Molecular analysis of starvation in rainbow trout

Nutrient deprivation, or starvation, is a commonly used intervention when analyzing the metabolic and physiologic capabilities of model organisms and tissues. Unfortunately, little information is available concerning the effects of starvation on physiology and nutrient metabolism in teleost fishes. Previous studies have employed such methods as enzyme activity assays and DNA micro arrays to assess the affect of starvation on muscle protein catabolism and metabolic fuel utilization in rainbow trout (Oncorhynchus mykiss). However, results from previous studies tend to differ regarding activation of proteolytic mechanisms and utilization of metabolic energy reserves, in particular the contribution of muscle protein to hepatic gluconeogenesis. This is likely due to the complex nature of nutrient metabolism and physiology of an obligate carnivorous fish species such as rainbow trout. I conducted a series of experiments to determine the effect of starvation on nutrient metabolism and physiology in rainbow trout using advanced proteomic and metabolomic analysis. Global proteomic and metabolomic analysis was conducted on serum, liver, muscle, gut intestinal tract (GIT), and pyloric ceca (PC) tissue from satiation fed (fed), half-satiation fed (half-fed), and starved rainbow trout. Proteomic analysis was performed using two-dimensional gel electrophoreses (2-DE) and matrix assisted laser desorption time-of-flight/time-of-flight (MALDI-TOF/TOF). Metabolomic analysis was conducted via two-dimensional gas chromatography/time of flight-mass spectrometry (GCxGC/TOF-MS). Proteomic analysis identified sixteen proteins that were significantly expressed (p \u3c 0.05) between treatments. Energy metabolism and stress-related enzymes accounted for the majority of these. Notably, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was variably expressed between treatments in liver and muscle tissue. A single isoform of GAPDH was determined to be significantly greater in liver of starved vs. fed fish. In contrast, three isoforms of GAPDH were significantly expressed in muscle tissue with two being significantly increased in fed and starved vs. half-fed fish. Stress-related proteins were expressed primarily in GIT and PC. Alpha-I-antiproteinase was significantly greater in GIT of half-fed vs. starved trout. Leukocyte elastase inhibitor was significantly greater in PC of fed and half-fed vs. starved trout. Also, copper/zinc-superoxide dismutase was significantly greater in PC of fed vs. starved individuals. Metabolomic analysis revealed that levels of gluconeogenic amino acids were significantly greater (P \u3c 0.05) in serum of fed and half-fed vs. starved fish. Fatty acids levels tended to vary greatly with tissue and diet. Octadecanoic acid was significantly greater in liver of fed vs. starved fish while being significantly greater in muscle of starved vs. fed fish. Other significantly different fatty acids included tetradecanoic acid, which was significantly greater in serum of fed vs. starved trout, but significantly greater in muscle of starved vs. fed trout. In addition to global analysis I conducted a follow-up study to determine if the significant increase in GAPDH protein expression in liver of starved vs. fed rainbow trout was associated with nitric oxide (NO)-induced apoptosis. Eleven fish were fed to apparent satiation once daily while another eleven fish were starved for the duration of a 4 wk trial. Nuclear and cytoplasmic fractions as well whole-cell lysates were processed from liver tissue of all fish. Cell fractions were subjected to targeted proteomic analysis using absolute quantitative analysis (AQUA) and multiple reaction monitoring (MRM). Nuclear and cytoplasmic fractions analyzed for expression GAPDH and cell lysate samples were analyzed for inducible nitric oxide synthase (iNOS) expression. Also dot blot and DNA fragmentation analysis were conducted to determine the level of protein nitrosylation and apoptosis, respectively. My data indicated that starvation promoted a significant decrease in hepato-somatic index ([liver weight/body weight]*100) but did not increase apoptosis. Also, starvation had no significant effect on iNOS expression or protein S-nitrosylation. Cytoplasmic GAPDH was 3.4-fold higher in liver of starved vs. fed rainbow trout. In summary, my results indicate that starving rainbow trout for 4 wk resulted in greater susceptibility of intestinal epithelia to physical and oxidative damage but did not promote increased muscle protein catabolism. This was confirmed by greater gluconeogenic amino acid level in serum of fed vs. starved fish. Also, varying tissue fatty acids levels indicate selective utilization of specific tissue long-chain fatty acids in food-deprived fish. Finally, starvation did not promote increased cellular NO-induced apoptosis in rainbow trout. Overall, my results indicate that starving rainbow trout for 4 wk promoted only minor physiologic and metabolic changes that were not associated with increased tissue protein catabolism or hepatic gluconeogenesis