Isolation and characterization of a mRNA encoding a novel insulin receptor (IR) subtype, IR2, from rainbow trout (Oncorhynchus mykiss) and patterns of expression of the four IR subtypes, IR1-IR4, in tissues and during embryonic development

Insulin (INS) plays a critical role in the growth, development, and metabolism of vertebrates. In this study, a cDNA encoding a novel insulin receptor (IR) subtype was isolated, cloned, and sequenced from the liver of rainbow trout. A 1525-bp cDNA encoding a partial amino acid sequence of the β-subunit including the transmembrane domain, the tyrosine kinase domain, and the 3′ untranslated region (UTR) was obtained and designated IR2 based on comparison with known IR subtypes, including the three previously reported IR subtypes of trout. Trout IR2 shares 90.0%, 82.8%, and 84.3% nucleotide identity with previously characterized trout IR1, IR3 and IR4, respectively. Quantitative real-time PCR revealed that the four IR mRNAs were differentially expressed, both in terms of distribution among tissues as well as in terms of abundance within selected tissues of juvenile trout. IR1 mRNA was most abundant in spleen, liver, kidney, and muscle (white, red and cardiac), but least abundant in adipose. IR3 mRNA was most abundant in liver, spleen, kidney, and pancreas; in other tissues, levels of IR3 mRNA were uniformly abundant. By contrast, levels of IR2 and IR4 mRNA were uniformly abundant in most tissues, except in spleen where levels of IR4 were significantly lower. All IR subtypes were detected over the course of embryonic development. In head and tail regions, levels of IR2 and IR3 mRNA declined from pre-hatch (29 days post-fertilization, dpf) to post-hatch (68-90 dpf), whereas levels of IR1 and IR4 remained relatively unchanged. These findings contribute to our understanding of the evolution, distribution, and function of insulin receptors.     

Differential expression of two somatostatin receptor subtype 1 mRNAs in rainbow trout (Oncorhynchus mykiss)

Somatostatins (SSs) play important roles in the growth, development and metabolism of vertebrates. In this study, cDNAs for two unique somatostatin receptor variants were cloned and sequenced from rainbow trout. The two cDNAs, one consisting of 1755 bp and the other of 1743 bp, share 63.6% identity in nucleotide sequence and 94.1% identity in deduced amino acid sequence and presumably arose through gene duplication. Each cDNA encodes for a putative 371-amino acid somatostatin receptor (one designated sst1A and the other sst1B) containing seven transmembrane domains. Rainbow trout sst1A and sst1B have 64.4 and 65.5% similarity respectively with human sst1 and only 43-60% similarity with other subtypes. Trout sst1 mRNAs are differentially expressed, both in terms of distribution among tissues as well as in terms of abundance within selected tissues. Both sst1A and sst1B mRNAs were present in brain, stomach, liver, pancreas, upper and lower intestine, pyloric cecum, kidney and muscle, whereas only sst1B mRNA was present in the esophagus. sst1A mRNA was more abundant than sst1B in the optic tectum, whereas sst1B mRNA was more abundant than sst1A in liver. sst1A and sst1B mRNAs were equally abundant in pancreas. These findings contribute to the understanding of the evolution of the SS signaling system and provide insight into the mechanisms that regulate the expression of SS receptors.     

The expression of insulin and insulin receptor mRNAs is regulated by nutritional state and glucose in rainbow trout (Oncorhynchus mykiss)

Many species of fish, including rainbow trout, possess multiple INS- and IR-encoding mRNAs. In this study, rainbow trout (Oncorhynchus mykiss) were used as a model to study the regulation of INS (INS1, INS2) and IR (IR1, IR2, IR3, and IR4) mRNA expression by nutritional state and glucose. In the nutritional state study, fish were either fed continuously, fasted (4 or 6 weeks), or fasted 4 weeks, then refed for 2 weeks. Nutritional state regulated INS and IR mRNA expression in a subtype- and tissue-specific manner. A 4-week fast reduced INS1 expression in endocrine pancreas (Brockmann body) and of INS1 and INS2 in brain, whereas a 6-week fast reduced the expression of both INS1 and INS2 in pancreas but only of INS1 in brain. Refeeding only restored INS2 levels in pancreas. In adipose tissue, by contrast, a 4-week fast increased INS1 expression, and a 6-week fast increased the expression of both INS1 and INS2. Nutritional state also modulated the pattern of IR mRNA expression. Fasting for 4 weeks resulted in no significant change in IR expression. Prolonged fasting (6 weeks) increased the expression of IR4 mRNA in the pancreas, adipose tissue, cardiac muscle, and gill; however, fasting decreased expression of IR3 mRNA in liver. Refeeding restored fasting-associated increases in IR4 expression in pancreas, adipose tissue, cardiac muscle, and gill, but had no effect on the fasting-associated decrease in IR3 expression in liver. Glucose differentially regulated the expression of INS and IR mRNAs in Brockmann bodies and liver pieces incubated in vitro, respectively. Low glucose (1 mM) reduced pancreatic expression of both INS1 and INS2 mRNAs compared to levels observed at 4 or 10 mM glucose. In the liver, IR1 and IR2 mRNA expression was insensitive to glucose concentration, whereas expression of IR3 and IR4 was attenuated at 1 and 10 mM compared to 4 mM glucose. These findings indicate that the pattern of INS and IR expression in selected tissues is regulated by nutritional state and glucose.     

