A quantitative real-time RT-PCR assay for salmon IGF-I mRNA, and its application in the study of GH regulation of IGF-I gene expression in primary culture of salmon hepatocytes.
The hormone insulin-like growth factor-I (IGF-I) regulates vertebrate growth. The liver produces most circulating IGF-I, under the control of pituitary growth hormone (GH) and nutritional status. To study the regulation of liver IGF-I production in salmon, we established a primary hepatocyte culture system and developed a TaqMan quantitative real-time RT-PCR assay for salmon IGF-I gene expression. A portion of the coho salmon acidic ribosomal phosphoprotein P0 (ARP) cDNA was sequenced for use as a reference gene. A systematic bias across the 96 well PCR plate was discovered in an initial IGF-I assay, which was corrected when the assay was redesigned. IGF-I mRNA levels measured with the validated assay correlated well with levels measured with an RNase protection assay, and were highest in liver compared with other tissues. We examined the time course of hepatocyte IGF-I gene expression over 48 h in culture, the response to a range of GH concentrations in hepatocytes from fed and fasted fish, and potential effects of variation in IGF-I in the medium. IGF-I gene expression decreased over time in culture in hepatocytes in plain medium, and in cells treated with 5 nM GH with or without a combination of metabolic hormones (1 microM insulin, 100 nM triiodothyronine, and 0.1 nM dexamethasone). GH stimulated IGF-I gene expression at all time points. In cells treated with GH plus metabolic hormones, IGF-I gene expression was intermediate between the controls and GH alone. Increasing concentrations of GH resulted in biphasic IGF-I gene expression response curves in cells from fed and fasted fish, with the threshold for stimulation from 0.5 to 2.5 nM GH, maximal response from 5 to 50 nM, and a reduced response at 500 nM. Medium IGF-I (5 nM) did not affect basal or GH stimulated IGF-I gene expression. This study shows that primary hepatocyte culture and the TaqMan IGF-I assay can be used to study the regulation of hepatic IGF-I gene expression in salmon, and provides the first evidence of a biphasic response to GH concentration in fish hepatocyte culture.
Pierce
A L
AL
School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA 98195, USA. piercea@u.washington.edu
Dickey
J T
JT
Larsen
D A
DA
Fukada
H
H
Swanson
P
P
Dickhoff
W W
WW
D000825:Animal Structures / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005215:Fasting / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D013006:Growth Hormone / Q000494:pharmacology / Q000502:physiology*; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D017684:Oncorhynchus kisutch / Q000235:genetics*; D010750:Phosphoproteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012269:Ribosomal Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D014284:Triiodothyronine / Q000494:pharmacology
D018076:DNA, Complementary; D007328:Insulin; D010750:Phosphoproteins; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C054675:ribosomal protein P0; D014284:Triiodothyronine; D007334:Insulin-Like Growth Factor I; D003907:Dexamethasone; D013006:Growth Hormone