Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B-C-A with full biological activity in boars

RLF (relaxin-like factor), also known as INSL3 (insulin-like peptide 3), is a novel member of the relaxin/insulin gene family that is expressed in testicular Leydig cells. Despite the implicated role of RLF/INSL3 in testis development, its native conformation remains unknown. In the present paper we demonstrate for the first time that boar testicular RLF/INSL3 is isolated as a monomeric structure with full biological activity. Using a series of chromatography steps, the native RLF/INSL3 was highly purified as a single peak in reverse-phase HPLC. MS/MS (tandem MS) analysis of the trypsinized sample provided 66% sequence coverage and revealed a distinct monomeric structure consisting of the B-, C- and A-domains deduced previously from the RLF/INSL3 cDNA. Moreover, the N-terminal peptide was four amino acid residues longer than predicted previously. MS analysis of the intact molecule and PMF (peptide mass fingerprinting) analysis at 100% sequence coverage confirmed this structure and indicated the existence of three site-specific disulfide bonds. RLF/INSL3 retained full bioactivity in HEK (human embryonic kidney)-293 cells expressing RXFP2 (relaxin/insulin-like family peptide receptor 2), the receptor for RLF/INSL3. Furthermore, RLF/INSL3 was found to be secreted from Leydig cells into testicular venous blood. Collectively, these results indicate that boar RLF/INSL3 is secreted from testicular Leydig cells as a B-C-A monomeric structure with full biological activity.     

Reproductive biology of the relaxin-like factor (RLF/INSL3)

The relaxin-like factor (RLF), which is the product of the insulin-like factor 3 (INSL3) gene, is a new circulating peptide hormone of the relaxin-insulin family. In male mammals, it is a major secretory product of the testicular Leydig cells, where it appears to be expressed constitutively but in a differentiation-dependent manner. In the adult testis, RLF expression is a good marker for fully differentiated adult-type Leydig cells, but it is only weakly expressed in prepubertal immature Leydig cells or in Leydig cells that have become hypertrophic or transformed. It is also an important product of the fetal Leydig cell population, where it has been demonstrated using knockout mice to be responsible for the second phase of testicular descent acting on the gubernaculum. INSL3 knockout mice are cryptorchid, and in estrogen-induced cryptorchidism, RLF levels in the testis are significantly reduced. RLF is also made in female tissues, particularly in the follicular theca cells of small antral follicles and in the corpus luteum of the cycle and pregnancy. The ruminant ovary has a very high level of RLF expression, and analysis of primary cultures of ovarian theca-lutein cells indicated that, as in the testis, expression is probably constitutive but differentiation dependent. Female INSL3 knockout mice have altered estrous cycles, where RLF may be involved in follicle selection, an idea strongly supported by observations on bovine secondary follicles. Recently, a novel 7-transmembrane domain receptor (LGR8 or Great) has been tentatively identified as the RLF receptor, and its deletion in mice leads also to cryptorchidism.     

Design, synthesis and pharmacological evaluation of cyclic mimetics of the insulin-like peptide 3 (INSL3) B-chain.

Insulin-like peptide 3 (INSL3) is a peptide hormone belonging to the relaxin-insulin superfamily of peptides that plays important roles in testes descent, oocyte maturation and the control of male germ cell apoptosis. These actions are mediated via a specific G-protein coupled receptor, LGR8. Previous structure-activity studies have shown that the key binding site of INSL3 is situated within its B-chain. Recent studies in our laboratory have led to the identification of a cyclic peptide mimetic 2 of the INSL3 B-chain, which we have shown to compete with the binding of [33P]-relaxin to LGR8 expressed in HEK293T cells, and to inhibit cAMP-mediated signaling in these cells, i.e. it is an antagonist of INSL3. In order to further define the structure-activity relationships of cyclic analogues of the INSL3 B-chain, we used a structure-based approach to design a series of cyclic, disulfide-constrained INSL3 B-chain mimetics. To do this, we first created a model of the 3D structure of INSL3 using the crystal structure of human relaxin as a template. This model of INSL3 was then used as a template to design a series of disulfide-constrained mimetics of the INSL3 B-chain. The peptides were synthesized by solid-phase peptide synthesis using pseudoproline dipeptides to improve the synthesis outcome. Of the seven prepared INSL3 B-chain mimetics, three compounds were found to have partial displacement activity, while four were able to completely displace [33P]-relaxin from LGR8, including compounds that were markedly shorter than compound 2. The best of these, mimetic 6, showed significantly greater affinity for LGR8 than compound 2, but still displayed around 1000-fold less affinity for LGR8 than native INSL3. Analysis of selected mimetics for their alpha-helical content using circular dichroism (CD) spectroscopy revealed that, generally, the mimetics showed less than expected helicity. The inability of the compounds to display true native INSL3 structure is likely contributing to their reduced receptor binding affinity. We are currently examining alternative INSL3 B-chain mimetics that might better present key receptor binding residues in the native INSL3-like conformation.     

