Insulin-like Peptide Research Articles

Insulin Stabilization Designs for Enhanced Therapeutic Efficacy and Accessibility.

ConspectusInsulin has remained indispensable in the treatment of diabetes since it was first discovered in 1921. Unlike small molecular drugs, insulin and other protein drugs are prone to degradation when exposed to elevated temperatures, mechanical agitation during transportation, and prolonged storage periods. Therefore, strict cold-chain management is crucial for the insulin supply, requiring significant resources, which can limit the access to insulin, particularly in low-income areas. Moreover, although insulin formulations have advanced tremendously in the last century, insulin treatment still imposes a challenging regimen and provides suboptimal outcomes for the majority of patients. There is an increasing focus on pursuing improved pharmacology, specifically on safer, more user-friendly insulin therapies that minimize the self-management burden. These challenges underscore the need for developing novel insulin formulations with improved stability that are compatible with advanced insulin therapy.Insulin stabilization can be achieved through either chemical modification of insulin or formulation component design. Inspired by insulin-like peptides from invertebrates, we have developed novel stable insulin analogs based on a fundamental understanding of the insulin receptor engagement for insulin bioactivity. We created a novel four-disulfide insulin analog with high aggregation stability and potency by introducing a fourth disulfide bond between a C-terminal extended insulin A-chain and residues near the C-terminus of the B-chain. In an effort to stabilize insulin in its monomeric state to develop ultrafast-acting insulin with rapid absorption upon injection, we have developed a series of structurally miniaturized yet fully active insulin analogs that do not form dimers due to the lack of the canonical B-chain C-terminal octapeptide. Additionally, our study provided strategies for expanding the scope of cucurbit[7]uril (CB[7])-assisted insulin stabilization by engineering safe and biodegradable CB[7]-zwitterionic polypeptide excipients. We also explored insulin N-terminal substitution methods to achieve pH-dependent insulin stabilization without prolonging the duration of action.This Account describes our exploration of engineering stable insulin analogs and formulation design strategies for stabilizing insulin in aqueous solutions. Beyond conventional stabilization strategies for insulin injections, the unmet challenges and recent innovations in insulin stabilization are discussed, addressing the growing demand for alternative, less invasive routes of insulin administration. Additionally, we aim to provide a thorough overview of insulin stabilization from the perspective of commercially available insulin drugs and common pharmaceutical engineering practices in the industry. We also highlight unresolved insulin stabilization challenges and ongoing research strategies. We anticipate that further emphasis on collective efforts of protein engineering, pharmaceutical formulation design, and drug delivery will inform the development of stable and advanced insulin therapy.

Sublethal concentration of λ-cyhalothrin inhibits insulin-like peptides and leads to reproductive toxicity in Chrysoperla sinica.

Insulin-like peptides (ILPs) act as crucial reproductive neuropeptides in insects, regulating insect reproduction through the insulin signaling pathway (ISP). Our previous studies have found that the sublethal concentrations (LC1 and LC10) of lambda-cyhalothrin (λCy) could induce severe reproductive toxicity in the lacewing, Chrysoperla sinica (Tjeder), but the toxicological mechanism remains unclear. This study discovered that λCy could inhibit CsILP transcription, leading to a decrease in insulin content and downregulation of C. sinica insulin receptor (CsInR) and C. sinica forkhead box O (CsFOXO) expression in ISP. Interference with CsILP expression resulted in downregulation of C. sinica vitellogenin (CsVg) and decreasing fecundity, while exogenous injection of bovine insulin promoted upregulation of CsVg expression and facilitated reproduction in female adults of C. sinica. Meanwhile, interference with FOXO of ILP downstream transcription factor could lead to downregulation of CsVg, hindering ovarian development and resulting in a decrease in egg production. However, exogenous injection of bovine insulin could remedy the effects caused by FOXO interference. In addition, ILP mediates juvenile hormone and 20-hydroxyecdysone biosynthesis by acting on their synthetic regulatory enzymes and influences the signal transduction of the 2 reproductive endocrine hormones, thereby regulating the reproductive endocrine environment in C. sinica. In conclusion, λCy inhibits CsILP expression, leading to disorder of ISP, leading to the reduced fecundity of C. sinica.

