Increased expression of GAPDH protein is not indicative of nitrosative stress or apoptosis in liver of starved rainbow trout (Oncorhynchus mykiss) |
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| Authors: | Bradley L Baumgarner Catherine P Riley Maria S Sepulveda Paul B Brown Jennifer L Meyer Jiri Adamec |
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| Institution: | (1) Department of Forestry and Natural Resources, Purdue University, 195 Marsteller Street, West Lafayette, IN 47907, USA;(2) Purdue Proteomics Facility, Bindley Bioscience Center, Purdue University, 1203 W. State Street, West Lafayette, IN 47907, USA;(3) Department of Biochemistry, University of Nebraska–Lincoln, Beadle Center, N151; 1901 Vine Street, Lincoln, NE 68588, USA;(4) Department of Physiology, University of Kentucky College of Medicine, MS-533 Medical Center, 800 Rose Street, Lexington, KY 40536, USA |
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| Abstract: | Short-term starvation has been linked to in vivo protein degradation in liver of rainbow trout (Oncorhynchus mykiss). However, it is unclear whether this proposed increase in protein degradation is followed by programmed cell death (apoptosis)
in liver of starved trout. A preliminary study in our laboratory revealed an isoform of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) protein that increased 4.5-fold in liver of starved trout. GAPDH is a glycolytic enzyme involved in other cellular
functions, including apoptosis. Increased intracellular nitric oxide (NO) promotes nuclear translocation of GAPDH that is
associated with increased apoptosis in mammals. If GAPDH protein is associated with apoptosis in rainbow trout, it could potentially
be used as a biomarker of cellular stress in liver of teleost fish species. The purpose of this study was to determine whether
increased GAPDH protein expression in liver of starved rainbow trout is associated with NO-induced apoptosis. Targeted proteomic
analysis using multiple reaction monitoring (MRM) was used to determine the level of GAPDH in nuclear and cytoplasmic fractions
and inducible nitric oxide synthase (iNOS) in cell lysates. Dot blot and DNA fragmentation analyses were conducted to evaluate
protein S-nitrosylation and apoptosis, respectively. Results showed that cytoplasmic GAPDH was 3.4-fold higher in liver of
starved versus fed rainbow trout but could not be detected in nuclear fractions. Starvation significantly reduced hepato-somatic
index but had no effect on iNOS protein expression, protein S-nitrosylation, or apoptosis. Our results indicate that starvation
promoted significant reduction in liver mass that was not associated with increased apoptosis or NO-induced stress and that
greater GAPDH concentration in liver of starved rainbow trout was located primarily in the cytoplasm. |
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