Polygenic expression of somatostatin in orange-spotted grouper (Epinephelus coioides): molecular cloning and distribution of the mRNAs encoding three somatostatin precursors

In the present study, three preprosomatostatin (PSS) cDNAs were characterized from hypothalamus of orange-spotted grouper Epinephelus coioides. The first cDNA encodes a 123-amino acid protein (PSSI) that contains the SS14 sequence at its C-terminal extremity and that is identical to that of PSSI of human and other vertebrates. The second cDNA encodes a 127-amino acid protein (PSSII) that contains the SS28 sequence with [Tyr7, Gly10]-SS14 at its C-terminus. The third cDNA encodes a 110-amino acid protein (PSSIII) that contains the somatostatin variant [Pro2]-SS14 at its C-terminal extremity. All these three PSS mRNAs were expressed in brain and pituitary with different mRNA levels. In peripheral tissues, PSSII was more widely distributed than PSSI and PSSIII. High mRNA levels of PSS were found in stomach, intestine and ovary. PSS mRNAs were detected throughout embryogeny and early larval development. Its levels increased with the embryonic development and maintained a higher level during larva developing. The mRNA distribution suggests that the three grouper PSS products play important physiological functions in adult fish as well as in cell growth and organ differentiation in embryo and larva development.     

Polygenic expression of somatostatin in rainbow trout, Oncorhynchus mykiss: evidence of a preprosomatostatin encoding somatostatin-14

Previously we reported the existence of two distinct cDNAs in rainbow trout that encode for separate preprosomatostatins (PPSS), each containing [Tyr7, Gly10]-somatostatin-14. In the present study, we used rainbow trout to further characterize the polygenic origin of somatostatins (SSs), a peptide hormone important in the regulation of growth, development, and metabolism of vertebrates. A two-phase rapid amplification of cDNA ends (RACE)-PCR was used for the isolation of selected cDNAs. We amplified and sequenced a ca. 350-bp 3' RACE-PCR fragment. Based upon this sequence we designed a second gene-specific primer for 5' RACE-PCR which yielded a 452-bp fragment. Sequence analysis revealed a 745-bp cDNA containing the complete 5'-untranslated region, a single initiation site 118 bases from the most 5' end, and a single putative polyadenylation site 17 bases from the most 3' end that was terminated with a polyadenylated tail. The deduced protein is a 114-amino acid PPSS molecule that contains a number of putative processing sites, potentially yielding a 26-amino acid peptide that could be processed further to a 14-amino acid peptide identical in structure to mammalian SS-14. Northern analysis revealed that PPSS-I was expressed in the pancreas, stomach, intestine, and brain of rainbow trout. These results suggest a polygenic origin of SS, possibly resulting from gene duplication events prior to the divergence of teleosts.     

