Identification of N-arachidonylglycine, U18666A, and 4-androstene-3,17-dione as novel insulin Secretagogues

https://doi.org/10.1016/j.bbrc.2005.06.005Get rights and content

Abstract

The glucose-induced insulin secretion is fine-tuned by numerous factors. To systematically identify insulinotropic factors, we optimized a primary β-cell-based functional assay to monitor intracellular Ca2+ flux ([Ca2+]i). By this assay system, we successfully identified several insulinotropic peptides including cholecystokinin, gastrin releasing peptide, vasopressin, and oxytocin from tissue extracts. Screening of an assortment of chemical compounds, we determined three novel insulin secretagogues: N-arachidonylglycine (NAGly), 3β-(2-diethylamino-ethoxy) androstenone hydrochloride (U18666A), and 4-androstene-3,17-dione. The NAGly increased [Ca2+]i through stimulation of the voltage-dependent Ca2+ channels and it was dependent on extracellular glucose level. On the other hand, U18666A and 4-androstene-3,17-dione increased [Ca2+]i in the presence of KATP channel opener diazoxide while it was inhibited by the presence of Ca2+ channel blocker nitrendipine, suggesting that their effects are independent of KATP channel. These unique features will be useful for further development of insulinotropic factors and drugs for treating type 2 diabetes.

Section snippets

Materials and methods

Rat islet cells preparation and culture. Islets were prepared from six male Wistar rats aged 6–7-weeks with free access to food and water. Pancreatic islets were isolated by collagenase treatment followed by Ficoll gradient separation. Briefly, pancreas was inflated by perfusion with 1 mg/ml of collagenase (Wako Pure Chemical Industries, Osaka, Japan) in Hanks’ solution (Sigma) containing 5.6 mM glucose. Pancreas was dissected and then incubated with collagenase at 37 °C for 17 min. After

[Ca2+]i imaging assay system and purification of peptide hormones

To establish a highly sensitive and reproducible physiological screening system for insulinotropic modulators, we employed primary rat pancreatic β-cells and optimized the single-cell calcium imaging technique as described under Materials and methods. This system allowed us to screen 150 samples in a single 5-h experiment, using islets from six animals. This throughput was sufficient to purify insulinotropic peptides from various tissue extracts and screen hundreds of chemical compounds. To

Discussion

We describe here a single-cell Ca2+ imaging assay in primary pancreatic β-cells that is sensitive, reproducible, and efficient enough to permit comprehensive screening of endogenous insulinotropic peptides from various tissue extracts. Our system allows us to screen ∼150 samples in a single 5-h experiment. The technique also allows us to monitor individual islet cells simultaneously and in parallel, which is crucial to avoid misleading false positive signals. However, our system may not permit

Acknowledgments

We thank Dr. Robert W. Rawson and Dr. Patrick C. Reid for helpful discussions, and Yasuyo Urashima, Aoi Uchida, and Satomi Takahashi for excellent technical assistance. This work was supported through Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government and by Exploratory Research for Advanced Technology/Japan Science and Technology Agency (Yanagisawa orphan receptor project). M.Y. is

References (33)

  • D.S. Ory

    Niemann–Pick type C: a disorder of cellular cholesterol trafficking

    Biochim. Biophys. Acta

    (2000)
  • J.P. Incardona et al.

    Cyclopamine inhibition of Sonic hedgehog signal transduction is not mediated through effects on cholesterol transport

    Dev. Biol.

    (2000)
  • F. Xia et al.

    Disruption of pancreatic beta-cell lipid rafts modifies Kv2.1 channel gating and insulin exocytosis

    J. Biol. Chem.

    (2004)
  • T. Tanaka et al.

    Activation of peroxisome proliferator-activated receptor delta induces fatty acid beta-oxidation in skeletal muscle and attenuates metabolic syndrome

    Proc. Natl. Acad. Sci. USA

    (2003)
  • R.H. Unger

    Minireview: weapons of lean body mass destruction: the role of ectopic lipids in the metabolic syndrome

    Endocrinology

    (2003)
  • K. Miyawaki et al.

    Glucose intolerance caused by a defect in the entero-insular axis: a study in gastric inhibitory polypeptide receptor knockout mice

    Proc. Natl. Acad. Sci. USA

    (1999)
  • Cited by (30)

    • Fecal metabolomics reveals the positive effect of ethanol extract of propolis on T2DM mice

      2022, Food Science and Human Wellness
      Citation Excerpt :

      N-arachidonoyl glycine (NAGly) is a product of an enzymatic synthesis of arachidonic acid and glycine followed by degradation by fatty acid amide hydrolase; the resulting fat amino acid promotes insulin secretion; it has a structural similarity with anandamide (AEA) found mainly in the spinal cord, small intestine, pancreas, and in a variety of animal tissues [48,49]. Ikeda et al. [50] found that in pancreatic beta cells, NAGly increased the [Ca2+]i through voltage-dependent Ca2+ channels (VDCC), resulting in exocytosis of insulin-containing vesicle, thereby promoting the release of insulin to maintain glucose levels in the body. This study was carried out in HFD joint induced by STZ; it is a typical pathological model of “three more and one less,” symptoms, weight loss, and “insulin secretion failure period”; NAGly level was lower in the T2DM group compared to Con (Fig. 6 (18)); following EEP intervention, it rose to a higher level, possibly by stimulating receptors on VDCC, increasing [Ca2+]i, and inhibiting the decomposition of peripheral adipose tissue to improve the body’s glucolipid metabolic disorders.

    • The cannabinoid acids, analogs and endogenous counterparts

      2014, Bioorganic and Medicinal Chemistry
    • N-Acyl taurines trigger insulin secretion by increasing calcium flux in pancreatic β-cells

      2013, Biochemical and Biophysical Research Communications
      Citation Excerpt :

      Expression of TRPV4 has been detected in mouse pancreas and MIN6 cells and may be involved in the increase in intracellular calcium in response to human islet amyloid polypeptide fibril formation [11]. Another N-acyl amino acid, N-arachidonoyl glycine (NAGly), has been identified as a novel insulin secretagogue in primary β-cells [12], although the insulinotropic action of NAGly in rat islet β-cells occurs via voltage-dependent Ca2+ channels (VDCC), rather than via activation mechanism of the TRPV1 channel [12]. The present study was carried out to examine if N-acyl taurines (specifically N-arachidonoyl taurine and N-oleoyl taurine) play a role in insulin secretion in pancreatic β-cells.

    • A synaptogenic amide N-docosahexaenoylethanolamide promotes hippocampal development

      2011, Prostaglandins and Other Lipid Mediators
      Citation Excerpt :

      A number of these N-acylamides have bioactivity. For example, N-arachidonoylglycine inhibits pain [47,48], is an insulin secretagogue [49], a ligand for the orphan receptors GPR18 and GPR92 [50,51], and a reversible inhibitor of the glycine transporter GLYT2a [52]. N-palmitoylglycine increases Ca2+ influx in a dorsal root ganglion-like cell line and is antinociceptive [53], and N-arachidonoylGABA also suppresses pain [47].

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text