Yasuaki Itoh

Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40.

Diabetes, a disease in which carbohydrate and lipid metabolism are regulated improperly by insulin, is a serious worldwide health issue. Insulin is secreted from pancreatic β cells in response to elevated plasma glucose, with various factors modifying its secretion. Free fatty acids (FFAs) provide an important energy source as nutrients, and they also act as signalling molecules in various cellular processes, including insulin secretion. Although FFAs are thought to promote insulin secretion in an acute phase, this mechanism is not clearly understood. Here we show that a G-protein-coupled receptor, GPR40, which is abundantly expressed in the pancreas, functions as a receptor for long-chain FFAs. Furthermore, we show that long-chain FFAs amplify glucose-stimulated insulin secretion from pancreatic β cells by activating GPR40. Our results indicate that GPR40 agonists and/or antagonists show potential for the development of new anti-diabetic drugs.

Cloning and sequence analysis of cDNA encoding the precursor of a human endothelium-derived vasoconstrictor peptide, endothelin: Identity of human and porcine endothelin

A cDNA encoding a human endothelium‐derived vasoconstrictor peptide, endothelin, was isolated from a human placenta cDNA library. The nucleotide sequence of this cDNA clone showed that the primary structure of the human preproendothelin has 212 amino acid residues and is highly homologous to porcine preproendothelin, and that human endothelin is identical with porcine endothelin.

Neurons expressing relaxin 3/INSL 7 in the nucleus incertus respond to stress

Relaxin 3/INSL 7 has recently been identified as a new member of the insulin/relaxin superfamily. Although it was reported to be dominantly expressed in the brain, its detailed distribution and function in the central nervous system are still obscure. In the present study we demonstrated that in the rat relaxin 3 was mainly expressed in neurons of the nucleus incertus (NI) of the median dorsal tegmental pons. Other relaxin 3‐expressing neurons were scattered in the pontine raphe nucleus, the periaqueductal gray and dorsal area to the substantia nigra in the midbrain reticular formation. Relaxin 3‐immunoreactive fibers projected particularly densely in the septum, hippocampus, lateral hypothalamus and intergeniculate leaflet of the thalamus. Ultrastructural examination revealed that relaxin 3 was localized in the dense‐cored vesicles in the perikarya and was also observed in the synaptic terminals of axons. As almost all relaxin 3‐containing neurons express corticotropin‐releasing factor (CRF) type 1 receptor in the NI, we examined the response of relaxin 3 neurons to intracerebroventricular administration of CRF; 65% of relaxin 3 neurons expressed c‐Fos 2 h after intracerebroventricular administration of 1 µg CRF. We then confirmed that c‐Fos was induced in 60% of relaxin 3 neurons in the NI and the expression of relaxin 3 mRNA increased significantly in the NI after water‐restraint stress. Collectively, these results suggest that relaxin 3 produced in the NI is released from nerve endings and is involved in the regulation of the stress response.

Synthesis of the vasoconstrictor peptide endothelin in kidney cells

The expression plasmid containing human prepro‐endothelin cDNA was constructed and introduced into COS‐7 cells. Mature endothelin, consisting of 21 amino acid residues, was secreted into the culture medium of the transfected cells and was also synthesized by non‐transfected COS‐7 cells. Normal kidney cells derived from other species also synthesized and secreted endothelins. Partial characterization of endothelins produced by kidney cells suggested the existence of new types of endothelin. This is the first report of the vasoconstrictor peptide endothelin being synthesized in kidney cells.

Characterization of murine PACAP mRNA

A murine PACAP precursor cDNA was isolated by screening a brain cDNA library. The amino acid sequence of the precursor was highly similar (from 81% to 93% similarity) to its rat, human, and ovine counterparts. The primary structure of murine PACAP is identical with those from sheep, humans, and rats, indicating that the mature PACAP is well conserved among mammals. Northern blot analysis revealed that the approximately 2.4 kb transcript for the PACAP precursor is expressed in murine brain. The verification that murine PACAP is identical to its human counterpart provides a rationale for physiological and pathophysiological studies of PACAP in mice.

Recombinant hepatitis B virus surface antigen carrying the pre-S2 region derived from yeast: purification and characterization

Abstract Modified hepatitis B virus surface antigen (HBsAg) carrying the pre-S2 region (HBsAg M-P31c) has been highly purified from the recombinant yeast Saccharomyces cerevisiae . The purified HBsAg M-P31c formed spherical particles with a diameter of about 20 nm, which were similar in both shape and size to human plasma-derived HBsAg small particles (h-HBsAg). The M-P31c particles were composed of two major glycoproteins with molecular masses of 37 kDa (GP37) and 34 kDa (GP34). GP37 and GP34 had identical polypeptide backbones (P31) of 31 kDa and had N-linked sugar chains with a molecular mass of about 3 kDa at the Asn 4 residue. GP37 has additional sugar chain(s) in the pre-S2 region. The sugar chains were composed of mannose and N -acetylglucosamine. Like h-HBsAg, the particles also contained phospholipids, triglycerides, and free fatty acids. On the other hand, little cholesterol was contained in the particles. M-P31c vaccine adsorbed on Al(OH) 3 gel elicited anti-pre-S2 antibodies in addition to anti-S antibodies. These results demonstrate that M-P31c particles are promising as an improved immunogen for the hepatitis B vaccine.

