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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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.
KeywordsRainbow trout Starvation GAPDH Proteomics Apoptosis
The authors thank Dr. Robert K. Swihart for his tremendous effort in helping to make this project a success. We also thank Jeff Weber and Robert Rode of the Purdue University Aquaculture Research Laboratory for their technical assistance. We are especially grateful to Douglas Sweet of the Ohio Department of Natural Resources London Fish Hatchery for kindly providing the animals for this study. And finally, the authors thank Drs. Ok-Kyong Park-Sarge and Jon J. McCarthy of the University Of Kentucky College Of Medicine for helping to review and edit this manuscript.
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