Primary cultured hepatocytes of the bony fish, Oreochromis mossambicus, the tilapia: a valid tool for physiological studies on IGF-I expression in liver (original) (raw)
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General and Comparative Endocrinology, 2004
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 lM 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.
Transgenic Research, 2007
Several lines of growth hormone (GH)-overexpressing fish have been produced and analysed for growth and fertility parameters. However, only few data are available on the growth-promoting hormone insulin-like growth factor I (IGF-I) that mediates most effects of GH, and these are contradictory. Using quantitative real-time RT-PCR, radioimmunoassay, in situ hybridization, immunohistochemistry, and radiochromatography we investigated IGF-I and IGF binding proteins (IGFBPs) in an adult (17 months old) transgenic (GH-overexpressing) tilapia (Oreochromis niloticus). The transgenics showed an around 1.5-fold increase in length and an approximately 2.3-fold higher weight than the non-transgenics. Using radioimmunoassay, the serum IGF-I levels were lower (6.22 ± 0.75 ng/ml) in transgenic than in wild-type (15.01 ± 1.49 ng/ml) individuals (P = 0.0012). Radioimmunoassayable IGF-I in transgenic liver was 4.2-times higher than in wild-type (16.0 ± 2.21 vs. 3.83 ± 0.71 ng/g, P = 0.0017). No hepatocytes in wild-type but numerous hepatocytes in transgenic liver contained IGF-I-immunoreactivity. RT-PCR revealed a 1.4-times higher IGF-I mRNA expression in the liver of the transgenics (10.51 ± 0.82 vs. 7.3 ± 0.49 pg/μg total RNA, P = 0.0032). In correspondence, in situ hybridization showed more IGF-I mRNA containing hepatocytes in the transgenics. A twofold elevated IGF-I mRNA expression was determined in the skeletal muscle of transgenics (0.33 ± 0.02 vs. 0.16 ± 0.01 pg/μg total RNA, P < 0.0001). Both liver and serum of transgenics showed increased IGF-I binding. The increased IGFBP content in the liver may lead to retention of IGF-I, and/or the release of IGF-I into the circulation may be slower resulting in accumulation of IGF-I in the hepatocytes. Our results indicate that the enhanced growth of the transgenics likely is due to enhanced autocrine/paracrine action of IGF-I in extrahepatic sites, as shown here for skeletal muscle.
Cell and Tissue Research, 2006
The cellular sites of insulin-like growth factor I (IGF-I) synthesis in the early developing tilapia (0-140 days post fertilization, DPF) were investigated. IGF-I mRNA and peptide appeared in liver as early as 4 DPF and in gastro-intestinal epithelial cells between 5-9 DPF. In exocrine pancreas, the expression of IGF-I started at 4 DPF and continued until 90 DPF. IGF-I production was detected in islets at 6 DPF in non-insulin cells and occurred throughout life. In renal tubules and ducts, IGF-I production started at 8 DPF. IGF-I production in chondrocytes had its onset at 4 DPF, was more pronounced in growing regions and was also found in adults. IGF-I mRNA and peptide appeared in the cytoplasm of skeletal muscle cells at 4 DPF. In gill chloride cells, IGF-I production started at 6 DPF. At 13 DPF, IGF-I was detected in cardiac myocytes. IGF-I-producing epidermal cells appeared at 5 DPF. In brain and ganglia, IGF-I was expressed in virtually all neurones from 6 to 29 DPF, their number decreasing with age. Neurosecretory IGF-I-immunoreactive axons were first seen in the neurohypophysis around 17 DPF. Endocrine cells of the adenohypophysis exhibited IGF-I mRNA at 28 DPF and IGF-I immunoreactivity at 40 DPF. Thus, IGF-I appeared early (4-5 DPF), first in liver, the main source of endocrine IGF-I, and then in organs involved in growth or metabolism. The expression of IGF-I was more pronounced during development than in juvenile and adult life. Local IGF-I therefore seems to have a high functional impact in early growth, metabolism and organogenesis.