Identification and characterization of a new member of the placental prolactin-like protein-C (PLP-C) subfamily, PLP-Cbeta

We have isolated a complementary DNA (cDNA) clone that encodes a new member of the PRL-like protein-C (PLP-C) subfamily of the PRL gene family. The clone was amplified from a 13.5-day-old mouse conceptus cDNA library by PCR using primers based on conserved regions of PLP-C sequences. The full-length cDNA encodes a predicted protein of 241 residues, which contains a putative signal sequence and 2 putative N-linked glycosylation sites. The predicted protein shares 55-66% amino acid identity with mouse PLP-Calpha and rat PLP-D, PLP-H, PLP-Cv, and PLP-C and also contains 6 homologously positioned cysteine residues. Thus, we named this protein PLP-Cbeta for consistency. We have also isolated rat PLP-Cbeta from rat placenta cDNA library. Surprisingly, two messenger RNA (mRNA) isoforms of rat PLP-Cbeta were isolated: one mRNA (rPLP-Cbeta) encodes a 241-amino acid product, but another mRNA (rPLP-Cbetadelta39) lacks 39 bases that encode for a region rich in aromatic amino acids. The 39-bp region corresponds to exon 3 of other PLP-C subfamily members, such as PLP-Calpha, PLP-Cv, and d/tPRP. It suggests that the two isoforms are probably generated by an alternative splicing from a single gene. RT-PCR analysis revealed that the rPLP-Cbeta form was dominantly expressed in placenta, although both isoforms are coexpressed during placentation. The mouse PLP-Cbeta mRNA expression, which was specific to the placenta, was first detected by Northern analysis on embryonic day 11.5 (E 11.5) and persisted until birth. However, in situ hybridization analysis revealed mPLP-Cbeta expression on E 10.5 in specific trophoblast subsets, such as giant cells and spongiotrophoblast cells. mPLP-Cbeta mRNA was detected in the labyrinthine zone on E 18.5, suggesting that spongiotrophoblast cells had penetrated the labyrinthotrophoblast zone. Consistent with the observed expression in trophoblast giant cells, PLP-Cbeta expression was also detected in in vitro differentiated Rcho-1 cells, which express the trophoblast giant cell phenotype. In summary, overall high amino acid identity (79%), the locations of cysteine residues, and consensus sites for N-linked glycosylation between mouse and rat PLP-Cbeta clearly indicate that PLP-Cbeta is a bona fide member of the PLP-C subfamily. The conservation between mouse and rat, the presence of alternative isoforms, and the pattern of expression during gestation suggest the biological significance of PLP-Cbeta during pregnancy.     

Growth hormone and insulin-like growth factor-1 differentially stimulate the expression of preprosomatostatin mRNAs in the Brockmann bodies of rainbow trout, Oncorhynchus mykiss

We previously characterized three cDNAs obtained from the endocrine pancreas (Brockmann body) of rainbow trout that encode for distinct preprosomatostatin (PPSS) molecules: PPSS I containing somatostain-14 (SS-14) at its C-terminus and two separate PPSS IIs, PPSS II' and PPSS II', containing [Tyr7,Gly10]-SS-14 at their C-termini. In this study, we examined the control of PPSS I, PPSS II', and PPSS II' mRNA expression by growth hormone (GH) and insulin-like growth factor-1 (IGF-1). Rainbow trout implanted with GH for 21 days displayed elevated pancreatic expression of all PPSS mRNAs compared to control animals. Growth hormone directly stimulated the expression of all pancreatic PPSS mRNAs in vitro in a dose-dependent manner; however, GH was a more potent stimulator of PPSS II' expression than of PPSS I or PPSS II' expression. Insulin-like growth factor-1 also directly stimulated the expression of PPSS mRNAs in a dose-dependent manner in Brockmann bodies incubated in vitro; IGF-1 was a more potent stimulator of PPSS I and PPSS II' expression than of PPSS II' expression. These results indicate that the expression of PPSS mRNAs in the Brockmann body of trout is differentially regulated by GH and IGF-1 and suggest that SS mediate the feedback regulation of GH and IGF-1.     

New insights into the signaling system and function of insulin in fish

Fish have provided essential information about the structure, biosynthesis, evolution, and function of insulin (INS) as well as about the structure, evolution, and mechanism of action of insulin receptors (IR). INS, insulin-like growth factor (IGF)-1, and IGF-2 share a common ancestor; INS and a single IGF occur in Agnathans, whereas INS and distinct IGF-1 and IGF-2s appear in Chondrichthyes. Some but not all teleost fish possess multiple INS genes, but it is not clear if they arose from a common gene duplication event or from multiple separate gene duplications. INS is produced by the endocrine pancreas of fish as well as by several other tissues, including brain, pituitary, gastrointestinal tract, and adipose tissue. INS regulates various aspects of feeding, growth, development, and intermediary metabolism in fish. The actions of INS are mediated through the insulin receptor (IR), a member of the receptor tyrosine kinase family. IRs are widely distributed in peripheral tissues of fish, and multiple IR subtypes that derive from distinct mRNAs have been described. The IRs of fish link to several cellular effector systems, including the ERK and IRS-PI3k-Akt pathways. The diverse effects of INS can be modulated by altering the production and release of INS as well as by adjusting the production/surface expression of IR. The diverse actions of INS in fish as well as the diverse nature of the neural, hormonal, and environmental factors known to affect the INS signaling system reflects the various life history patterns that have evolved to enable fish to occupy a wide range of aquatic habitats.     

