Human Insulin-like Peptide 3 Research Articles

Expression and Role of INSL3 in the Fetal Testis.

Insulin-like peptide 3 (INSL3) is a small peptide hormone of the insulin-relaxin family which is produced and secreted by the fetal Leydig cells in the testes only. It appears to be undetectable in female fetuses. In the human fetus INSL3 synthesis begins immediately following gonadal sex determination at weeks 7 to 8 post coitum and the peptide can be detected in amniotic fluid 1 to 2 weeks later. INSL3 acts through a unique G-protein-coupled receptor, called RelaXin-like Family Peptide receptor 2 (RXFP2), which is expressed by the mesenchymal cells of the gubernacular ligament linking the testes to the inguinal wall. The role of INSL3 in the male fetus is to cause a thickening of the gubernaculum which then retains the testes in the inguinal region, while the remainder of the abdominal organs grow away in an antero-dorsal direction. This represents the first phase of testis descent and is followed later in pregnancy by the second inguino-scrotal phase whereby the testes pass into the scrotum through the inguinal canal. INSL3 acts as a significant biomarker for Leydig cell differentiation in the fetus and may be reduced by maternal exposure to endocrine disrupting chemicals, such as xenoestrogens or phthalates, leading to cryptorchidism. INSL3 may have other roles within the fetus, but as a Leydig cell biomarker its reduction acts also as a surrogate for anti-androgen action.

Insulin-Like Peptide 3 (INSL3) Serum Concentration During Human Male Fetal Life.

Context: Insulin-like peptide 3 (INSL3), a protein hormone produced by Leydig cells, may play a crucial role in testicular descent as male INSL3 knockout mice have bilateral cryptorchidism. Previous studies have measured human fetal INSL3 levels in amniotic fluid only.Objective: To measure INSL3 serum levels and mRNA in fetal umbilical cord blood and fetal testes, respectively.Design: INSL3 concentrations were assayed on 50 μl of serum from male human fetal umbilical cord blood by a non-commercial highly sensitive and specific radioimmunoassay. For secondary confirmation, quantitative real-time PCR was used to measure INSL3 relative mRNA expression in 7 age-matched human fetal testes.Setting: UT Southwestern Medical Center, Dallas, TX and Medical University of South Carolina, Charleston, SC.Patients or other Participants: Twelve human male umbilical cord blood samples and 7 human male testes were obtained from fetuses 14–21 weeks gestation. Male sex was verified by leukocyte genomic DNA SRY PCR.Interventions: None.Main Outcome Measures: Human male fetal INSL3 cord blood serum concentrations and testicular relative mRNA expression.Results: INSL3 serum concentrations during human male gestational weeks 15–20 were 2–4 times higher than published prepubertal male levels and were 5–100 times higher than previous reports of INSL3 concentrations obtained from amniotic fluid. Testicular fetal INSL3 mRNA relative expression was low from weeks 14–16, rose significantly weeks 17 and 18, and returned to low levels at week 21.Conclusions: These findings further support the role of INSL3 in human testicular descent and could prove relevant in uncovering the pathophysiology of cryptorchidism.

INSL3 stimulates spermatogonial differentiation in testis of adult zebrafish (Danio rerio).

INSL3 (insulin-like peptide 3) is a relaxin peptide family member expressed by Leydig cells in the vertebrate testis. In mammals, INSL3 mediates testicular descent during embryogenesis but information on its function in adults is limited. In fish, the testes remain in the body cavity, although the insl3 gene is still expressed, suggesting yet undiscovered, evolutionary older functions. Anti-Müllerian hormone (Amh), in addition to inhibiting spermatogonial differentiation and androgen release, inhibits the Fsh (follicle-stimulating hormone)-induced increase in insl3 transcript levels in zebrafish testis. Therefore, the two growth factors might have antagonistic effects. We examine human INSL3 (hINSL3) effects on zebrafish germ cell proliferation/differentiation and androgen release by using a testis tissue culture system. hINSL3 increases the proliferation of type A undifferentiated (Aund) but not of type A differentiating (Adiff) spermatogonia, while reducing the proliferation of Sertoli cells associated with proliferating Aund. Since the area occupied by Aund decreases and that of Adiff increases, we conclude that hINSL3 recruits Aund into differentiation; this is supported by the hINSL3-induced down-regulation of nanos2 transcript levels, a marker of single Aund spermatogonia in zebrafish and other vertebrates. Pulse-chase experiments with a mitosis marker also indicate that hINSL3 promotes spermatogonial differentiation. However, hINSL3 does not modulate basal or Fsh-stimulated androgen release or growth factor transcript levels, including those of amh. Thus, hINSL3 seems to recruit Aund spermatogonia into differentiation, potentially mediating an Fsh effect on spermatogenesis.