7942 Evolution of Insulin, Insulin-like Growth Factor and Their Cognate Receptors in Vertebrates, Invertebrates and Viruses

Abstract Disclosure: M. Chrudinova: None. Y.D. Dogru: None. R. Huang: None. R. Reiners: None. P. De Meyts: None. J. DaCosta: None. E. Altindis: None. The insulin/insulin-like growth factor (IGF) signaling system is an evolutionarily conserved system and controls central processes including metabolism, cellular growth, proliferation, and differentiation in vertebrates. On the other hand, invertebrate insulin-like peptides (ILPs) regulate growth, stress responses, longevity and sex-related functions. In addition, we recently showed that six viruses possess viral insulin-like peptides (VILPs) that can bind to the receptors and stimulate post-receptor signaling. The evolution of these hormones and the origin of VILPs are not completely understood. Here, we collected vertebrate and invertebrate insulin, IGF and their cognate receptor sequences to analyze their evolutionary relations. We analyzed 177 vertebrate insulins, 119 vertebrate IGF-1s, 127 vertebrate IGF-2s, 197 invertebrate ILPs and 6 viral insulin/IGF-1 like peptides. We also analyzed 214 insulin receptor (IR), 160 IGF receptor (IGF1R) and 39 invertebrate receptor sequences to identify conservation and the evolutionary trajectory to reveal conserved motifs. Through phylogenetic analyses, we observed a separate branching of invertebrate ILPs and vertebrate insulins, IGF-1s and IGF-2s with notable exceptions including VILPs. IGF-1 and IGF-2 ligands were evolutionally better conserved compared to insulin ligands. Insulin C-peptide which is processed and cleaved was not well conserved compared to the functional A- and B-chain residues. In contrast, C-peptide of IGF-1 that plays an important role in receptor binding was very well conserved. IGF-1 C-domain showed higher variability in IGF1R binding residues compared to the A and B-domains, while residues important for binding of IGF-2 to IGF1R were well conserved. Insulin residues interacting with IR binding Site 1 were more conserved than those interacting with IR binding Site 2, while residues important for insulin dimerization and hexamerization residues are well conserved across species. We also showed that VILPs contain 14 amino acids that are conserved in insulin and IGFs, indicating their functional importance. Phylogenetic relationships of the receptor sequences suggest vertebrate IRs and IGF1Rs evolved from a common ancestor during early vertebrate evolution. Interestingly, receptor subunits revealed a closer relation of amphibian IR α-subunits to Mammalia IR α-subunits than Aves and Reptilians. The conservation rate of receptor residues essential for ligand-receptor interaction revealed that these residues are best conserved in the L1 domain for IR and IGF1R, with a higher rate of variation in other domains for both receptors. Taken together, our study provides a comprehensive analysis of insulin, IGFs, VILPs and their cognate receptors’ evolution, and opens new avenues to better understand the evolution, function, and structure relations of this complex signaling system. Presentation: 6/3/2024

Antagonistic insulin-like signaling influencing glucose regulation in C. elegans

The insulin/ IGF-signaling (IIS) is an evolutionary conserved signaling pathway within eukaryotic organisms that regulates glucose metabolism and cell division. The nematode, C. elegans is an excellent model for exploring IIS as they have 40 insulin-like peptides that act on a single receptor, DAF-2. We aim to explore the relationships between IIS and glucose metabolism, focusing on the antagonistic insulin-like peptide, INS-17 and its influence over FDGT-1 (facilitated glucose transporter 1). FDGT-1 is expressed throughout the nematode, including the basolateral membrane of intestinal cells and where it is theorized to export glucose to the surrounding somatic cells rather than absorb it from the intestinal tract. We hypothesize that IIS controls transcription of FDGT-1 and antagonistic peptides should lead to a decrease in FDGT-1 transcription, resulting in the increased lifespan and increased fat accumulation observed in daf-2(lf) loss of function and ins-17(oe) over expressing strains. In this study, survival assays are used to assess the impact which high-glucose content has on lifespan, along with Oil Red O (ORO) lipid staining to visualize lipid accumulation amongst conditions and protein quantification of FDGT-1:GFP to evaluate FDGT-1 protein levels. Our findings are consistent with the idea of low insulin signaling leading to extended lifespan as daf-2(lf) lived significantly longer than WT in both 0% and 2% glucose conditions and ins-17(oe) living significantly longer than wildtype in 2% glucose conditions. The ORO lipid staining revealed that high glucose conditions and low insulin signaling result in lipid accumulation within the nematode. Using the fdgt-1:gfp strain, we were able to determine an increase in the FDGT-1 in the presence of glucose, suggesting FDGT-1 expression is influenced by glucose. These findings aim to further our understanding of IIS regulating glucose uptake and metabolism, along with the significance of the FDGT-1 glucose transporter.

