Physiological function, expression pattern, and transcriptional regulation of a Caenorhabditis elegans insulin-like peptide, INS-18

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Abstract

In Caenorhabditis elegans, insulin/insulin-like growth factor (IGF)-1 signaling (IIS) is an important pathway that controls larval diapause and adult lifespan. The IIS pathway is modulated by many insulin-like peptides (ILPs) through the DAF-2 receptor, the sole insulin/IGF-1 receptor-like protein in C. elegans. We previously identified the ILP, INS-18, and predicted its tertiary structure to be similar to the crystal structures of human insulin and IGF-1. In this study, the physiological function of INS-18 was first examined by gene disruption and overexpression, and we identified INS-18 as a DAF-2 antagonist required for larval diapause and longevity. Analysis of the INS-18 expression pattern using a reporter gene showed it to be expressed in nerve cells, including hermaphrodite-specific neurons (HSNs) at the adult stage. Other ILP expressions have not been previously observed in HSNs, and we believe that INS-18 expression in these cells may contribute to longevity by regulating reproduction. Loss of the DAF-16 transcription factor located downstream of the IIS pathway completely blocked ins-18 expression. We propose a positive feedback model for the regulation of ins-18 expression in which an antagonist binding to the DAF-2 receptor increases ins-18 gene expression, thus leading to increased INS-18 protein levels and increased DAF-2 receptor binding. Thus, this study provides a new insight into the hormonal regulation of insulin, an important and widespread process in the animal kingdom.

Highlights

► INS-18, one of the insulin like-peptides, functions as an antagonist. ► INS-18 modulates larval diapause and adult lifespan through DAF-2 receptor. ► ins-18 Expression may be regulated by a positive feedback loop.

Introduction

Environmental conditions affect the development of most species. In Caenorhabditis elegans (C. elegans), environmental conditions determine whether the worm develops directly into an adult or is arrested at an alternative L3 larval stage to form a dauer larva. Dauer larvae are induced by harsh environments such as starvation, a high concentration of dauer pheromones secreted by crowded worms, or high temperatures [1], [2]. Worms can survive these adverse conditions because of their distinctive adaptive morphological, behavioral, and metabolic features. When environmental conditions improve, worms resume normal growth. This survival system, known as larval diapause, is controlled by many signaling cascades including the insulin/insulin-like growth factor (IGF)-1 signaling (IIS) pathway. This pathway contains the sole C. elegans insulin/IGF-1 receptor-like protein, DAF-2 [3], and the forkhead box O (FOXO) transcription factor homolog, DAF-16 [4], [5]. DAF-2 inactivation results in DAF-16 nuclear translocation [6], [7], [8], which induces larval diapause and leads to increased longevity [9], [10], [11]. Thus, IIS is an important pathway controlling larval diapause and adult lifespan in worms.

The activation or inactivation of this signaling pathway is controlled by DAF-2-ligands, the insulin-like peptides (ILPs). Agonistic ILPs are thought to promote signaling and antagonistic ILPs to suppress signaling through the DAF-2 receptor. To date, 40 insulin-like genes have been identified in the C. elegans genome and predicted peptides encoded by the genes are classified into types α, β, and γ according to their disulfide bond pattern [12], [13]. Type-γ ILPs have three canonical disulfide bonds that are conserved among vertebrate insulin family peptides. We previously identified INS-18 as a type-γ ILP and predicted its tertiary structure to be similar to the crystal structures of human insulin and IGF-1. In addition, we speculated that INS-18 may function as a DAF-2 agonist because ins-18 knockdown in wild-type animals slightly increases longevity [14]. In contrast, Pierce and coworkers showed that a high ins-18 gene dosage enhances the daf-2 mutant phenotype, indicating that INS-18 may function as a DAF-2 antagonist [13]. Thus, the function of INS-18 remains unclear.

In this study, we first determined whether INS-18 functions as a DAF-2 agonist or antagonist. We subsequently examined the spatio-temporal expression of INS-18 and investigated the transcriptional regulation of ins-18. Our data suggests that INS-18 is a DAF-2 antagonist that functions mainly in nerve cells. In addition, our data also allows us to propose a positive feedback model for the regulation of INS-18 expression.

Section snippets

Strains

C. elegans were grown on standard nematode growth medium (NGM) [15]. Unless otherwise stated, NGM plates were seeded with E. coli OP50 bacteria and maintained at 20 °C [15]. Worm strains and alleles used in this study were described in the Supplemental Manuscript.

Construction of plasmids and transgenic worms

Amplified genomic fragments were subcloned into pBluescript SK(+) (Stratagene, Santa Clara, CA) or pPD_venus (provided by Dr. Ishihara of Kyushu University) vectors. A plasmid overexpressing ins-18 was constructed to rescue the ins-18

INS-18 functions as a DAF-2 antagonist to modulate larval diapause and adult lifespan

To elucidate the physiological functions of INS-18, we first mutated the ins-18 gene using the TMP/UV method [21] followed by sib-screening. A 1.4 kb ins-18 deletion removed half of the second and the entire third exon, indicating that the mutant could not synthesize the INS-18 peptide. This deletion mutant was named tm339 (after the reference list [22]) and used in subsequent experiments.

We first examined whether INS-18 modulates larval diapause. At 20 °C and 25 °C, ins-18(tm339) animals showed

Acknowledgments

We are grateful to Dr. Andrew Fire of Stanford University for providing us with the RNAi vector L4440. We thank Dr. Takeshi Ishihara of Kyusyu University for providing us with the reporter plasmid pPD_venus. We also thank Dr. James M. Kramer of Northwestern University for providing us with the maker plasmid pRF4. We thank Caenorhabditis Genetic Center for providing us with the E. coli strain HT115 and, the C. elegans strains daf-2(e1370) and daf-16(mu86). We thank Mr. Kenji Gouda and Ms. Yukari

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