Absence of direct regulation of prolactin cells by estradiol-17 beta in rainbow trout (Oncorhynchus mykiss).

The effects of estradiol-17 beta (E2) implants on plasma prolactin (PRL) concentrations, pituitary PRL content and pituitary PRL mRNA levels were examined in rainbow trout (Oncorhynchus mykiss). Intact immature fish treated with 1 mg estradiol-17 beta did not show significant changes in both PRL mRNA levels and pituitary PRL content after 3 days of treatment. In a similar experiment, no changes were observed in plasma PRL levels followed during 7 days. Similarly, lack of estradiol-17 beta effect on plasma PRL levels and on final PRL pituitary content was observed in ovariectomized female rainbow trout treated during 48 days with 25 mg estradiol-17 beta and in mature male fish over a 3-week treatment period. Localization of estradiol receptor (ER) mRNAs in the pituitary was carried out by Northern blot analysis using a full-length rainbow trout estrogen receptor (rtER) cDNA as a probe. The rostral pars distalis of the pituitary which contained mostly PRL cells showed the lower amount of rtER mRNA when compared to other parts of the pituitary. Moreover, two mRNAs of different size (3.5 and 1.4 kb) were detected in different parts of the pituitary. Further hybridization experiments using probes containing part of the rtER cDNA (E domain or C and D domains) indicated that the small-sized mRNA (1.4 kb) probably encodes a truncated ER protein lacking hormone binding domain or an ER-related protein. Thus, only the 3.56 kb mRNA appeared to be involved in the regulation of pituitary function by estradiol. In situ hybridization analysis allowed a more precise localization of this rtER mRNA in the pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expression of a novel seven transmembrane domain protein in epididymal fat from aged and diabetic mice.

To identify novel seven transmembrane domain proteins from 3T3-L1 adipocytes, we used PCR to amplify 3T3-L1 adipocyte complementary DNA (cDNA) with primers homologous to the N- and C-termini of pancreatic glucagon-like peptide-1 (GLP-1) receptor. We screened a cDNA library prepared from fully differentiated 3T3-L1 adipocytes using a 500-bp cDNA PCR product probe. Herein describes the isolation and characterization of a 1.6-kb cDNA clone that encodes a novel 298-amino acid protein that we termed TPRA40 (transmembrane domain protein of 40 kDa regulated in adipocytes). TPRA40 has seven putative transmembrane domains and shows little homology with the known GLP-1 receptor or with other G protein-coupled receptors. The levels of TPRA40 mRNA and protein were higher in 3T3-L1 adipocytes than in 3T3-L1 fibroblasts. TPRA40 is present in a number of mouse and human tissues. Interestingly, TPRA40 mRNA levels were significantly increased by 2- to 3-fold in epididymal fat of 24-month-old mice vs. young controls as well as in db/db and ob/ob mice vs. nondiabetic control littermates. No difference in TPRA40 mRNA levels was observed in brain, heart, skeletal muscle, liver, or kidney. Furthermore, no difference in TPRA40 expression was detected in brown fat of ob/ob mice when compared with age-matched controls. Taken together, these data suggest that TPRA40 represents a novel membrane-associated protein whose expression in white adipose tissue is altered with aging and type 2 diabetes.     

Cloning and cDNA sequence of the dihydrolipoamide dehydrogenase component human alpha-ketoacid dehydrogenase complexes.

cDNA clones comprising the entire coding region for human dihydrolipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4) have been isolated from a human liver cDNA library. The cDNA sequence of the largest clone consisted of 2082 base pairs and contained a 1527-base open reading frame that encodes a precursor dihydrolipoamide dehydrogenase of 509 amino acid residues. The first 35-amino acid residues of the open reading frame probably correspond to a typical mitochondrial import leader sequence. The predicted amino acid sequence of the mature protein, starting at the residue number 36 of the open reading frame, is almost identical (greater than 98% homology) with the known partial amino acid sequence of the pig heart dihydrolipoamide dehydrogenase. The cDNA clone also contains a 3' untranslated region of 505 bases with an unusual polyadenylylation signal (TATAAA) and a short poly(A) track. By blot-hybridization analysis with the cDNA as probe, two mRNAs, 2.2 and 2.4 kilobases in size, have been detected in human tissues and fibroblasts, whereas only one mRNA (2.4 kilobases) was detected in rat tissues.     