Lactobacillus Firm-5-derived succinate prevents honeybees from having diabetes-like symptoms

The gut microbiome plays an important role in honeybee hormonal regulation and growth, but the underlying mechanisms are poorly understood. Here, we showed that the depletion of gut bacteria resulted in reduced expression of insulin-like peptide gene (ilp) in the head, accompanied by metabolic syndromes resembling those of Type 1 diabetes in humans: hyperglycemia, impaired lipid storage, and decreased metabolism. These symptoms were alleviated by gut bacterial inoculation. Gut metabolite profiling revealed that succinate, produced by Lactobacillus Firm-5, played deterministic roles in activating ilp gene expression and in regulating metabolism in honeybees. Notably, we demonstrated that succinate modulates host ilp gene expression through stimulating gut gluconeogenesis, a mechanism resembling that of humans. This study presents evidence for the role of gut metabolite in modulating host metabolism and contributes to the understanding of the interactions between gut microbiome and bee hosts.

Glut1 Functions in Insulin-Producing Neurons to Regulate Lipid and Carbohydrate Storage in Drosophila.

Obesity remains one of the largest health problems in the world, arising from the excess storage of triglycerides (TAGs). However, the full complement of genes that are important for regulating TAG storage is not known. The Glut1 gene encodes a Drosophila glucose transporter that has been identified as a potential obesity gene through genetic screening. Yet, the tissue-specific metabolic functions of Glut1 are not fully understood. Here, we characterized the role of Glut1 in the fly brain by decreasing neuronal Glut1 levels with RNAi and measuring glycogen and TAGs. Glut1RNAi flies had decreased TAG and glycogen levels, suggesting a nonautonomous role of Glut1 in the fly brain to regulate nutrient storage. A group of hormones that regulate metabolism and are expressed in the fly brain are Drosophila insulin-like peptides (Ilps) 2, 3, and 5. Interestingly, we observed blunted Ilp3 and Ilp5 expression in neuronal Glut1RNAi flies, suggesting Glut1 functions in insulin-producing neurons (IPCs) to regulate whole-organism TAG and glycogen storage. Consistent with this hypothesis, we also saw fewer TAGs and glycogens and decreased expression of Ilp3 and Ilp5 in flies with IPC-specific Glut1RNAi. Together, these data suggest Glut1 functions as a nutrient sensor in IPCs, controlling TAG and glycogen storage and regulating systemic energy homeostasis.

P-664 Insulin-like peptide 3 as a potential biomarker in predicting IVF success and ovarian reserve in women with unexplained infertility and diminishing ovarian reserve

