Date of Award


Document Type

Open Access Thesis


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School of Medicine

First Advisor

Norma Frizzell


Background. Diabetes is a widespread public health concern that alters the metabolism of adipocytes through high glucose stress and hormonal dysregulation. Under diabetic conditions there are excess nutrients available and subsequently an accumulation of tricarboxylic acid cycle intermediates such as fumarate. Fumarate can irreversibly react with protein cysteine residues to form S-2-succinocysteine (2SC, protein succination). Succinated proteins can be turned over by autophagy, a mechanism of removing damaged proteins and organelles that have accumulated due to metabolic stress.

Purpose. The basal flux through autophagy in adipocytes matured in non-diabetic and diabetic conditions was examined to determine the turnover of succinated proteins. Additionally, fumarase content was reduced using a shRNA lentiviral knockdown to elevate fumarate levels. This model of enhanced succination was used to assess autophagic flux and succinated protein turnover in the total homogenate and enriched fractions of the cytosol, mitochondria, and nucleus.

Methods.3T3-L1 fibroblasts were differentiated to adipocytes then matured in normal or high glucose conditions. Using total cell lysates, I assessed 2SC and autophagic flux via western blotting. Additionally, I knocked down fumarase (Fh k/d) in 3T3-L1 fibroblasts, differentiated them to adipocytes, and evaluated differences in 2SC and autophagic flux both in the total homogenate and in individual cellular fractions.

Conclusions. I conclude that succinated proteins are turned over by autophagy, but that flux through autophagy is reduced under high glucose conditions. Similarly, when succination is enhanced in adipocytes by Fh k/d, autophagic flux is reduced. Despite this, succinated protein turnover by autophagy occurs at a faster rate in the mitochondrial enriched fraction verses other fractions of Fh k/d adipocytes. These results provide insight on how metabolic dysregulation and protein modification may contribute to reduced autophagy under diabetic conditions and confirm the utility of 2SC as a marker for high glucose stress