Lack of GPR40/FFAR1 does not induce diabetes even under insulin resistance condition.

AIMS: G protein-coupled receptor/free fatty acid receptor 1 (GPR40/FFAR1 ) regulates free fatty acid-induced insulin secretion. This study has been performed to clarify whether or not loss of GPR40/FFAR1 function exacerbates diabetes, that is, whether GPR40 has an essential physiological role in the development of diabetes or not. METHODS: We generated GPR40/FFAR1 knockout (KO) mice and analysed their phenotypes in vitro and in vivo under the condition of dietary or genetically induced insulin resistance. RESULTS: GPR40/FFAR1 KO mice kept on a high-fat diet became obese, developed glucose intolerance to a similar degree as GPR40/FFAR1 wild-type (WT) mice. In addition, the phenotype of KO mice harbouring diabetogenic KK background genes showed glucose intolerance at a level similar to level for control KK mice. In both mouse models with insulin resistance, insulin secretion after oral glucose load and homeostasis model assessment-insulin resistance (HOMA-IR) did not change between GPR40/FFAR1 KO and WT mice. Although glucose-induced insulin secretion under high palmitate concentration was significantly lower in KO than in WT islets, pancreatic insulin content and insulin secretion stimulated with glucose alone were not different between KO and WT mice. CONCLUSIONS: GPR40/FFAR1 has a major role in regulating fatty-acid-mediated insulin secretion, but the lack of GPR40/FFAR1 does not exacerbate glucose intolerance and insulin resistance induced by high-fat diet or diabetogenic KK gene. Our findings indicate that loss of GPR40/FFAR1 function does not play an important role in inducing or exacerbating diabetes.

Cloning and expression of a cDNA encoding a novel human neurotrophic factor.

A cDNA encoding a novel human neurotrophic factor (designated nerve growth factor-2; NGF-2) was cloned from a human glioma cDNA library using a synthetic DNA corresponding to human nerve growth factor (NGF). The cloned cDNA encodes a polypeptide composed of 257 amino acid residues including a prepro-sequence of 138 residues and a mature region of 119 residues. The amino acid sequence of human NGF-2 exhibits 58% similarity with that of human NGF. Conditioned medium of COS-7 cells transfected with an expression plasmid for human NGF-2 cDNA supported the survival of sensory neurons isolated from dorsal root ganglia of embryonic chicks. A 1.5 kb of NGF-2 mRNA can be detected from an early development stage in rat brain, by Northern blotting analysis.

Analysis of neurotrophin-3 expression using the lacZ reporter gene suggests its local mode of neurotrophic activity.

We replaced the mouse neurotrophin-3 gene with the Escherichia coli-derived lacZ gene by means of homologous recombination. The mice with this mutation were useful models for studying the distribution of neurotrophin-3 expression in vivo, because visualization by 5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside (X-Gal) staining was simple and rapid compared with in situ hybridization or immunohistochemistry. Whole-mount staining of mutant embryos at embryonic day 10 revealed that lacZ, a reporter for the neurotrophin-3 gene, was expressed in the mesencephalon, mandibular arch and somites. In the embryos at days 13-17, lacZ was markedly expressed in the peripheral target tissues of sensory and sympathetic neurons. We also found that spinal motor neurons and sensory neurons in trigeminal and dorsal root ganglia express lacZ. Some of these X-Gal staining regions overlapped with the sites expressing trkC, a high-affinity receptor for neurotrophin-3. The distribution of X-Gal staining in heterozygotes and homozygotes was similar to that of neurotrophin-3 messenger RNA detected by in situ hybridization. However, there was less lacZ expression in the dorsal root ganglia of homozygotes than neurotrophin-3 expression in wild-type mice. These results suggest that the neurotrophin-3 produced in the dorsal root ganglia also plays a role in the survival of some of the neurotrophin-3-positive neurons and that the local mode of neurotrophic activity is widely distributed.

Identification of the functional regulatory region of the neurotrophin-3 gene promoter.