Abstract Study question Could insulin-like peptide 3 (INSL3) predict ovarian reserve and in vitro fertilization (IVF) success in women with unexplained infertility (UI) and diminishing ovarian reserve (DOR)? Summary answer In women with DOR, reduced intrafollicular and serum INSL3 levels correspond with low AMH and high FSH levels, similar to established ovarian reserve markers. What is known already INSL3, belonging to the insulin-like peptide family, is produced by theca interna cells within antral follicles and the corpora lutea. It is hypothesized that INSL3 is integral to the initial development and function of antral follicles, specifically through its regulatory effect on androgen biosynthesis in the thecal cells of these follicles. Moreover, INSL3 is implicated in the modulation of the ovarian microenvironment, which is essential for facilitating the maturation of oocytes. Study design, size, duration This research was carried out at the IVF centers of two tertiary university hospitals, assessing 49 infertile women with either UI (n = 24) or DOR (n = 25) during IVF treatment. A control group (n = 26) comprised women with normal ovarian reserve undergoing IVF due to male or tubal factors. Outcome parameters are ovarian reserve markers (serum FSH, estradiol, AMH, INSL3 concentration and antral follicle count [AFC]), positive pregnancy rate and live birth rate (LBR) (gestational age ≥28 weeks). Participants/materials, setting, methods Women aged between 20-40 with a body mass index (BMI)<30 kg/m² were included. Serum FSH, estradiol, AMH, INSL3 levels and AFC were evaluated on menstrual cycle days 2 or 3, and INSL3 in follicular fluid from dominant follicles (>14 mm) were evaluated during oocyte retrieval. Exclusions included endocrine abnormalities (e.g., polycystic ovary syndrome, hyperprolactinemia, hypo and hyperthyroidism) cycle cancellation due to lack of a viable embryo, uncorrected Mullerian anomalies, recurrent miscarriage history and hydrosalpinx presence. Main results and the role of chance The mean age in the study groups was 28.41±3.5 (UI), 32.40±4.21 (DOR), and 29.65±3.91 (control). In the DOR group, female age (p = 0.001), BMI (p = 0.049), duration of infertility (p = 0.006), previous IVF attempts (p < 0.001), basal FSH (p < 0.001), and basal estradiol (p < 0.001) were higher, while AMH (p < 0.001), AFC (p < 0.001), and follicular fluid INSL3 (p < 0.001) were lower. Serum INSL3 in DOR was lower but not statistically significant. No correlation was found between serum INLS, follicular fluid INSL3, ovarian reserve markers, positive pregnancy rates, miscarriage, and LBR. DOR group had longer stimulation durations (p = 0.017), but lower max estradiol (p < 0.001), fewer oocytes (p < 0.001), mature follicles (p < 0.001), and PN (p < 0.001). Pregnancy rates were 54.1% (UI), 24% (DOR), and 53.84% (control); LBRs were 54.1% (UI), 20% (DOR), and 42.3% (control), with significant differences in pregnancy tests (p = 0.04) and LBR (p = 0.03), both lower in DOR. Univariate analysis identified duration of infertility (p = 0.019) and basal FSH (p = 0.009) as significant LBR predictors. Multivariable analysis confirmed basal FSH (p = 0.033) as significant, with increased duration of infertility and basal FSH level reducing LBR probability. Limitations, reasons for caution The limited sample size of our study potentially undermines the robustness of circulating INSL3 as a predictive biomarker for DOR, evidenced by the non-significant decrease in serum INSL3 levels in the DOR group. Moreover, the lack of INSL3 level assessment during oocyte retrieval further constrains our findings. Wider implications of the findings Our research explored INSL3 as a viable biomarker for predicting ovarian reserve and IVF success in UI and DOR cases. Reduced INSL3 levels in circulation and follicular fluid in DOR patients could be beneficial in clinical practice. Trial registration number not applicable

Identification of Two Insulin Receptors from the Swimming Crab Portunus trituberculatus: Molecular Characterization, Expression Analysis, and Interactions with Insulin-Like Androgenic Gland Hormone.

AbstractThe insulin-like androgenic gland hormone is a crucial sexual regulator that is involved in the masculine sexual differentiation of crustaceans. As an insulin-like peptide, the insulin-like androgenic gland hormone has been proposed to act through the insulin receptor-mediated pathway. The present study cloned and characterized two insulin receptors (PtIR1 and PtIR2) from the swimming crab Portunus trituberculatus hallmarked with a conserved intracellular tyrosine kinase catalytic domain and several other typical insulin receptor domains in their deduced amino acid sequences. Both insulin receptors were predominately expressed in the testis and the insulin-like androgenic gland hormone-producing organ androgenic gland. Their testicular expression during the annual cycle suggested that they may play critical roles in spermatogenesis. By using the protein colocalization analysis in HEK293 cells, interactions of PtIAG with the two PtIRs were further confirmed. In addition, the insulin receptor antagonist was found to attenuate the stimulatory effects of androgenic gland homogenate on the phosphorylated MAPK levels in testis explants, suggesting that the insulin receptor-dependent MAPK pathway may be essential for insulin-like androgenic gland hormone functions.

Drosophila melanogaster Limostatin and Its Human Ortholog Promote West Nile Virus Infection.