General and Comparative Endocrinology, 2004
We developed a one-tube two-temperature real-time RT-PCR that allows to absolutely quantify the gene expression of hormones using the standard curve method. As our research focuses on the expression of the insulin-like growth factors (IGFs) in bony Wsh, we established the technique for IGF-I and IGF-II using the tilapia (Oreochromis niloticus) as model species. As approach, we used primer extension adding a T7 phage polymerase promoter (21 nt) to the 5Јend of the antisense primers. This procedure avoids the disadvantages arising from plasmids. Total RNA extracted from liver was subjected to conventional RT-PCR to create templates for in vitro transcription of IGF-I and IGF-II cRNA. Correct template sizes including the T7 promoter were veriWed (IGF-I: 91 nt; IGF-II: 94 nt). The PCR products were used to create IGF-I and IGF-II cRNAs which were quantiWed in dot blot by comparison with deWned amounts of standardised kanamycin mRNA. Standardised threshold cycle (C t) values for IGF-I and IGF-II mRNA were achieved by real-time RT-PCR and used to create standard curves. To allow sample normalisation the standard curve was also established for-actin as internal calibrator (template: 86 nt), and validation experiments were performed demonstrating similar ampliWcation eYciencies for target and reference genes. Based on the standard curves, the absolute amounts of IGF-I and IGF-II mRNA were determined for liver (IGF-I: 8.90 § 1.90 pg/ g total RNA, IGF-II: 3.59 § 0.98 pg/ g total RNA) and extrahepatic sites, such as heart, kidney, intestine, spleen, gills, gonad, and brain considering the diVerent lengths of cRNAs and mRNAs by correction factors. The reliability of the method was conWrmed in additional experiments. The ampliWcation of descending dilutions of cRNA and total liver RNA resulted in parallel slopes of the ampliWcation curves. Furthermore, ampliWcation plots of the standard cRNA and the IGF-I and IGF-II mRNAs showed signals starting at the expected C t values. Thus, the one-tube RT-PCR described here is highly sensitive (detection level »2 pg/ g total RNA) and allows precise absolute quantiWcation. The method is rapid as there are neither separate reverse transcriptions nor post-ampliWcation steps, and can be executed with low risk of contamination. Therefore, it will be helpful when investigating gene expression in any species and tissue whenever absolute levels are of concern.
General and …, 2007
Like IGF-I, progranulin (pgrn) is a growth factor involved in tumorigenesis and wound healing. We report here the identiWcation and characterization of pgrn cDNA in tilapia and the regulation of its expression by growth hormone (GH). The tilapia pgrn cDNA was cloned by RT-PCR ampliWcation, using gene speciWc oligonucleotides as ampliWcation primers. The cDNA contains an open reading frame encoding a peptide of 206 amino acid residues (aa) that contains a presumptive signal peptide (23 aa) and two repeat units of granulin (grn, 51 and 52 aa, respectively) franked by a GAP of 49 aa and the carboxyl terminus with 31 aa. The two predicted grn peptides are arranged in tandem repeats interrupted by a GAP peptide. RT-PCR analysis revealed that high levels of prgn mRNA were present in several tissues such as spleen, gastric cecum, intestine, fat tissue, gill, kidney, eye and pancreas, and lower levels in liver, muscle, heart, brain, skin and stomach. Administration of a single dose (500 ng/g body weight) of recombinant seabream growth hormone (rbGH) by intraperitoneal (ip) injection into one-month-old tilapia resulted in an obvious increase of IGF-I and pgrn mRNA (2.7-fold and 2.5-fold, respectively) in the liver at three hours post-GH treatment. The peptide levels of pgrn in the liver of GH-treated Wsh also were substantially induced over controls at 12 h post-GH treatment as detected by western immuno-blot analysis. The co-induction of IGF-I and pgrn following GH treatment may suggest the involvement of pgrn in GH regulated growth in tilapia.
Aquaculture, 2006
IGF-I is a mitogenic polypeptide that is an important regulator of growth in fish. The potential of IGF-I mRNA abundance asa rapid growth indicator in the Nile tilapia, Oreochromis niloticus, was evaluated. Hepatic IGF-I cDNA was isolated and partially cloned. The partial sequence having 539 bases encodes for the signal peptide, mature protein and a portion of the Edomain. The deduced 68 amino acid sequence for mature IGF-I showed 84–90% and 77–79% sequence identity with fish andmammalian counterparts, respectively. The deduced amino acid sequence for domains B and A was most conserved (93–97%)relative to other fishes. A sensitive TaqMan real time qRT-PCR assay for O. niloticus was developed based on the mature IGF-I peptide for measures of hepatic IGF-I mRNA levels. Hepatic IGF-I mRNA levels were found to be significantly correlated withgrowth rate of fish reared under different feeding regimes and temperature conditions. Higher feed consumption and water temperature produced faster-growing fish and increased hepatic IGF-I mRNA expression. These findings suggest that hepatic-derived IGF-I plays a key role in controlling growth in O. niloticus and indicates that IGF-I mRNA quantification could proveuseful for the rapid assessment of growth rate in this species.
Molecular and Cellular Endocrinology, 2010
Contradictory studies suggest IGF-I in fish liver and gills is involved in osmoregulation, but nothing is known about the kidney and intestine's role nor about IGF-II's role in any organ. Tilapia were transferred from freshwater (FW) to seawater (SW) for 1 week (wk) and retransferred to FW for another week. At 4 h, 1 d, 2 d, 3 d and 1 wk after SW-transfer and FW-retransfer IGF-I, IGF-II and growth hormone receptor (GHR1) mRNA were measured by real-time PCR. Hepatic IGF-I, IGF-II and GHR1 mRNA were downregulated in parallel after SW-transfer, recovered and were again downregulated after FWretransfer. In gills, IGF-I, IGF-II and GHR1 were upregulated synchronously after SW-transfer and, partially also after FW-retransfer. The renal genes were downregulated after SW-transfer and partially upregulated after FW-retransfer. Persisting upregulation in intestinal IGF-I mRNA occurred after FW-retransfer. Thus, endocrine and auto/paracrine IGF-I and IGF-II seem to be involved in fish osmoregulation in an organ-specific manner.