Date of Award

8-9-2014

Document Type

Open Access Thesis

Department

Biological Sciences

First Advisor

Norma Frizzell

Abstract

We previously identified the chemical modification S-(2-succino)cysteine (2SC), which is formed when the Krebs cycle metabolite fumarate reacts with protein cysteine residues, also termed protein succination. Protein succination is increased in the adipose tissue of ob/ob and db/db mice in vivo and in 3T3-L1 adipocytes grown in high glucose in vitro. The increase in 2SC in the 3T3-L1 adipocyte occurs as a direct result of glucotoxicity and increased mitochondrial stress. We have shown that uncoupling agents, which lower mitochondrial stress, prevent the increase in succinated proteins.

In this study we examined the relationship between increased succination and mitochondrial bioenergetics in adipocytes matured in 30 mM (high) vs. 5 mM (normal) glucose. In addition we examined if sodium phenylbutyrate (PBA), which lowers protein succination, might be acting as an uncoupling agent in adipocytes cultured in high glucose. We observed that adipocytes matured in high glucose had a decreased spare respiratory capacity, increased proton leak across the inner membrane and increased non-mitochondrial respiration, consistent with increased mitochondrial stress. We also determined that PBA was not acting as an uncoupling agent but instead appeared to lower the mitochondrial respiratory protein content, thereby reducing respiration and protein succination.

The unfolded protein response (UPR) and endoplasmic reticulum (ER) stress have previously been documented in the adipose tissue during diabetes and during adipocyte maturation in high glucose. We assessed the development of ER stress in adipocytes matured in 5 mM vs. 30 mM glucose and observed that although most ER stress markers were unchanged with glucose concentration, the terminal ER stress marker CHOP was consistently elevated in high glucose and this occurred in parallel with increased 2SC.

The use of PBA as a therapeutic agent for the treatment of T2DM is currently being investigated in human subjects as it has been shown to reduce ER stress in the adipose tissue of animal models of T2DM. We examined the effects of PBA administration for 8 weeks in db/db mice. We observed reduced serum glucose and reduced triglyceride (TG) levels after PBA treatment; however, there was no effect of PBA on 2SC or ER stress markers in the adipose tissue of db/db mice. Unexpectedly, PBA treatment increased ER stress and TG deposition in the liver of db/db mice.

Considering that 2SC is an irreversible and stable protein modification we wanted to determine if succinated proteins could be degraded intracellularly. We demonstrated that succinated proteins are degraded by the lysosome and are released from the cell as a free amino acid (2SC) and bound in a peptide.

Overall, these studies have further extended our knowledge of the relationship between mitochondrial stress, protein succination and ER stress. In addition, we have determined the mechanism of turnover of succinated proteins in the adipocyte in vitro.

Rights

© 2014, Ross Tanis

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