The arbovirus West Nile virus (WNV) is a danger to global health. Spread primarily by mosquitoes, WNV causes about 2000 cases per year in the United States. The natural mosquito immune response controls viral replication so that the host survives but can still transmit the virus. Using the genetically malleable Drosophila melanogaster model, we previously dissected innate immune pathways used to control WNV infection. Specifically, we showed that insulin/IGF-1 signaling (IIS) activates a JAK/STAT-mediated immune response that reduces WNV. However, how factors that regulate IIS in insects control infection has not been identified. D. melanogaster Limostatin (Lst) encodes a peptide hormone that suppresses insulin secretion. Its mammalian ortholog, Neuromedin U (NMU), is a peptide that regulates the production and secretion of insulin from pancreatic beta cells. In this study, we used D. melanogaster and human cell culture models to investigate the roles of these insulin regulators in immune signaling. We found that D. melanogaster Lst mutants, which have elevated insulin-like peptide expression, are less susceptible to WNV infection. Increased levels of insulin-like peptides in these flies result in upregulated JAK/STAT activity, leading to protection from infection. Treatment of human cells with the insulin regulator NMU results in increased WNV replication. Further investigation of methods to target Lst in mosquitoes or NMU in mammals can improve vector control methods and may lead to improved therapeutics for human and animal infection.

Unraveling the Roles of Neuropeptides in the Chemosensation of the Root-Knot Nematode Meloidogyne javanica.

The identification of novel drug targets in plant-parasitic nematodes (PPNs) is imperative due to the loss of traditional nematicides and a lack of replacements. Chemosensation, which is pivotal for PPNs in locating host roots, has become a focus in nematode behavioral research. However, its underlying molecular basis is still indistinct in such a diverse group of PPNs. To characterize genes participating in chemosensation in the Javanese root-knot nematode Meloidogyne javanica, RNA-sequencing of the second-stage juveniles (J2s) treated with tomato root exudate (TRE) for 1 h and 6 h was performed. Genes related to chemosensation in M. javanica mainly responded to TRE treatment at 1 h. Moreover, a gene ontology (GO) analysis underscored the significance of the neuropeptide G protein-coupled receptor signaling pathway. Consequently, the repertoire of putative neuropeptides in M. javanica, including FMRFamide-like peptides (FLPs), insulin-like peptides (ILPs), and neuropeptide-like peptides (NLPs), were outlined based on a homology analysis. The gene Mjflp-14a, harboring two neuropeptides, was significantly up-regulated at 1 h TRE treatment. Through peptide synthesis and J2 treatment, one of the two neuropeptides (MjFLP-14-2) was proven to influence the J2 chemotaxis towards tomato root tips. Overall, our study reinforces the potential of nematode neuropeptides as novel targets and tools for root-knot nematode control.

Molecular characterization and functional identification of the insulin-like peptides receptor gene in the oriental river prawn Macrobrachium nipponense

The insulin-like signaling pathway plays diverse biological roles in regulating metabolism, immune responses, growth, and development in invertebrates. The insulin-like peptides (ILP) receptor (ILPR) is the pivotal protein in any insulin-like signaling pathway. In our previous study, we cloned the Mn-ILP gene from the oriental river prawn Macrobrachium nipponense and elucidated its function. However, the ILPR has not yet been described in M. nipponense. In the present study, we further examined the function of Mn-ILPR in M. nipponense. The full length of the Mn-ILPR mRNA was 4790 bp, encoding 1412 amino acids. Mn-ILPR was a transmembrane receptor under a subfamily of receptor tyrosine kinases. The Mn-ILPR mRNA was predominantly expressed in muscle and hepatopancreas. To elucidate the function of Mn-ILPR, we investigated its expression in different growth stages of prawns of both sexes, in prawns of different sizes at the same time, and in prawns of the same size at different stages of the molt cycle using qRT-PCR. The expression of Mn-ILPR was markedly higher: (1) in the rapid growth stage than in other stages of male individuals; (2) in the normal growth stage than in the gonad development stage of female individuals; (3) in large male individuals than in small male individuals; and (4) in the intermolt stage than in other stages of the molt cycle. Furthermore, the long-term knockdown of Mn-ILPR by RNAi effectively delayed the growth speed of M. nipponense. Meanwhile, the short-term knockdown of Mn-ILPR revealed that the ILP-ILPR signal pathway played a part in the process of growth of M. nipponense. These findings provide a greater understanding of the molecular mechanisms underlying growth and development in crustaceans.

Developmental and conditional regulation of DAF-2/INSR ubiquitination in Caenorhabditis elegans.