The 5'-flanking region of the human neurotrophin-3 (NT-3) gene was isolated from a human placental genomic library using the oligonucleotide corresponding to the 5'-noncoding region of the NT-3 cDNA as a probe. A 3.8 kbp genomic fragment containing the 5'-flanking region, the first exon and a portion of the first intron was isolated and sequenced. The transcriptional initiation site, identified by S1 nuclease mapping, was located 27 bp downstream from the TATA-like sequence. Several plasmids, in which the NT-3 promoter regions were fused to the chloramphenicol acetyltransferase (CAT) gene, were constructed. Transient expression in human glioma Hs683 cells demonstrated that a fragment of about 0.1 kbp from the transcriptional initiation site was sufficient for promoter activity. While, in human plasma cell leukemia ARH77 cells, in which NT-3 mRNA was not detected, the region upstream from -65 functioned to silence CAT activity. It is suggested that this region contains the transcriptional regulatory element for the specific expression of the NT-3 gene.

Tissue and cell type specificity of the human neurotrophin-3 promoter region in transgenic mice.

To examine the tissue and cell type specificity of the human neurotrophin-3 (NT-3) promoter, we generated transgenic mice bearing the 3.3 kbp upstream region of the human NT-3 gene linked to the bacterial chloramphenicol acetyltransferase (CAT) gene as a reporter. Eight independent founders of transgenic mice were obtained, and four of them transmitted the transgene to their offsprings. Among three lines of four transgenic mice at 6 weeks of age, a high level of production of CAT protein was detected in the spleen and a low level in the brain. By in situ hybridization analysis, CAT gene expression was detected in the hippocampal neurons and in the cerebellar granular neurons of the transgenic mouse brain. These results suggest that the 3.3 kbp 5' flanking region of the human NT-3 gene has adequate promoter activity in vivo and that its expression pattern resembles the endogenous gene.

The novel compound TDN-345 induces synthesis/secretion of nerve growth factor in C6-10A glioma cells.

A novel compound, TDN-345, not bearing catechol moiety, induced NGF synthesis/secretion in C6-10A glioma cells. Both intracellular and extracellular nerve growth factor (NGF) protein levels increased within 3 h and reached a maximum around 12 h after the addition of TDN-345. The induction of NGF synthesis/secretion by TDN-345 occurred in a concentration-dependent manner, beginning with about 0.1 microM and reaching a maximum at 10 microM. The ED50 was 0.88 microM. The induction was accompanied by an increase in NGF mRNA but not beta-actin mRNA. In a time-course study, the NGF mRNA level was found to reach a maximum 2-3 h after the addition of TDN-345 and then to return to control levels. The induction occurred dose-dependently. The catecholaminergic compound epinephrine, which induces NGF synthesis/secretion, increased the intracellular cyclic AMP content by more than 1000-times at 10 microM. In contrast, TDN-345 did not cause such a prominent increase in cAMP even at 100 microM. These results indicate that TDN-345 induces NGF synthesis/secretion by increasing NGF mRNA expression, and the action of TDN-345 clearly differs from that of epinephrine, as it does not seem to involve cAMP as a second messenger. The results of the present study suggest the existence of a signal transduction pathway for NGF synthesis/secretion which is not mediated by cAMP.

Developmental changes of neurotrophin-3 level in the mouse brain detected by a highly sensitive enzyme immunoassay.

Levels of neurotrophin-3 (NT-3) in the mouse brain were measured by a highly sensitive enzyme immunoassay (EIA). The monoclonal antibody, 3W3, was labeled with beta-galactosidase, followed by measurement of galactosidase activity. The detection limit of the EIA system was 0.4 pg/well (4 pg/ml). At 1 and 8 weeks of age, the highest level of NT-3 was detected in the hippocampus, a relatively high level also observed in the cerebellum. In contrast, in the cortex, the striatum, the diencephalon, the midbrain, and the brainstem, NT-3 levels were low. Furthermore, we examined the developmental changes of NT-3 level in the hippocampus and the cerebellum. In the hippocampus, the NT-3 levels were more than 20 ng/g tissue from 1 week to 14 weeks of age, but at 20 weeks of age the level decreased to about half. In the cerebellum, although the NT-3 level was high at 1 week of age, the levels were gradually decreased to one-fourth by 20 weeks of age. In peripheral tissues, a large amount of NT-3 protein was observed in the heart.

Expression of neurotrophin genes in the brain of senescence-accelerated mouse (SAM) during postnatal development.