Identification of Key Residues Essential for the Structural Fold and Receptor Selectivity within the A-chain of Human Gene-2 (H2) Relaxin

Human gene-2 (H2) relaxin is currently in Phase III clinical trials for the treatment of acute heart failure. It is a 53-amino acid insulin-like peptide comprising two chains and three disulfide bonds. It interacts with two of the relaxin family peptide (RXFP) receptors. Although its cognate receptor is RXFP1, it is also able to cross-react with RXFP2, the native receptor for a related peptide, insulin-like peptide 3. In order to understand the basis of this cross-reactivity, it is important to elucidate both binding and activation mechanisms of this peptide. The primary binding mechanism of this hormone has been extensively studied and well defined. H2 relaxin binds to the leucine-rich repeats of RXFP1 and RXFP2 using B-chain-specific residues. However, little is known about the secondary interaction that involves the A-chain of H2 relaxin and transmembrane exoloops of the receptors. We demonstrate here through extensive mutation of the A-chain that the secondary interaction between H2 relaxin and RXFP1 is not driven by any single amino acid, although residues Tyr-3, Leu-20, and Phe-23 appear to contribute. Interestingly, these same three residues are important drivers of the affinity and activity of H2 relaxin for RXFP2 with additional minor contributions from Lys-9, His-12, Lys-17, Arg-18, and Arg-22. Our results provide new insights into the mechanism of secondary activation interaction of RXFP1 and RXFP2 by H2 relaxin, leading to a potent and RXFP1-selective analog, H2:A(4-24)(F23A), which was tested in vitro and in vivo and found to significantly inhibit collagen deposition similar to native H2 relaxin.

Insulin-like peptide 3 stimulates testosterone secretion in mouse Leydig cells via cAMP pathway

Testicular Leydig cells secrete insulin-like peptide 3 (INSL3) and express its receptor, RXFP2. However, the effects of INSL3 on endocrine function of Leydig cells are unknown. The present study examines the effects of INSL3 on mouse Leydig cells taking testosterone and cAMP secretions as endpoints. Leydig cells were isolated from testicular interstitial cells obtained from 8-week-old male mice. Cells were then plated in the presence or absence of mouse, human, canine or bovine INSL3 (0–100ng/ml) for 18h in multiwell-plates (96 wells) in different cell densities (2500, 5000, 10,000 or 20,000 cells per well). The effects of bovine INSL3 (100ng/ml) on testosterone secretion by Leydig cells were examined in the presence or absence of, an adenylate cyclase inhibitor, SQ 22536 (1μM) or INSL3 antagonist (bovine and human; 100ng/ml). Testosterone and cAMP in spent medium were measured by enzyme immunoassay. All INSL3 species tested significantly stimulated the testosterone secretion in Leydig cells, and the maximum stimulation was observed with 100ng/ml bovine INSL3 at the lowest Leydig cell density (2500 cells per well). Moreover, bovine INSL3 (100ng/ml) significantly stimulated the cAMP production from Leydig cells maximally at 1h, and remained significantly elevated even at 18h. SQ 22536 and INSL3 antagonists (bovine and human) significantly reduced INSL3-stimulated testosterone secretion from Leydig cells. Taken together, stimulatory effects of INSL3 on testosterone secretion in Leydig cells are exerted via the activation of cAMP, suggesting a new autocrine function of INSL3 in males.