Insulin/IGF signaling (IIS) regulates developmental and metabolic plasticity. Conditional regulation of insulin-like peptide expression and secretion promotes different phenotypes in different environments. However, IIS can also be regulated by other, less-understood mechanisms. For example, stability of the only known insulin/IGF receptor in C. elegans, DAF-2/INSR, is regulated by CHIP-dependent ubiquitination. Disruption of chn-1/CHIP reduces longevity in C. elegans by increasing DAF-2/INSR abundance and IIS activity in adults. Likewise, mutation of a ubiquitination site causes daf-2(gk390525) to display gain-of-function phenotypes in adults. However, we show that this allele displays loss-of-function phenotypes in larvae, and that its effect on IIS activity transitions from negative to positive during development. In contrast, the allele acts like a gain-of-function in larvae cultured at high temperature, inhibiting temperature-dependent dauer formation. Disruption of chn-1/CHIP causes an increase in IIS activity in starved L1 larvae, unlike daf-2(gk390525). CHN-1/CHIP ubiquitinates DAF-2/INSR at multiple sites. These results suggest that the sites that are functionally relevant to negative regulation of IIS vary in larvae and adults, at different temperatures, and in nutrient-dependent fashion, revealing additional layers of IIS regulation.

Comparative transcriptome analysis provides a glance into the regulatory of insulin-like peptide 1 gene in the Pacific white shrimp Litopenaeus vannamei

Insulin-like peptides (ILPs) have been reported to exert significant and diverse regulatory effects through the insulin signaling pathway in invertebrates. However, there is a large research gap in the functional analysis of ILP genes in crustaceans. In this study, we combined RNA interference (RNAi) technology with transcriptome sequencing to investigate the regulatory effects of the ILP1 gene in the Pacific white shrimp Litopenaeus vannamei. After two weeks of continuous RNAi of LvILP1, the shrimp in the dsLvILP1 group exhibited reduced body length gain, weight gain, molting rate, and hemolymph glucose concentration compared to the control dsEGFP group, indicating that knockdown of LvILP1 slowed shrimp growth, molting, and metabolism. Comparative transcriptome analysis of three tissues revealed that knockdown of LvILP1 inhibited muscle growth and development by modulating the mTOR and MAPK signaling pathways, and prevented the renewal of epidermal tissue through the Hippo signaling pathway. Meanwhile, it also suppressed certain growth signals in the hepatopancreas, such as juvenile hormone (JH) synthesis and 20-hydroxyecdysteroid (20E) signal activation. Additionally, it promoted the conversion pathway of some sugars into amino acid metabolism instead of energy metabolism after glycolysis in the hepatopancreas. Interestingly, knockdown of LvILP1 also affected carbohydrate metabolism by regulating the intracellular and extracellular transport of glucose and trehalose, as well as regulating the expression levels of key metabolic genes in three shrimp tissues. This disruption led to a decrease in blood glucose levels and suppressed energy production, ultimately providing less energy for shrimp growth and molting. This study provides valuable insights into the underlying genetic mechanism through which ILP1 regulates glucose metabolism and growth in shrimp. It enhances our understanding of the structure and function of the insulin pathway in crustaceans and serves as an important reference for the identification of candidate genes in molecular genetic breeding of shrimp.

A nutrient responsive lipase mediates gut-brain communication to regulate insulin secretion in Drosophila

Pancreatic β cells secrete insulin in response to glucose elevation to maintain glucose homeostasis. A complex network of inter-organ communication operates to modulate insulin secretion and regulate glucose levels after a meal. Lipids obtained from diet or generated intracellularly are known to amplify glucose-stimulated insulin secretion, however, the underlying mechanisms are not completely understood. Here, we show that a Drosophila secretory lipase, Vaha (CG8093), is synthesized in the midgut and moves to the brain where it concentrates in the insulin-producing cells in a process requiring Lipid Transfer Particle, a lipoprotein originating in the fat body. In response to dietary fat, Vaha stimulates insulin-like peptide release (ILP), and Vaha deficiency results in reduced circulatory ILP and diabetic features including hyperglycemia and hyperlipidemia. Our findings suggest Vaha functions as a diacylglycerol lipase physiologically, by being a molecular link between dietary fat and lipid amplified insulin secretion in a gut-brain axis.

