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DIETARY CARBOHYDRATE LEVEL AFFECTS EXPRESSION OF HEPATIC GLUCOSE TRANSPORTERS IN RAINBOW TROUT (Oncorhynchus mykiss) Jon J. Amberg1, Gordon K. Murdoch2, Barrie D. Robison3, Madison S. Powell4, Kenneth J. Rodnick5, Rodney A. Hill2 & Ronald W. Hardy4 1Department of Forestry and Natural Resources, Purdue University, West Lafayette, IN 47907 2Department of Animal and Veterinary Sciences, University of Idaho, Moscow, ID 83844 3 Department of Biological Sciences, University of Idaho, Moscow, ID 83844 4 University of Idaho, Aquaculture Research Institute, Hagerman, ID 83332 5 Department of Biological Sciences, Idaho State University, Pocatello, ID 83209 PROBLEM Sustainability • Alternative protein sources Plant-based higher levels of dietary carbohydrates Rainbow Trout • Intolerant of CHO levels higher than 20 percent (Hung et al. 1994; Panserat et al. 2000; Panserat et al. 2001) Leads to a hyperglycemia (Kirchner et al. 2008) Poor growth (Hung et al. 1994; Kirchner et al. 2008) • Carnivores are unable to regulate glucose uptake (Karasov et al. 1983; Buddington 1987; Buddington et al. 1987a) Purpose • Develop stage specific diets & more economical feeding strategies RESEARCH QUESTION Do carnivorous rainbow trout transcriptionally regulate hepatic glucose transporters in response to manipulated dietary glucose levels? GLUCOSE TRANSPORTERS Facilitative Glucose Transporter 1 (GLUT1) – Ubiquitous distribution – Basal transporter – Glucose dependent Facilitative Glucose Transporter 2 (GLUT2) – Glucose dependent – Dominate Facilitative Glucose Transporter 4 (GLUT4) – Muscle and adipocytes – Glucose independent - Insulin responsive EXPERIMENTAL DESIGN Fish • 30-g mixed sex (CSI strain) 12 tanks Diets • • • • Iso-nitrogenous & Iso-lipidic Diff. % gelatinized starch (0, 15, 25 & 35%) Inert filler (diatomaceous earth & α-cellulose) Triplicate tanks Sampling • Liver • 10 fish/tank/at weeks 4, 8 & 12 Analysis • ANCOVA Multiple reference genes (18S rRNA & α-actin) EXPRESSION GLUT 1 0 Relative transcript abundance 50 15 25 35 40 30 20 10 0 0 5 10 Week N = 12 Reference genes: 18S rRNA & α-actin 15 EXPRESSION GLUT 4 0 Relative transcript abundance 25 15 25 35 20 15 10 5 0 -5 0 5 10 Week N = 12 Reference genes: 18S rRNA & α-actin 15 EXPRESSION Relative transcript abundance GLUT 2 (mean transcript abundance during 12 week study) A 100 90 80 70 60 50 40 30 20 10 0 AB -5 5 N = 12 Reference genes: 18S rRNA & α-actin 15 B B 25 35 % CHO inclusion PHYSIOLOGICAL EFFECTS P ≤ 0.05 N = 12 PHYSIOLOGY Balanced Blood Glucose Ito Cell GLUT4 GLUT2 GLUT2 BLOOD CELL Hepatocyte PHYSIOLOGY Low Blood Glucose GLUT2 GLUT2 GLUT2 GLUT2 BLOOD CELL PHYSIOLOGY Elevated Blood Glucose X GLUT4 GLUT2 BLOOD CELL IN SILICO ANALYSIS Are there differences in functional amino acids/domains among vertebrates? Methods Sequences: 3-spine stickleback Rainbow trout Human Medaka Atlantic cod Mouse Zebrafish Fugu Horse • Align using CLUSTAL W • Putative Transmembrane Domains (putative) TopPred II software (von Heijne 1992; Claros et al. 1994) • Phosphorylation sites KinasePhos 2.0 (Huang et al. 2005; Wong et al. 2007) AMINE-END OF GLUT2 TMD #1 Lacking 18-20 AA TMD #2 CARBOXYL-END OF GLUT2 TMD #12 Phosphorylation sites SUMMARY • GLUT1 expression did not respond to changing blood glucose levels • GLUT4 expression increased early and then decreased Possible storage in Ito cells • GLUT2 expression increased with low blood glucose levels Differs from mammalian GLUT2 • Piscine GLUT2 appears to be missing ~20 amino acids in the extracellular loop between TMDs 1 & 2. • RBT GLUT2 carboxyl end is truncated & lacking phosphorylation sites -- unable to turn off? IMPORTANCE • RBT appear to attempt to regulate blood glucose levels • Modifications to the amino acid sequence may play an important role in the function of GLUT2 Chimeric studies? • Incorporate into genetic selection programs Decrease protein content in RBT diets Development of more sustainable RBT feeds ACKNOWLEDGEMENTS Thanks to… University of Idaho Fish Physiology Group Hagerman Experimental Fish Culture Station Aquaculture Research Institute Idaho EPSCoR This project is supported by National Science Foundation, Idaho EPSCoR under grant #EPS0447689