIP140-mediated cellular changes observed in our experiments. |
| Keyword | metabolism; RIP140; skeletal muscle; L6 cells |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m |
| Contributing entity | University of Southern California |
| Rights | Constantinescu, Silvana |
| Access conditions | The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given. |
| Repository name | University of Southern California Digital Library |
| Repository address | USC Digital Library, University of Southern California, University Park Campus MC 7002, 106 University Village, Los Angeles, California 90089-7002, USA |
| Repository email | cisadmin@usc.edu |
| Archival file | uscthesesreloadpub_Volume3/etd-Constantin-558.pdf |
Description
| Title | Page 1 |
| Contributing entity | University of Southern California |
| Full text | GENETIC MANIPULATION OF RECEPTOR INTERACTING PROTEIN (RIP140) UNCOVERS ITS CRITICAL ROLE IN THE REGULATION OF METABOLISM, GENE EXPRESSION AND INSULIN SIGNALING IN SKELETAL MUSCLE CELLS by Silvana Constantinescu A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (INTEGRATIVE AND EVOLUTIONARY BIOLOGY) May 2012 Copyright 2012 Silvana Constantinescu |