Sex-specific developmental gene expression atlas unveils dimorphic gene networks in C. elegans

Sex-specific traits and behaviors emerge during development by the acquisition of unique properties in the nervous system of each sex. However, the genetic events responsible for introducing these sex-specific features remain poorly understood. In this study, we create a comprehensive gene expression atlas of pure populations of hermaphrodites and males of the nematode Caenorhabditis elegans across development. We discover numerous differentially expressed genes, including neuronal gene families like transcription factors, neuropeptides, and G protein-coupled receptors. We identify INS-39, an insulin-like peptide, as a prominent male-biased gene expressed specifically in ciliated sensory neurons. We show that INS-39 serves as an early-stage male marker, facilitating the effective isolation of males in high-throughput experiments. Through complex and sex-specific regulation, ins-39 plays pleiotropic sexually dimorphic roles in various behaviors, while also playing a shared, dimorphic role in early life stress. This study offers a comparative sexual and developmental gene expression database for C. elegans. Furthermore, it highlights conserved genes that may underlie the sexually dimorphic manifestation of different human diseases.

Expression of Insl3 Protein in Adult Danio rerio.

Insulin-like peptide 3 (INSL3) is a biomarker for Leydig cells in the testes of vertebrates, and it is principally involved in spermatogenesis through specific binding with the RXFP2 receptor. This study reports the insl3 gene transcript and the Insl3 prepropeptide expression in both non-reproductive and reproductive tissues of Danio rerio. An immunohistochemistry analysis shows that the hormone is present at a low level in the Leydig cells and germ cells at all stages of Danio rerio testis differentiation. Considering that the insl3 gene is transcribed in Leydig cells, our results highlight an autocrine and paracrine function of this hormone in the Danio rerio testis, adding new information on the Insl3 mode of action in reproduction. We also show that Insl3 and Rxfp2 belonging to Danio rerio and other vertebrate species share most of the amino acid residues involved in the ligand-receptor interaction and activation, suggesting a conserved mechanism of action during vertebrate evolution.

Gene redundancy and gene compensation of insulin-like peptides in the oocyte development of bean beetle.

Bean beetle (Callosobruchus maculatus) exhibits clear phenotypic plasticity depending on population density; However, the underlying molecular mechanism remains unknown. Compared to low-density individuals, high-density individuals showed a faster terminal oocyte maturity rate. Four insulin-like peptide (ILP) genes were identified in the bean beetle, which had higher expression levels in the head than in the thorax and abdomen. The population density could regulate the expression levels of CmILP1-3, CmILP2-3, and CmILP1 as well as CmILP3 in the head, thorax, and abdomen, respectively. RNA interference results showed that each CmILP could regulate terminal oocyte maturity rate, indicating that there was functional redundancy among CmILPs. Silencing each CmILP could lead to down-regulation of some other CmILPs, however, CmILP3 was up-regulated in the abdomen after silencing CmILP1 or CmILP2. Compared to single gene silencing, silencing CmILP3 with CmILP1 or CmILP2 at the same time led to more serious retardation in oocyte development, suggesting CmILP3 could be up-regulated to functionally compensate for the down-regulation of CmILP1 and CmILP2. In conclusion, population density-dependent plasticity in terminal oocyte maturity rate of bean beetle was regulated by CmILPs, which exhibited gene redundancy and gene compensation.

Dietary starch level regulates the growth and glycolipid metabolism through the insulin-PI3K-AKT signaling pathway of juvenile mud crab (Scylla paramamosain)

