Glucose uptake and glucose transporters in the rainbow trout (Oncorhynchus mykiss) ventricle

My dissertation examines glucose uptake and glucose transporters in a novel experimental model: male and female rainbow trout (Oncorhynchus mykiss) ventricles.;One of the questions that I addressed was the effect of hypoxia on glucose uptake. It has been shown in both mammalian striated muscle and fish cardiac muscle that hypoxia can increase glucose uptake via GLUT type glucose transport proteins. Given the structure of the rainbow trout cardiovascular system and the potential for low oxygen levels in its environment, we hypothesized that hypoxia may stimulate glucose uptake. We addressed this question using the glucose analog 2-deoxyglucose (2-DG) to measure the uptake of glucose into the ventricle tissue. These experiments also incorporated cytochalasin B (CB), a specific GLUT type glucose transporter inhibitor. Interestingly, CB significantly decreased glucose uptake approx. 60% (P < 0.001), however hypoxia did not increase glucose uptake above the oxygenated controls. Additional experiments showed that hypoxia increased lactate efflux approx. five-fold (P < 0.001) over oxygenated controls. Interestingly, both glycogen and free glucose were significantly less in males (P < 0.0001; n = 3-5 for each sex) for all conditions with no treatment effects found in the 2-way ANOVA analysis.;The results from the 2-DG uptake experiments with CB supported the existence of GLUT type glucose transporter proteins in the ventricle tissue. In order to verify this, we commissioned the production of a specific GLUT transporter protein antibody and probed for GLUT type transporters in the rainbow trout ventricle. Our Western Blots did indeed demonstrate GLUT1 and GLUT4 type glucose transport proteins in both male and female rainbow trout ventricle tissue just below a molecular weight of 50 kD.;Given that our model expressed GLUTs but did not increase glucose uptake in response to hypoxia, we followed with several experiments designed to better define glucose uptake kinetics in the rainbow trout ventricle. Here we show that only high (100 mM and 50 mM) D-glucose competitively inhibits 2-DG uptake significantly (P < 0.001 and P < 0.05, respectively). Data from 2-DG Concentration-response assay (1mM and 40 mM) was evaluated with the Michaelis-Menten equation and fit a rectangular hyperbola. However, calculated Km was extrapolated beyond concentrations used in the assay. When temperature is decreased from 14°C to 4°C, total and GLUT mediated 2-DG uptake is decreased 51 +/- 6.5% (P < 0.001; n=6) and 31 +/- 20% (P < 0.05; n = 6) respectively, consistent with a facilitated diffusion model. The decrease in 2-DG uptake xvi between 24°C and 4°C was significant with CB (65 +/- 5.3%; P < 0.01; n = 6); without CB (51 +/- 6.5%; P < 0.001; n = 6); and in the GLUT mediated fraction (31 +/- 20%; P < 0.05; n = 6). Taken together, we conclude that glucose enters rainbow trout ventricle tissue by a combination of facilitated diffusion via GLUTs and another, yet to be identified route. Additionally, our findings support that the rainbow trout ventricle GLUT transporters are regulated differently than those found in mammalian and some other fish models. These novel findings expand our understanding of the variety of glucose transporter function and glucose uptake in a non-pathological, non-mammalian model. (Abstract shortened by UMI.)...