Western ligand blotting of salmon serum typically reveals three insulin-like growth factor (IGF) binding proteins (IGFBPs) at 22, 28 and 41 kDa. Physiologic regulation of the 22 kDa IGFBP is similar to that of mammalian IGFBP-1; it is increased in catabolic states such as fasting and stress. On the other hand, its molecular mass on Western ligand blotting is closest to mammalian IGFBP-4. The conflict between physiology and molecular mass makes it difficult to determine the identity of the 22 kDa IGFBP. This study therefore aimed to identify the 22 kDa IGFBP from protein and cDNA sequences. The 22 kDa IGFBP was purified from chinook salmon serum by a combination of IGF-affinity chromatography and reverse-phase chromatography. The N-terminal aminoacid sequence of the purified protein was used to design degenerate primers. Degenerate PCR with liver template amplified a partial IGFBP cDNA, and full-length cDNA was obtained by 5′- and 3′-rapid amplification of cDNA ends (RACE). The 1915-bp cDNA clone encodes a 23.8 kDa IGFBP, and its N-terminal amino-acid sequence matched that of purified 22 kDa IGFBP. Sequence comparison with six human IGFBPs revealed that it is most similar to IGFBP-1 (40% identity and 55% similarity). These findings indicate that salmon 22 kDa IGFBP is IGFBP-1. Salmon IGFBP-1 mRNA is predominantly expressed in the liver, and its expression levels appear to reflect circulating levels. The 3′-untranslated region of salmon IGFBP-1 mRNA contains four repeats of the nucleotide sequence ATTTA, which is involved in selective mRNA degradation. In contrast, amino-acid sequence analysis revealed that salmon IGFBP-1 does not have an Arg-Gly-Asp (RGD) integrin recognition sequence nor a Pro, Glu, Ser and Thr (PEST)-rich domain (a segment involved in rapid turnover of protein), both of which are characteristic of mammalian IGFBP-1. These findings suggest that association with the cell surface and turnover rate may differ between salmon and mammalian IGFBP-1.

Liver production of insulin-like growth factor-I (IGF-I) is a major point of control in the growth hormone (GH)/IGF axis, the endocrine system regulating body growth in fishes and other vertebrates. Pituitary GH stimulates hepatocyte production of IGF-I; however, in catabolic states, hepatocyte GH resistance results in decreases in liver IGF-I production. To investigate endocrine mechanisms leading to the development of hepatocyte GH resistance, we examined the regulation of IGF-I mRNA level by GH and metabolic hormones in primary culture of salmon hepatocytes. Cells were cultured in RPMI medium, and exposed to insulin (Ins, 10⁻⁶ M), glucagon (Glu, 10⁻⁶ M), triiodothyronine (T₃, 10⁻⁷ M), dexamethasone (Dex, 10⁻⁶ M) and glucagon-like peptide (GLP, 10⁻⁶ M), in the presence and absence of GH (5×10⁻⁹ M). GH always increased IGF-I mRNA. None of the other hormones tested alone affected IGF-I mRNA. However, Dex, Ins and Glu reduced the response to GH. The response to GH was inhibited by Dex at concentrations of 10⁻¹² M and above, by Ins at 10⁻⁹ M and above, and by Glu only at 10⁻⁶ M. Inhibition of GH response by glucocorticoids is found in other vertebrates. Salmon hepatocytes were very sensitive to Dex, suggesting that glucocorticoids may play an important role in salmon growth regulation even in unstressed conditions. Inhibition of GH response by Ins is the opposite of what is found in mammals and chickens, suggesting that the role of Ins in growth regulation may differ between fishes and tetrapods. To examine mechanisms for modulation of GH sensitivity, we measured hepatocyte GH receptor (GHR) mRNA levels. Ins inhibited and Dex stimulated GHR mRNA, suggesting that different mechanisms mediate the inhibition of GH response by these hormones. This study shows that glucocorticoids, Ins, and Glu induce GH resistance in cultured salmon hepatocytes.