In Vitro Degradation of Insulin-like Peptide 3 by Insulin-degrading Enzyme

Insulin-like peptide 3 (INSL3) is an insulin superfamily peptide hormone, primarily expressed in the testes and playing a key role in the fetus testes descent and suppression of male germ cell apoptosis. Insulin-degrading enzyme (IDE) is a zinc-metalloprotease, responsible for in vivo degradation of insulin, Abeta, and other peptide hormones. IDE has high expression level in the testes, implying it might be involved in INSL3 turnover in vivo. In present work, we studied in vitro degradation of INSL3 by IDE. Recombinant human IDE degraded human INSL3, but its degradation rate for INSL3 is more than a magnitude lower than that for insulin. However, IDE bound INSL3 and insulin with almost same affinity. IDE cleaved the peptide bond between B26R and B27W of INSL3, and released a pentapeptide, WSTEA, from the C-terminal of B-chain. Our present work suggested that IDE might play a role in INSL3 degradation in vivo.

Recombinant expression of an insulin-like peptide 3 (INSL3) precursor and its enzymatic conversion to mature human INSL3

Insulin-like peptide 3 (INSL3), which is primarily expressed in the Leydig cells of the testes, is a member of the insulin superfamily of peptide hormones. One of its primary functions is to initiate and mediate descent of the testes of the male fetus via interaction with its G protein-coupled receptor, RXFP2. Study of the peptide has relied upon chemical synthesis of the separate A- and B-chains and subsequent chain recombination. To establish an alternative approach to the preparation of human INSL3, we designed and recombinantly expressed a single-chain INSL3 precursor in Escherichia coli cells. The precursor was solubilized from the inclusion body, purified almost to homogeneity by immobilized metal-ion affinity chromatography and refolded efficiently in vitro. The refolded precursor was subsequently converted to mature human INSL3 by sequential endoproteinase Lys-C and carboxypeptidase B treatment. CD spectroscopic analysis and peptide mapping showed that the refolded INSL3 possessed an insulin-like fold with the expected disulfide linkages. Recombinant human INSL3 demonstrated full activity in stimulating cAMP activity in RXFP2-expressing cells. Interestingly, the activity of the single-chain precursor was comparable with that of the mature two-chain INSL3, suggesting that the receptor-binding region within the mid- to C-terminal of B-chain is maintained in an active conformation in the precursor. This study not only provides an efficient approach for mature INSL3 preparation, but also resulted in the acquisition of a useful single-chain template for additional structural and functional studies of the peptide.

Lysosomal Acid Hydrolases of the Cathepsin Family Are Novel Targets of INSL3 in Human Thyroid Carcinoma Cells

Insulin-like peptide 3 (INSL3) is present in hyperactive and neoplastic thyrocytes, but the functional role of this relaxin-like peptide hormone during carcinogenesis in the thyroid gland is currently unknown. We generated new cell models of stable transfectants of the human follicular thyroid carcinoma cell line FTC-133 expressing and secreting bioactive human INSL3. These transfectants displayed higher intracellular ATP levels, but INSL3 failed to act as a promoter of growth. The acquisition of an invasive tumor cell phenotype with local tissue invasion represents the beginning of a number of events leading to metastasis, the major cause of fatal outcome in cancer patients. Here we demonstrate a function of INSL3 in elastin degradation, which is considered an early step during basal membrane penetration and tissue invasion by tumor cells. INSL3 markedly increased the production of the lysosomal enzymes cathepsin-L and cathepsin-D. Enhanced secretion of the elastinolytic cathepsin-L was associated with increased elastinolytic activity of FTC-133-INSL3 transfectants. Thus, we provide the first evidence that the INSL3 peptide can promote early tumor cell invasiveness in human thyroid carcinoma cells by enhancing their metabolic activity and elastin-degrading potential.

Nuclear Receptors, Testosterone, and Posttranslational Modifications in Human INSL3 Promoter Activity in Testicular Leydig Cells

Insulin-like peptide 3 (INSL3) is a hormone produced by fetal and adult Leydig cells of the mammalian testis. During embryonic life INSL3 is required for testicular descent, whereas in adults it is involved in bone metabolism and male germ cell survival. Despite these important roles, the molecular mechanisms regulating INSL3 expression remain poorly understood. So far, two transcription factors have been implicated in INSL3 transcription: the nuclear receptors SF1 and NUR77. Circumstantial evidence also points to a role for androgens. Using transient transfections in MA-10 Leydig cells, we found that testosterone regulates in a time- and dose-dependent manner the human INSL3 promoter. The INSL3 promoter, however, does not contain a classical androgen-responsive element. Testosterone responsiveness was found to be mediated through an element located in the proximal INSL3 promoter, which also contains a NUR77-SF1-binding site. Furthermore, we found that posttranslational modifications, such as phosphorylation and acetylation, modulate transcription factor activity and therefore also contribute to INSL3 promoter activity in Leydig cells. All together, these data provide new insights into the molecular mechanisms regulating INSL3 expression in the mammalian testis.