Effect of helix-promoting strategies on the biological activity of novel analogues of the B-chain of INSL3

Insulin-like 3 (INSL3) is a novel circulating peptide hormone that is produced by testicular Leydig cells and ovarian thecal and luteal cells. In males, INSL3 is responsible for testicular descent during foetal life and suppresses germ cell apoptosis in adult males, whereas in females, it causes oocyte maturation. Antagonists of INSL3 thus have significant potential clinical application as contraceptives in both males and females. Previous work has shown that the INSL3 receptor binding region is largely confined to the B-chain central α-helix of the hormone and a conformationally constrained analogue of this has modest receptor binding and INSL3 antagonist activity. In the present study, we have employed and evaluated several approaches for increasing the α-helicity of this peptide in order to better present the key receptor binding residues and increase its affinity for the receptor. Analogues of INSL3 with higher α-helicity generally had higher receptor binding affinity although other structural considerations limit their effectiveness.     

Melanin concentrating hormone (MCH): synthesis and bioactivity studies of MCH fragment analogues

Nineteen analogues of melanin concentrating hormone (MCH) were synthesized and tested for their skin-lightening activities in the in vitro eel skin (Synbranchus marmoratus) bioassay. All the analogues synthesized were fragments of the native sequence: Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val with sequential elimination of substituents from both the carboxy- and amino-termini. All the analogues that contained tryptophan in position 15 were found to be full agonists and equipotent to MCH. In the absence of Trp15, full agonist activity was maintained but potency was reduced ten-fold or more. The minimal fragment analogue possessing equipotency to the parent peptide, MCH, was the MCH(5-15) sequence. These observations coupled with results from work reported previously by our laboratories suggest the importance of the Trp15 residue for interaction with the MCH receptor in this assay system.     

Chemical synthesis and biological activity of rat INSL3.

The recently identified protein, insulin 3 (INSL3), has structural features that make it a bona fide member of the insulin superfamily. Its predicted amino acid sequence contains the classic two-peptide chain (A- and B-) structure with conserved cysteine residues that results in a disulphide bond disposition identical to that of insulin. Recently, the generation of insl3 knockout mice has demonstrated that testicular descent is blocked due to the failure of a specific ligament, the gubernaculum, to develop. The mechanism by which INSL3 exerts its action on the gubernaculum is currently unknown. The purpose of this study was to, for the first time, synthesize rat INSL3 and test its action on organ cultures of foetal rat gubernaculum. INSL3 also contains a cassette of residues Arg-X-X-X-Arg within the B-chain, a motif that is essential for characteristic activity of another related member of the superfamily, relaxin. Hence, the relaxin activity of rat INSL3 was also tested in two different relaxin bioassays. The primary structure of rat INSL3 was determined by deduction from its cDNA sequence and successfully prepared by solid phase peptide synthesis of the two constituent chains followed by their combination in solution. Following confirmation of its chemical integrity by a variety of analytical techniques, circular dichroism spectroscopy confirmed the presence of high beta-turn and alpha-helical content, with a remarkable spectral similarity to the synthetic ovine INSL3 peptide and to synthetic rat relaxin. The synthetic rat INSL3 bound with very low affinity to rat relaxin receptors and had no activity in a relaxin bioassay. Furthermore, it did not augment or antagonize relaxin activity. The rat INSL3 did however induce growth of foetal rat gubernaculum in whole organ cultures demonstrating that INSL3 has a direct action on this structure.     

Effect of ovine prolactin on serum somatomedin bioactivity in hypophysectomized female rats.

Ovine prolactin (oPRL) increased serum somatomedin (SM) bioactivity in hypophysectomized female rats. ACTH, in small but not large doses, augmented this oPRL effect. These results suggest that in the female rat PRL may regulate SM production. Adrenal factors may variably modulate SM production or serum SM bioactivity.     

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.     

Structure of the transmembrane signal initiation site of the relaxin-like factor (RLF/INSL3)

We have discovered the signal initiation structure of the relaxin-like factor and shown its function to be independent of the amino acid side chains in the contact region. Evidence presented in this article suggests that signal induction is a function of the peptide bond and that completion of the signaling contact is initiated by ligand binding to the leucine-rich repeat G-protein coupled receptor 8 (LGR8). The specific mode of binding forces certain peptide bonds into a signaling position. This observation implies that the receiving structures are equally nonspecific so that signaling should occur at any peptide bond of the receptor or the trans-membrane loop that is within reach of the signaling wires of the receptor-bound ligand. Our observations offer an explanation for ligand cross-talk as well as for the ability of some antibodies to elicit the biological response normally associated with a specific ligand.     

New antitumour cyclic astin analogues: synthesis, conformation and bioactivity.