A six-week feeding trial was conducted to investigate the effects of dietary starch levels on growth performance, glucose and lipid metabolism and insulin signaling pathway of juvenile Scylla paramamosain. Three isonitrogenous (41.51% ± 0.31%) and isolipidic (8.68% ± 0.53%) experimental diets were formulated to contain 10%, 20% and 30% starch levels, respectively. A total 96 crabs were divided into 3 treatment groups. There were 4 replicates per treatment group and 8 crabs per replicate (initial weight 5.12 ± 0.05 g). The crabs fed the diet with 20% starch showed the best growth performance, with higher weight gain rate (WGR), specific growth rate (SGR), protein efficiency ratio (PER), and feed efficiency (FE) compared to the other diets. Increasing starch levels in the diet resulted in higher levels of glycogen and lipid in the muscles and hepatopancreas. The levels of glucose (GLU), triglyceride (TG), and free fatty acid (FFA) displayed a significant increase in both hemolymph and hepatopancreas with the escalating dietary starch concentrations. In contrast, as the dietary starch levels increased, the concentrations of crustacean hyperglycemic hormone (CHH) and insulin-like peptides (ILPs) in hemolymph and hepatopancreas demonstrated a notable decline. The glycolysis and pentose phosphorylation pathways were significantly promoted, while the gluconeogenic pathway was significantly inhibited, with dietary starch levels increasing from 10% to 30%. Additionally, increasing dietary starch levels led to a significant down-regulation of genes related to the lipid synthesis, lipolysis and insulin signaling pathway. Histological results showed that hepatopancreas fat accumulation increased with the increase of dietary starch level. Overall, the findings of this study suggest that adding 20% starch to the diet can promote growth and improve nutrient utilization. In addition, the level of dietary starch had significant effects on the glucose and lipid metabolism and insulin signaling pathway of mud crabs, thus affecting the deposition of lipid reserves in hepatopancreas.

Fucoidan alleviates high sucrose-induced metabolic disorders and enhances intestinal homeostasis through modulation of Notch signaling

IntroductionThe therapeutic potential of fucoidan (FUC), a natural polysaccharide, in metabolic disorders is recognized, yet its underlying mechanisms remain unclear. MethodsWe conducted investigations into the therapeutic mechanisms of FUC sourced from Sargassum fulvellum concerning metabolic disorders induced by a high-sucrose diet (HSD), employing Drosophila melanogaster and mice models. Drosophila larvae were subjected to HSD exposure to monitor growth inhibition, reduced pupation, and developmental delays. Additionally, we examined the impact of FUC on growth- and development-related hormones in Drosophila. Furthermore, we assessed the modulation of larval intestinal homeostasis by FUC, focusing on the regulation of Notch signaling. In mice, we evaluated the effects of FUC on HSD-induced impairments in intestinal epithelial barrier integrity and gut hormone secretion. ResultsFUC supplementation significantly enhanced pupal weight in Drosophila larvae and effectively countered HSD-induced elevation of glucose and triglyceride levels. It notably influenced the expression of growth- and development-related hormones, particularly augmenting insulin-like peptides production while mitigating larval growth retardation. FUC also modulated larval intestinal homeostasis by negatively regulating Notch signaling, thereby protecting against HSD-induced metabolic stress. In mice, FUC ameliorated HSD-induced impairments in ileum epithelial barrier integrity and gut hormone secretion. ConclusionsOur findings demonstrate the multifaceted therapeutic effects of FUC in mitigating metabolic disorders and maintaining intestinal health. FUC holds promise as a therapeutic agent, with its effects attributed partly to the sulfate group and its ability to regulate Notch signaling, emphasizing its potential for addressing metabolic disorders.

Developing insulin-like peptide 5-based antagonists for the G protein-coupled receptor, RXFP4

Human insulin-like peptide 5 (INSL5) is a gut hormone produced by colonic L-cells, and its biological functions are mediated by Relaxin Family Peptide Receptor 4 (RXFP4). Our preliminary data indicated that RXFP4 agonists are potential drug leads for the treatment of constipation. More recently, we designed and developed a novel RXFP4 antagonist, A13-nR that was shown to block agonist-induced activity in cells and animal models. We showed that A13-nR was able to block agonist-induced increases in colon motility in mice of both genders that express the receptor, RXFP4. Our data also showed that colorectal propulsion induced by intracolonic administration of short-chain fatty acids was antagonized by A13-nR. Therefore, A13-nR is an important research tool and potential drug lead for the treatment of colon motility disorders, such as bacterial diarrhea. However, A13-nR acted as a partial agonist at high concentrations in vitro and demonstrated modest antagonist potency (∼35 nM). Consequently, the primary objective of this study is to pinpoint novel modifications to A13-nR that eliminate partial agonist effects while preserving or augmenting antagonist potency. In this work, we detail the creation of a series of A13-nR-modified analogues, among which analogues 3, 4, and 6 demonstrated significantly improved RXFP4 affinity (∼3 nM) with reduced partial agonist activity, enhanced antagonist potency (∼10 nM) and maximum agonist inhibition (∼80 %) when compared with A13-nR. These compounds have potential as candidates for further preclinical evaluations, marking a significant stride toward innovative therapeutics for colon motility disorders.