Differential expression of preprosomatostatin- and somatostatin receptor-encoding mRNAs in association with the growth hormone-insulin-like growth factor system during embryonic development of rainbow trout (Oncorhynchus mykiss)

Rainbow trout were used to evaluate the relationship between the somatostatin (SS) signaling and the growth hormone (GH)-insulin-like growth factor (IGF) systems during pre-hatch and post-hatch embryonic development. The expression of preprosomatostatins (PPSS), SS receptors (SSTR), GH receptors (GHR), IGF-1, IGF-2, and IGF type 1 receptors (IGFR1) was examined in various regions at the eyed-egg (29 days post-fertilization, dpf;), post-hatch (53dpf), swim-up (68dpf), and complete yolk-absorbed (90dpf) stages. In head, PPSSI mRNA abundance increased during development while that of PPSSII' decreased and that of PPSSII' remained unchanged. In body and tail, mRNA abundance of all PPSSs remained unchanged except that of PPSSII' which declined in the tail. SSTR expression increased as development progressed in all regions with the exception of SSTR1A mRNA which remained unchanged. mRNA levels of GHR1 declined in all regions of post-hatch embryos, whereas those of GHR2 remained unchanged. Expression of IGF-1 and IGF-2 in head and tail regions increased immediately after hatching, and then declined, whereas the expression of neither IGF changed during development in the body. The expression of IGFR1 mRNAs declined in all regions, reaching their lowest levels at 90dpf, with the exception of IGFR1A mRNA in the body which remained unchanged. The general decline in the expression of GH-IGF system components during development appears inversely related to a general increase in the expression of SS system elements, and suggests that these two systems interact to regulate the tissue expansion and tissue regression of embryogenesis.     

Identification and tissue distribution of mRNAs encoding salmon-type calcitonins-IV and -V in the rainbow trout

Four types of calcitonin are produced in salmonid fish, although their functional diversity is almost unknown. To explore the significance of these isoforms, we have characterized salmon-type calcitonin (sCT) mRNAs in the rainbow trout (Oncorhynchus mykiss), and examined their tissue distribution. In addition to the previously isolated sCT-I cDNAs, two new forms of sCT cDNA were cloned from the ultimobranchial gland, and one of them (sCT-IV cDNA) was predicted to encode an N-terminal peptide of 80 amino acid residues, a putative cleavage site Lys-Arg, sCT-IV, a cleavage and amidation sequence Gly-Lys-Lys-Arg, and a C-terminal peptide of 18 amino acids. The sCT-IV precursor was 78% identical with the rainbow trout sCT-I precursors. The other cloned cDNA encoded a precursor for a novel CT, sCT-V. The sCT-V peptide was different from sCT-IV by only one amino acid residue: Val at position 8 in the latter was replaced by Met. The sCT-V precursor had 80 and 90% identity with the sCT-I and -IV precursors respectively. No cDNA clones were obtained for sCTs-II or -III.Tissue distribution of sCT-I, -IV and -V mRNAs was examined by RT-PCR and specific cleavage with restriction enzymes. An amplified fragment from sCT-I mRNA was detected not only in the ultimobranchial gland, but also in the gills, testis and ovary. RT-PCR analysis coupled to restriction digestion further revealed that sCT-IV mRNA was expressed in both the testis and the ultimobranchial gland. The expression sites of sCT-IV mRNA were localized to the Leydig cells of the testis and to the parenchymal cells of the ultimobranchial gland, by in situ hybridization histochemistry. Although the amino acid sequence of sCT-V peptide was nearly the same as that of sCT-IV, the sCT-V gene showed a much wider pattern of expression: the band amplified by RT-PCR was detected in all the tissues examined except the kidney, gills and blood cells. The sCT-V mRNA was shown to be localized in the parenchymal cells of the ultimobranchial gland, but not in other tissues at the cellular level, suggesting very low expression of sCT-V mRNA in those tissues. Our results show different patterns of tissue expression of three types of sCT genes in the rainbow trout, suggesting that sCTs-I, -IV and -V might differ in their local actions.