Astins, antitumour cyclic pentapeptides, were isolated from the Aster tataricus. Their chemical structures, consist of a 16-membered ring system containing a unique beta,gamma-dichlorinated proline [Pro(Cl)2], other non-coded amino acid residues and a cis conformation in one of the peptide bonds. The astin backbone conformation, along with the cis peptide bond in which the beta,gamma-dichlorinated proline residue is involved, was considered to play an important role in their antineoplastic activities on sarcoma 180A and P388 lymphocytic leukaemia in mice, but the scope and potential applications of this activity remain unclear. With the aim at improving our knowledge of the conformational properties influencing the bioactivity in this class of compounds, new astin-related cyclopeptides were synthesized differing from the natural products by the presence of some non-proteinogenic amino acid residues: Aib, Abu, -(S)beta3-hPhe and a peptide bond surrogate (-SO2-NH-). The analogues prepared c(-Pro-Thr-Aib-beta3-Phe-Abu-), c[Pro-Thr-Aib-(S)beta3-hPhe-Abu], c[Pro-Abu-Ser-(S)beta3-hPhe psi(CH2-SO2-NH)-Abu] and c[Pro-Thr-Aib-(S)beta3-hPhe psi(CH2-SO2-NH)-Abu] were synthesized by classical methods in solution and tested for their antitumour effect. These molecules were studied by crystal-state x-ray diffraction analysis and/or solution NMR and MD techniques.     

Synthesis and bioactivity of 2,4-diacyl analogues of paclitaxel

The 2,4-diacyl paclitaxel analogues 8a-8r were prepared from paclitaxel by acylation of 4-deacetyl-2-debenzoylpaclitaxel 1,2-carbonate (3) followed either by hydrolysis of the carbonate and acylation or by direct treatment of the carbonate with an aryllithium. Some of the resulting derivatives showed significantly improved tubulin assembly activity and cytotoxicity as compared with paclitaxel; in some cases this improvement was especially significant for paclitaxel-resistant cell lines.     

Synthesis and bioactivity studies of two isosteric acyclic analogues of melanin concentrating hormone.

Salmon melanin concentrating hormone (MCH) is a cyclic heptadecapeptide. MCH stimulates perinuclear aggregation of melanosomes within integumental melanocytes of teleost fishes resulting in skin blanching. MCH contains a disulfide bridge forming a 10-residue ring [sequence: see text]. It has been proposed that the ring is necessary for maintenance of potency. In order to test this proposal, we have synthesized two pseudo-isosteric analogues of MCH that cannot cyclize. They differed only in the polarity of the side chain group of positions 5 and 14. Serine was substituted for Cys5 and Cys14 in one peptide and L alpha-aminobutyrate (Abu) was the substitution at the two positions in the other peptide. Using a fish skin bioassay we determined that these analogues exhibit less than 1/10,000th the potency of the native hormone. These results suggest that the disulfide bridge is necessary to maintain the correct conformational and topographical features of the hormone for receptor binding and transmembrane signal transduction.     

Insulin and (or) dexamethasone effects on leptin production and metabolic activities of ovine adipose tissue explants

The in vitro effects of insulin and/or dexamethasone (DEX) on leptin production were studied on adipose tissue (AT) from adult non-lactating, non-pregnant ewes. Perirenal AT explants were incubated for 2 or 4 days and leptin production was determined using a specific ovine RIA. The effects of these hormones were simultaneously measured on glucose and acetate utilisation and on lipogenic enzyme activities. A preliminary dose-response study showed a maximal leptin production by the addition in the incubation medium of 2 mIU x mL(-1) of insulin and 100 nM of DEX. By using these concentrations, insulin or DEX increased leptin production by ovine AT explants whatever the incubation duration and the effects of these two hormones were additive. Insulin also increased substrate utilisation as well as lipogenic enzyme activities while DEX decreased substrate utilisation and did not change the lipogenic enzyme activities. To conclude, leptin response to DEX is specific and largely independent of the overall metabolic or lipogenic activity.     

Insulin analogues - state of the art

The use of insulin analogues is rapidly expanding. At present, there are two short-acting analogues available for practical use, insulin aspart and insulin lispro, and one long-acting analogue, insulin glargine. Another long-acting analogue, insulin detemir, is still under development. The time action profile of short-acting analogues is both much more rapid and shorter than that of human insulin; the prominent feature of the long-acting analogues is their peakfree and fairly constant action. Insulin analogues offer alternative options for the whole spectrum of insulin therapy in type 1 and type 2 diabetes patients. The perception by many patients is strikingly positive, in particular regarding the overall quality of life. In objective efficacy terms, however, the potential to improve the degree of metabolic control appears to be only minor, yet demonstrable, provided the analogues are used according to their specific time action profile. This ensured, analogues are instrumental in minimizing the side effects of insulin therapy, i.e. the risk of (nocturnal) hypoglycaemia or problems with body weight control. Although there are no indications of safety concerns with insulin analogues, the availability of long-term outcome data based upon observations in human patients would be very valuable.