We compared the expression patterns of neurotrophin genes in the brain of senescence-accelerated mouse (SAMP8) which shows age-related impairment of learning behavior, with SAMR1 control which shows normal aging. By Northern blot analysis, NT-3 mRNA levels in the cortex were higher in SAMP8 than in SAMR1 mice during development, whereas in the midbrain, hippocampus and forebrain, NT-3 expression levels in SAMP8 were lower than those in SAMR1. At early stages, although NGF mRNA levels in SAMP8 were lower than those in SAMR1, BDNF mRNA levels were almost equivalent in both strains. By in situ hybridization analysis, NT-3 mRNA signals in the CA1 and CA2 regions in SAMP8 were shown to be reduced at early stages. However, BDNF mRNA signals were almost equivalent in both SAMR1 and SAMP8.

Targeted disruption of the neurotrophin-3 gene with lacZ induces loss of trkC-positive neurons in sensory ganglia but not in spinal cords.

We have replaced the NT-3 gene with Escherichia coli-derived lacZ gene by means of homologous recombination in embryonic stem cells and thus produced null mutant mice. Mice homozygous for this mutation developed to birth, but most of them could not suck well and died within 2 days after birth. The surviving homozygous mutant mice displayed movement disorder similar to ataxia. The expression of lacZ was widely distributed in the target tissues of peripheral nerves, spinal motor neurons, lumbar dorsal root ganglia and trigeminal ganglia during the prenatal periods. A neuroanatomical examination revealed that there was marked cell reduction present in trigeminal and lumbar dorsal root ganglia in the developing homozygous mutant mice. In these tissues, the expression of trkC, a high-affinity receptor for NT-3, was markedly reduced. In contrast, we did not find any morphological abnormalities, significant cell loss or decreased levels of trkC expression in the motor neurons present in the ventral horn of the spinal cord. These results indicate that the absence of the NT-3 gene leads to a defect in the sensory nervous system, but it may be complemented by other neurotrophins in the motor nervous system during the development.

Nerve growth factor promoter driven neurotrophin-3 overexpression in the mouse and the protective effect of transgene on age-related behavioral deficits.

To clarify the biological function of neurotrophin-3 (NT-3) at the postnatal stage, we created a line of transgenic mice overexpressing NT-3 under the control of the mouse nerve growth factor gene promoter. Transgenic mice showed high-level NT-3 expression in the hippocampus and several tissues. We performed behavioral tests in young-adult (7-months-old) and aged (25-months-old) mice. Although aged non-transgenic mice exhibited spatial learning impairments in the Morris water maze, overexpression of NT-3 protected against these age-dependent spatial learning impairments in mice.

Neurotrophin-3 is expressed in the posterior lobe of mouse cerebellum, but does not affect the cerebellar development.

We replaced the neurotrophin-3 (NT-3) gene with Escherichia coli-derived lacZ via homologous recombination in embryonic stem (ES) cells and generated the mutant mice. Here we show the in vivo expression of NT-3 in the cerebellum during the postnatal period. A high level of lacZ expression was found in the granular layer of posterior lobe (lobules VII to X) in the postnatal NT-3(+/-) cerebellum. The expression in these regions was reduced with age. Although the Purkinje cells are considered to be a target of NT-3 and the NT-3(-/-) mice displayed severe moving disorders like ataxia, no histological abnormality was observed in their cerebellum. These findings suggest that the NT-3 expressed in the cerebellum gives some trophic effects primarily to the posterior lobe, however, the deficiency does not affect its development.

[A case of suprasellar arachnoid cyst followed up for a long time].

We followed a case of suprasellar arachnoid cyst for 12 years. The patient was a sixteen-year-old girl without particular problems in her general condition. She showed optic atrophy in both eyes and optic nerve hypoplasia with an inferotemporal quandranopsia in the left eye. A suprasellar arachnoid cyst communicating with the tubarachnoid space was found to extend into the sella turcica as an empty sella. A cyst wall was resected and a cyst-peritoneal shunt performed. After 12 years from the operation, sensitivity was slightly depressed in the visual field where it had already been disturbed. Although there are few reports in the literature on involvement of the optic nerves and chiasma by suprasellar arachnoid cysts, papilledema and optic atrophy are often found in children, and infero-temporal quandranopsia or homonymous hemianopsia have been reported. Visual field defects were most likely caused by compression of the optic nerve by cyst or prolonged papilledema. We also suspect that some kind of disturbance to the optic nerve occurred during extension of the arachnoid cyst as an empty sella, or during formation of arachnoid cyst in the fetus stage.

Increase in gene expression by respiratory-deficient mutation.