Development of Lanthanide-Labeled Human INSL3 as an Alternative Probe to Radioactively Labeled INSL3 for Use in Bioassays

The use of radioisotope-labeled peptides and proteins in bioassays is diminishing as a consequence of the emergence of fluorescent lanthanide-labeled probes. We describe a simple, single-step labeling of synthetic human insulin-like peptide 3 (INSL3) with a commercially available activated europium chelator. The labeled peptide was then used for the development of a robust and highly reproducible receptor-binding assay which is amenable to automated high-throughput screening of ligands. Europium-labeled INSL3 showed a similar binding profile to radioactively labeled INSL3.

Leucine-rich repeat-containing G-protein-coupled receptor 8 in the rat brain: Enrichment in thalamic neurons and their efferent projections

Leucine-rich repeat-containing G-protein-coupled receptor 8 (LGR8; also classified as relaxin family peptide 2 receptor; RXFP2) has been identified as a cognate receptor for the peptide hormone, insulin-like peptide 3 (INSL3) and INSL3-LGR8 signaling plays an essential role in testis descent and germ cell development in human and rodents. Lgr8 mRNA has been detected in human tissues including testis, kidney and brain, but its regional and cellular distribution in these tissues in human or other species is largely unknown. In an initial step to elucidate the physiological function of a putative INSL3-LGR8 system in rat brain, the localization of Lgr8 mRNA was investigated using in situ hybridization histochemistry, revealing a discrete distribution in forebrain, with expression highly enriched in the thalamus. High densities were detected in the parafascicular nucleus (Pf), the dorsolateral, ventrolateral and posterior thalamic nuclei, and in the medial habenula. Lgr8 transcripts were also detected in frontal and motor cortices. The comparative distribution of LGR8 (receptor protein) was examined by autoradiography of [ 125I]-human INSL3 binding sites, with high densities detected in the thalamus, especially in Pf, and in the entire striatum—the caudate putamen (CPμ), islands of Calleja, olfactory tubercle, nucleus accumbens—with lower levels in distinct layers of cerebral cortex. Notably, these areas also receive dopaminergic projections. These findings demonstrate the existence of LGR8 in neuronal soma in the thalamus and axons/terminals in thalamic target areas such as the striatum and frontal cortex. LGR8 was also detected throughout the medial habenula–fasciculus retroflexus–interpeduncular nucleus pathway, further indicating that the receptor is transported from mRNA-expressing soma to remote axonal/terminal sites. These findings suggest the existence of a broadly distributed LGR8 signaling system in the rat involved in sensorimotor, limbic and cognitive functions. Further studies are now required to elucidate the precise function of LGR8, under normal and pathological conditions, as importantly, several of the equivalent receptor-positive areas in human brain are part of the pathology of neurodegenerative conditions including Parkinson's disease.

Solid-Phase Synthesis of Europium-Labeled Human INSL3 as a Novel Probe for the Study of Ligand−Receptor Interactions

