Master of Science (MS)
Semester of Degree Completion
Michael A. Menze
Galactose is a simple sugar that at supraphysiological concentrations accelerates aging and age-related complications, which lead to impaired mitochondrial functions. MitoNEET is a small mitochondrial membrane protein with a molecular mass of 12.2 kDa that functions in diabetes, iron metabolism, regulation of oxidative phosphorylation (OXPHOS) and reactive oxygen species (ROS) homeostasis. MitoNEET knockdown increases cellular respiration rates and ROS levels similar to galactose treatment. Pioglitazone, an antidiabetic drug, binds to mitoNEET and ameliorates galactose toxicity. Cellular mitoNEET levels, exposure of cells to galactose medium, and pioglitazone treatment directly influence cellular respiration. To elucidate the role of mitoNEET in galactose induced toxicity (aging), we measured cellular mitoNEET levels using immunoblotting technique in galactose and pioglitazone treated human liver cancer cells (HepG2), as well as isolated synaptosomes from old and young mice. We also monitored liver cancer cell bioenergetics, using respirometry and calorimetry, of galactose and pioglitazone treated cells. Immunoblotting revealed treatment with galactose reduced endogenous mitoNEET levels and those of a chimeric mitoNEET protein fused to a green fluorescent protein (mNT-GFP), under control of the human cytomegalovirus (CMV) promotor. Furthermore, to determine whether mitoNEET mediates pioglitazone ameliorated galactose toxicity, cells were treated with pioglitazone. Pioglitazone rescued galactose reduced mitoNEET levels in a dose dependent manner. A concentration of 60 μM pioglitazone reduced cellular mitoNEET levels compared to controls but not mNT-GFP levels. However, pioglitazone at lower concentrations partially restored mitoNEET levels observed with galactose treatment. Since the synthesis of endogenous mitoNEET and mNT-GFP are regulated by different promoters, D-galactose treatment likely increased degradation rates of mitoNEET. Moreover, unlike galactose treatment in HepG2 cells, immunoblotting revealed higher mitoNEET levels in synaptosomes isolated from aged mice compared to their younger counterparts. We hypothesized that galactose and pioglitazone interact with mitoNEET and alter cellular bioenergetics. Acute treatment with pioglitazone significantly reduced complex I respiration in HepG2 and HepG2-mNT-GFP cells independent of treatment with galactose. Surprisingly, galactose treated HepG2-mNT-GFP cells were less sensitive to pioglitazone treatment. HepG2 cells treated with galactose and HepG2-mNT-GFP cells independent of treatment showed higher OXPHOS activity compared to controls in absence of galactose, but HepG2-mNTGFP cells did not respond to galactose treatment. Unlike respiration, galactose treatment significantly reduced cellular heat flow in HepG2 and HepG2-mNT-GFP cells measured via calorimetry. However, HepG2-mNT-GFP and pioglitazone treated cells showed higher heat dissipation compared to control HepG2 cells without galactose. Our results show that mitoNEET overexpression increases respiration rates and overall energy transduction in HepG2 cells but does not impact the response to pioglitazone treatment.
Paudel, Sudip, "D-Galactose Decreases MitoNEET Levels in Liver Cancer Cells: Impact on Cellular Bioenergetics" (2015). Masters Theses. 2384.