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First published online November 1, 2006
Journal of Experimental Biology 209, 4490-4502 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02532
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Sequence of Atlantic cod (Gadus morhua) GLUT4, GLUT2 and GPDH: developmental stage expression, tissue expression and relationship to starvation-induced changes in blood glucose

Jennifer R. Hall, Connie E. Short and William R. Driedzic*

Ocean Sciences Centre, Memorial University of Newfoundland, St John's, Newfoundland, A1C 5S7, Canada


Figure 1
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  		Fig. 1. Phylogenetic analysis of Atlantic cod GLUT4 and GLUT2. A phylogenetic tree of protein sequences of Atlantic cod GLUT4 and GLUT2 and GLUT1-GLUT4 from other vertebrates. SwissProt accession numbers are as follows: GLUT1 common carp (AAF75683), GLUT1 rainbow trout (AAF75681), GLUT1 Atlantic cod (AAS17880), GLUT1 chicken (AAB02037), GLUT1 human (AAA52571), GLUT1 rabbit (P13355), GLUT1 cow (P27674), GLUT1 rat (P11167), GLUT1 mouse (AAA37752), GLUT2 chicken (Q90592), GLUT2 rainbow trout (AAK09377), GLUT2 human (AAA59514), GLUT2 mouse (P14246), GLUT2 rat (P12336), GLUT2 Atlantic cod (AAV63984), GLUT3 cow (AAK70222), GLUT3 dog (P47842), GLUT3 human (AAB61083), GLUT3 mouse (AAH34122), GLUT3 rat (Q07647), GLUT3 chicken (AAA48662), GLUT3 grass carp (AAP03065), GLUT3 Atlantic cod (AAT67456), GLUT4 cow (Q27994), GLUT4 human (AAA59189), GLUT4 mouse (P14142), GLUT4 rat (P19357), GLUT4 coho salmon (AAM22227), GLUT4 brown trout (AAG12191), GLUT4 Atlantic cod (AAZ15731), Pacific hagfish (AAL27090). The tree was constructed as described in the text. The scale bar represents the number of substitutions per amino acid site.

 

Figure 2
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  		Fig. 2. (A) Alignment of protein sequences for class I GLUTs from Atlantic cod. The single letter code is used for the amino acids. Gaps in the amino acid sequences are indicated with a dash (-). Positions where there is a single, fully conserved residue are indicated with an asterisk (*), positions with a `strong' group fully conserved are indicated with a colon (:), and positions with a `weaker' group fully conserved are indicated with a dot (.). Numbered lines above the sequences indicate the approximate positions of the predicted membrane spanning helices. Residues that are highly conserved in all glucose transporters are highlighted in grey, and those that are specific to class I glucose transporters are highlighted in black. Residues discussed with respect to GLUT4 are indicated by a grey box and those to GLUT2 with a white box. (B) Alignment of GPDH protein sequences from Atlantic cod and other fish species.

 

Figure 3
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  		Fig. 3. Developmental expression patterns of (A) GLUT4, (B) GLUT2 and (C) GPDH in Atlantic cod. Atlantic cod were analyzed for GLUT4, GLUT2 and GPDH expression from eggs to larval fish by qRT-PCR. Each value represents a single analysis of a biomass of approximately 100 mg. Expression levels of the target gene were normalized to 18S rRNA. Units represent the relative expression of normalized target gene with respect to a calibrator sample, which in this case is the tissue that expresses the target gene at the lowest detectable level. Sampling times are based on developmental stages and changes in diet. Day 0, 45 degree days; HH, halfway to hatching; Hatch, 105 degree days. Post-hatch samples: Yolk sac, day 2; Rotifers, day 19; Rot/Atr (rotifers/Artemia), day 47; Artemia, day 52; Art/DF (Artemia/dry food), day 54; Dry food, day 60.

 

Figure 4
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  		Fig. 4. Tissue distribution of (A) GLUT4, (B) GLUT2 and (C) GPDH in Atlantic cod. Tissues from a single fed juvenile Atlantic cod were analyzed by qRT-PCR for expression of GLUT4, GLUT2 and GPDH. Expression levels of the target gene were normalized to 18S rRNA. Units represent the relative expression of normalized target gene with respect to a calibrator sample, which in this case is the tissue that expresses the target gene at the lowest detectable level.

 

Figure 5
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  		Fig. 5. The effects of fasting/re-feeding on morphometrics of Atlantic cod. (A) Body mass; (B) length; (C) condition factor (CF); (D) hepatosomatic index (HIS). Fish were deprived of food for 1 or 2 months with control fed fish being sampled at the same time. One group was re-fed for 3 weeks following 2 months of starvation. N=6 in all cases. Columns with different letters are significantly different (P<0.05).

 

Figure 6
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  		Fig. 6. The effects of fasting/re-feeding on blood glucose and liver glycogen levels of Atlantic cod. (A) Blood glucose; (B) liver glycogen; (C) blood glucose versus liver glycogen from individual fish (y=0.29x+3.4; R2=0.20, P=0.017); fish were fed (N=10 for glucose; N=12 for glycogen) or deprived of food for 1 month (N=6) or 2 months (N=6). Starved fish were then re-fed (N=6) for 3 weeks. Columns with different letters are significantly different (P<0.05).

 

Figure 7
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  		Fig. 7. The effects of fasting/re-feeding on total lipid and triglyceride levels in liver of Atlantic cod. (A) Total lipid; (B) triglyceride. Fish were fed (N=12) or deprived of food for 1 month (N=6) or 2 months (N=6).

 

Figure 8
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  		Fig. 8. The effects of fasting/re-feeding on GLUT4, GLUT2 and GPDH expression in Atlantic cod tissues. Atlantic cod were fed or deprived of food for 2 months. Starved fish were then re-fed for 3 weeks. GLUT4 levels in heart and white muscle, GPDH levels in heart and liver, and GLUT2 in liver were analyzed by qRT-PCR. Target gene levels were normalized to 18S rRNA. Units represent the relative expression of normalized target gene with respect to a calibrator sample, which in these studies is fed fish. N=6 in all cases. Columns with different letters are significantly different (P<0.05).

 

Figure 9
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  		Fig. 9. The relationship between GLUT expression and blood glucose level in Atlantic cod. (A) Heart GLUT4 versus blood glucose (y=0.048x+0.57; R2=0.28, P=0.035); (B) liver GLUT2 versus blood glucose (y=0.29x+0.28; R2=0.46, P=0.0037); (C) white muscle GLUT4 versus blood glucose. Values are for individual fish from fasting/re-feeding experiments.

 

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© The Company of Biologists Ltd 2006