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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