An efficient solid-phase synthesis protocol has been developed which, together with regioselective sequential formation of the three disulfide bonds, enabled the preparation of specifically monolanthanide (europium)-labeled human insulin-like peptide 3 (INSL3) for the study of its interaction with its G-protein-coupled receptor, RXFP2, via time-resolved fluorometry. A commercially available chelator, diethylene triamine pentaacetic acid (DTPA), was coupled to the N-terminus of the INSL3 A-chain on the solid phase, and then a coordination complex between europium ion and DTPA was formed using EuCl 3 to protect the chelator from production of an unidentified adduct during subsequent combination of the A- and B-chains. The labeled peptide was purified in high yield using high-performance liquid chromatography with nearly neutral pH buffers to prevent the liberation of Eu (3+) from the chelator. Using time-resolved fluorometry, saturation binding assays were undertaken to determine the binding affinity (p K d) of labeled INSL3 for RXFP2 in HEK-293T cells stably expressing RXFP2. The dissociation constant of DTPA-labeled INSL3 (9.05 +/- 0.03, n = 3) that was obtained from saturation binding experiments was comparable to that of (125)I-labeled INSL3 (9.59 +/- 0.09, n = 3). The receptor binding affinity (p K i) of human INSL3 was determined to be 9.27 +/- 0.06, n = 3, using Eu-DTPA-INSL3 as a labeled ligand, which again is similar to that obtained when (125)I-INSL3 was used as labeled ligand (9.34 +/- 0.02, n = 4). This novel lanthanide-coordinated, DTPA-labeled INSL3 has excellent sensitivity, stability, and high specific activity, properties that will be particularly beneficial in high-throughput screening of INSL3 analogues in structure-activity studies.

Defining the LGR8 Residues Involved in Binding Insulin-Like Peptide 3

The peptide hormone insulin-like peptide 3 (INSL3) is essential for testicular descent and has been implicated in the control of adult fertility in both sexes. The human INSL3 receptor leucine-rich repeat-containing G protein-coupled receptor 8 (LGR8) binds INSL3 and relaxin with high affinity, whereas the relaxin receptor LGR7 only binds relaxin. LGR7 and LGR8 bind their ligands within the 10 leucine-rich repeats (LRRs) that comprise the majority of their ectodomains. To define the primary INSL3 binding site in LGR8, its LRRs were first modeled on the crystal structure of the Nogo receptor (NgR) and the most likely binding surface identified. Multiple sequence alignment of this surface revealed the presence of seven of the nine residues implicated in relaxin binding to LGR7. Replacement of these residues with alanine caused reduced [(125)I]INSL3 binding, and a specific peptide/receptor interaction point was revealed using competition binding assays with mutant INSL3 peptides. This point was used to crudely dock the solution structure of INSL3 onto the LRR model of LGR8, allowing the prediction of the INSL3 Trp-B27 binding site. This prediction was then validated using mutant INSL3 peptide competition binding assays on LGR8 mutants. Our results indicated that LGR8 Asp-227 was crucial for binding INSL3 Arg-B16, whereas LGR8 Phe-131 and Gln-133 were involved in INSL3 Trp-B27 binding. From these two defined interactions, we predicted the complete INSL3/LGR8 primary binding site, including interactions between INSL3 His-B12 and LGR8 Trp-177, INSL3 Val-B19 and LGR8 Ile-179, and INSL3 Arg-B20 with LGR8 Asp-181 and Glu-229.

Solution Structure and Characterization of the LGR8 Receptor Binding Surface of Insulin-like Peptide 3

Insulin-like peptide 3 (INSL3), a member of the relaxin peptide family, is produced in testicular Leydig cells and ovarian thecal cells. Gene knock-out experiments have identified a key biological role in initiating testes descent during fetal development. Additionally, INSL3 has an important function in mediating male and female germ cell function. These actions are elicited via its recently identified receptor, LGR8, a member of the leucine-rich repeat-containing G-protein-coupled receptor family. To identify the structural features that are responsible for the interaction of INSL3 with its receptor, its solution structure was determined by NMR spectroscopy together with in vitro assays of a series of B-chain alanine-substituted analogs. Synthetic human INSL3 was found to adopt a characteristic relaxin/insulin-like fold in solution but is a highly dynamic molecule. The four termini of this two-chain peptide are disordered, and additional conformational exchange is evident in the molecular core. Alanine-substituted analogs were used to identify the key residues of INSL3 that are responsible for the interaction with the ectodomain of LGR8. These include Arg(B16) and Val(B19), with His(B12) and Arg(B20) playing a secondary role, as evident from the synergistic effect on the activity in double and triple mutants involving these residues. Together, these amino acids combine with the previously identified critical residue, Trp(B27), to form the receptor binding surface. The current results provide clear direction for the design of novel specific agonists and antagonists of this receptor.