Synthesis in yeast of hepatitis B virus surface antigen modified P31 particles by gene modification

The pre-S2 portion of hepatitis B virus surface antigen P31 gene was modified to make gene products resistant to trypsin-like proteases in Saccharomyces cerevisiae. The coding sequence for 6 amino acids (Ser44 - Thr49) including Arg48 was removed, and the altered gene was inserted into an expression vector. The modified HBsAg P31 (M-P31c) gene products, consisting of GP37 and GP34, formed particles having both HBsAg antigenicity and polymerized-albumin receptor activity. Since the M-P31c particles can elicite two kinds of protective antibodies against hepatitis B virus, anti-S and anti-pre-S2 antibodies, the M-P31c particles are expected to be potentially effective to S-nonresponders.

Expression of hepatitis B virus surface antigen P31 gene in yeast

The hepatitis B virus surface antigen (HBsAg) P31 gene has been expressed in yeast Saccharomyces cerevisiae. The gene products were shown to be glycoproteins with molecular sizes of 37,000 and 34,000 daltons (GP37 and GP34) containing polymerized albumin receptors. Successfully detecting these proteins depended on the extraction procedures. In the extract without protein denaturants and inhibitors, these products were degraded rapidly by proteases to yield smaller size derivatives lacking polymerized albumin receptors. As is the case in human serum-derived HBsAg, yeast HBsAg consisting of GP37 and GP34 was found to be particles or aggregates having a buoyant density of 1.2 g/cc; these particles bound to polymerized human serum albumin in species-specific manner.

Anatomical Transcriptome of G Protein-Coupled Receptors Leads to the Identification of a Novel Therapeutic Candidate GPR52 for Psychiatric Disorders

Many drugs of abuse and most neuropharmacological agents regulate G protein-coupled receptors (GPCRs) in the central nervous system (CNS)_ENREF_1. The striatum, in which dopamine D1 and D2 receptors are enriched, is strongly innervated by the ventral tegmental area (VTA), which is the origin of dopaminergic cell bodies of the mesocorticolimbic dopamine system_ENREF_3 and plays a central role in the development of psychiatric disorders_ENREF_4. Here we report the comprehensive and anatomical transcript profiling of 322 non-odorant GPCRs in mouse tissue by quantitative real-time PCR (qPCR), leading to the identification of neurotherapeutic receptors exclusively expressed in the CNS, especially in the striatum. Among them, GPR6, GPR52, and GPR88, known as orphan GPCRs, were shown to co-localize either with a D2 receptor alone or with both D1 and D2 receptors in neurons of the basal ganglia. Intriguingly, we found that GPR52 was well conserved among vertebrates, is Gs-coupled and responsive to the antipsychotic drug, reserpine. We used three types of transgenic (Tg) mice employing a Cre-lox system under the control of the GPR52 promoter, namely, GPR52-LacZ Tg, human GPR52 (hGPR52) Tg, and hGPR52-GFP Tg mice. Detailed histological investigation suggests that GPR52 may modulate dopaminergic and glutamatergic transmission in neuronal circuits responsible for cognitive function and emotion. In support of our prediction, GPR52 knockout and transgenic mice exhibited psychosis-related and antipsychotic-like behaviors, respectively. Therefore, we propose that GPR52 has the potential of being a therapeutic psychiatric receptor. This approach may help identify potential therapeutic targets for CNS diseases.

Cloning and sequence analysis of a snake, Atractaspis engaddensis gene encoding sarafotoxin S6c.

A 469 base pair genomic DNA, which encodes the mature region of a snake cardiotoxic peptide, sarafotoxin S6c, was isolated from the liver of the burrowing asp, Atractaspis engaddensis. The nucleotide sequence encoding the mature peptide region showed a high sequence homology with those of mammalian vasoconstrictor peptides, endothelin family as expected from the high homology of their amino acid sequences. In contrast, both of the upper and lower flanking sequences of sarafotoxin gene and the deduced amino acid sequence of the sarafotoxin precursor were quite different from those of endothelin family. These results suggest that the ancestral gene and biosynthetic pathway of sarafotoxins are different from those of endothelin.