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First published online July 14, 2008
Journal of Experimental Biology 211, 2510-2518 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.018374
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Insulin regulates the expression of several metabolism-related genes in the liver and primary hepatocytes of rainbow trout (Oncorhynchus mykiss)

Elisabeth Plagnes-Juan, Marine Lansard, Iban Seiliez, Françoise Médale, Geneviève Corraze, Sadasivam Kaushik, Stéphane Panserat and Sandrine Skiba-Cassy*

INRA, UMR 1067 Nutrition Aquaculture and Génomique, Pôle d'hydrobiologie, CD 918, F-64310 Saint Pée-sur-Nivelle, France


Figure 1
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  		Fig. 1. Plasma glucose levels 2, 4 and 6 h after intraperitoneal administration of insulin (10 u kg–1; I) or saline solution (S) to 48 h-fasted control rainbow trout (C). Results are expressed as means ± s.e.m. (N=8) and were analyzed by two-way ANOVA followed by Student–Newman–Keuls multiple comparison test. *Significant difference between insulin- and saline-treated fish (P<0.05). {dagger}Significant difference between 48 h-fasted control fish (C) and insulin (I)- or saline (S)-treated fish (P<0.05).

 

Figure 2
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  		Fig. 2. (A) Western blot analysis of Akt phosphorylation in rainbow trout liver 2, 4 and 6 h after intraperitoneal administration of insulin (I) or saline solution (S) as well as in 48 h-fasted control rainbow trout (C). p-Akt, phosphorylated form of Akt. The gel was loaded with 20 µg of total protein per lane. The figure is a representative blot. Western blots were performed on six individual samples and similar results were obtained. (B) Effects of intraperitoneal administration of insulin (I) or saline solution (S) on the level of expression of mRNA encoding hepatic genes. Pyruvate kinase (PK), glucose 6-phosphatase isoforms 1 and 2 (G6Pase 1 and 2), phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6-bisphosphatase (FBPase), carnitine palmitoyltransferase 1 isoform A and B (CPT1 A and B) and fatty acid synthase (FAS) mRNA levels were estimated using real-time RT-PCR. Expression values were normalized with elongation factor 1{alpha} (EF1{alpha})-expressed transcripts and are indicated as fold variation of the saline solution-treated group. Results are expressed as means ± s.e.m. (N=6) and were analyzed by one-way ANOVA followed by Student–Newman–Keuls comparison test. *Significant difference (P<0.05).

 

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  		Fig. 3. Expression of hepatic nuclear factor 4 (HNF4), albumin precursor (AP), serotransferin precursor (STP), tyrosine aminotransferase (TAT), glucose transporter 2 (GLUT2) and insulin receptor (InsR) assessed by conventional RT-PCR in cell suspension (CS) and hepatocytes after 4, 24, 48 and 72 h of culture. Expected lengths of the fragments are indicated in the left margin. For InsR, a larger fragment (509 bp) was also detected.

 

Figure 4
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  		Fig. 4. Induction of Akt phosphorylation in rainbow trout hepatocyte cell culture 5, 15, 30 and 60 min after insulin stimulation. The gel was loaded with 20 µg of total protein per lane. The figure is a representative blot. Western blots were performed on six individual samples and similar results were obtained. p-Akt, phosphorylated form of Akt.

 

Figure 5
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  		Fig. 5. Effects of insulin and glucose on the expression level of mRNA encoding hepatic genes. Glucokinase (GK), PK, G6Pase 1 and 2, PEPCK, FBPase and FAS mRNA levels were estimated using real-time RT-PCR. The experiment was conducted in 48 h-cultured hepatocytes of rainbow trout incubated for an additional 24 h with (Insulin, filled bars) or without (Control, hatched bars) insulin (4x10–9 mol l–1) and in the presence of 3 or 20 mmol l–1 glucose (glc). Expression values are normalized with elongation factor 1{alpha} EF1{alpha})-expressed transcripts and are indicated as fold variation of 3 mmol l–1 glucose control condition. Results are expressed as means +s.e.m. (N=6) and were analyzed by one-way ANOVA followed by Student–Newman–Keuls multiple comparison test (P<0.05). *Significant effect of insulin. {dagger}Significant effect of glucose.

 

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© The Company of Biologists Ltd 2008