Analogs of Insulin-like Peptide 3 (INSL3) B-chain Are LGR8 Antagonists in Vitro and in Vivo

Insulin-like peptide 3 (INSL3) is a member of the insulin superfamily that plays an important role in mediating testes descent during fetal development. More recently, it has also been demonstrated to initiate oocyte maturation and suppress male germ cell apoptosis. These actions are mediated via a specific G-protein-coupled receptor, LGR8. Little is known regarding the structure and function relationship of INSL3, although it is believed that the principal receptor binding site resides within its B-chain. We subsequently observed that the linear B-chain alone (INSL3B-(1-31)) bound to LGR8 and was able to antagonise INSL3 stimulated cAMP accumulation in HEK-293T cells expressing LGR8. Sequentially N- and C-terminally shortened linear analogs were prepared by solid phase synthesis and subsequent assay showed that the minimum length required for binding was residues 11-27. It was also observed that increased binding affinity correlated with a corresponding increase in alpha-helical content as measured by circular dichroism spectroscopy. Molecular modeling studies suggested that judicious placement of a conformational constraint within this peptide would increase its alpha-helix content and result in increased structural similarity to the B-chain within native INSL3. Consequently, intramolecularly disulfide-linked analogs of the B-chain showed a potentiation of INSL3 antagonistic activity, as well as exhibiting increased proteolytic stability, as assessed in rat serum in vitro. Administration of one of these peptides into the testes of rats resulted in a substantial decrease in testis weight probably due to the inhibition of germ cell survival, suggesting that INSL3 antagonists may have potential as novel contraceptive agents.

Synthesis, conformation, receptor binding and biological activities of monobiotinylated human insulin-like peptide 3.

Biotin-avidin immobilization has been routinely used as a tool to study peptide-receptor and peptide-antibody interactions. Biotinylated peptides can also be employed to localize cells that express the peptides' receptor, and to analyse ligand-receptor binding. Insulin-like peptide 3 (INSL3) is a peptide hormone which contains A- and B-chains connected by two disulphide bonds and plays a role in testicular descent during sexual development. In order to study the interaction of INSL3 with its receptor LGR8, a G protein-coupled receptor, we chemically synthesized Nalpha-mono-biotinylated human INSL3 (B-hINSL3) and compared it structurally and biologically with hINSL3. Both peptides exhibited similar, but high, receptor binding affinities on human foetal kidney fibroblast 293T cells transfected human LGR8 based on a competition radioreceptor assay with 33P-labelled relaxin H2 (B33). The modified B-hINSL3 showed full biological activity as determined by the stimulation of gubernacular cell proliferation. The labelled B-hINSL3 contains a higher alpha-helix content, and this increased helical structure is accompanied by an increase in ability to stimulate cAMP accumulation in 293T cells expressing LGR8. Our results suggest that the N-terminal region of the A-chain is not involved in the interaction of INSL3 with its receptor. However, the introduction of biotin onto the N-terminus of the A-chain promoted conformational stability which, in turn, permitted better receptor activation.

Structural requirements for the interaction of sheep insulin-like factor 3 with relaxin receptors in rat atria

Relaxin is a peptide with various reproductive and nonreproductive functions. The site for the peptide–receptor interaction contains two arginines (Arg) and an isoleucine (Ile) or valine (Val) residue in the B-chain with a configuration of -Arg-X-X-X-Arg-X-X-Ile/Val-X-. The sheep insulin-like peptide 3 (INSL3), a structural homologue of relaxin, also contains the n, n+4 arginines in the B-chain but they are displaced towards the carboxyl terminus by four residues (-X-X-X-X-Arg-X-X-Val-Arg-). Human INSL3 increases the activity of human relaxin in mouse bioassays. Here, we investigated whether sheep synthetic INSL3 affects the relaxin activity in rat atria. INSL3 lacked relaxin-like agonist activity but blocked the activity of relaxin and competed for relaxin binding sites at high concentrations. We also synthesized analogues of INSL3, with amino acid substitutions in the arginine-binding region. Analogues A, D and E, which have the arginines in positions identical to relaxin, showed weak relaxin-like agonist activity. These results suggest that other sites in the relaxin molecule are involved in high-affinity peptide–receptor interaction for the production of the relaxin biological responses.