Respiratory-deficient mutants (rho- cells) of Saccharomyces cerevisiae produced about 10 times as much human (h-) lysozyme as did wild-type strains (rho+ cells) when the GAL10 promoter was used in an expression plasmid with the h-lysozyme gene. Introduction of intact mitochondria into the rho- cells resulted in a significant decrease in the production of h-lysozyme, indicating that the rho- mutation increased the expression of the h-lysozyme gene. The copy number of the expression plasmid was not responsible for the increased expression. The level of h-lysozyme mRNA in the rho- cells was also much higher than that in the rho+ cells especially at the stationary phase. The increased expression of the h-lysozyme gene was also observed when a glyceraldehyde-3-phosphate dehydrogenase gene promoter and the PHO5 promoter were used in the expression plasmid. The rho- mutation also increased the expression of the PHO5 gene under the control of the HIS5 promoter in a plasmid and the ACT1 gene in the yeast chromosome, but did not increase the expression of the ribosomal RNA gene. In contrast to the rho- mutants, pet mutants did not show higher gene expression compared with wild-type strains.

Effect of NGF-2 on the survival of chick sensory ganglion neurons in vitro.

The biological activity of NGF-2, the third member of the NGF family, was investigated. Conditioned medium of COS cells transfected with expression plasmid for human NGF-2 cDNA promoted the survival of sensory neurons from dorsal root ganglia (DRG), and nodose ganglia (NG). Supernatant of mock transfected COS cells was less effective for the survival of DRG neurons and ineffective for the survival of NG neurons. These results suggest that NGF-2 is a novel neurotrophic factor whose biological activity is distinct from NGF.

Characterization of the 5'-flanking region of the human brain-derived neurotrophic factor gene.

The 5'-flanking region of the human brain-derived neurotrophic factor (BDNF) gene was isolated from a human placental genomic library using the cDNA fragment for the 5'-noncoding region of human BDNF as a probe. A 3.2 Kbp genomic fragment containing the 5'-flanking region, the first exon and a portion of the first intron was isolated and sequenced. The transcriptional initiation site, identified by S1 nuclease mapping, was located 26 bp downstream from the TATA-like sequence. Several expression plasmids, in which the BDNF promoter regions were fused to the chloramphenicol acetyltransferase (CAT) gene, were constructed. Transient expression in human glioma Hs683 cells demonstrated that a fragment of about 0.5 Kbp from the transcriptional initiation site was sufficient for promoter activity.

Secretion in yeast of human lysozymes with different specific activities created by replacing valine-110 with proline by site-directed mutagenesis.

Computer graphics indicate that a steric hindrance exists between valine-110 side chain of human lysozyme (EC 3.2.1.17) and an acetyl group of a modified substrate that contains N6,O-diacetylmuramic acid. To alter the substrate specificity of human lysozyme to be effective on the modified substrate, we replaced the valine-110 residue with various amino acids by site-directed mutagenesis. One of the mutant proteins (valine residue replaced with proline:P110) was secreted in Saccharomyces cerevisiae as at least four components (P110-A, P110-B, P110-C, and P110-D) with different specific activities. Two components, P110-B and P110-D, were isolated in a pure form and structurally characterized. The results suggest that this mutation lowered the lytic activity against Micrococcus lysodeikticus by changing a local conformation of the catalytic site while keeping almost the same substrate binding sites. Our results also indicate that cis/trans isomerization of prolyl peptide bonds probably occurs in vivo and that the conformational change of protein as well as point mutations in genes might influence the molecular evolution of the protein.

Purification and characterization of biologically active recombinant human neurotrophin-3 produced by expression of a chimera gene in Chinese hamster ovary cells.

In order to obtain high-level expression of recombinant human neurotrophin-3 (NT-3), we constructed several types of expression plasmids and examined several cell lines for expression of the human NT-3 gene. The highest level production of the recombinant protein was attained in Chinese hamster ovary cells transfected with an expression plasmid that contains a chimera gene encoding the human nerve growth factor (NGF) prepro-region and human NT-3 mature-region under control of a murine leukemia virus-derived long terminal repeat (MuLV-LTR). This cell line can produce more than 1 mg recombinant human NT-3/1 conditioned medium. The recombinant protein was purified to apparent homogeneity with a cation exchange column, a gel filtration column and a reversed-phase HPLC column with a recovery of about 30%. The purified NT-3, at a concentration as low as 0.2 ng/ml, induced neurite out-growth in neurons prepared from 8-day-old chick embryonic dorsal root ganglia; however, it showed little neurotrophic effect on rat PC12 pheochromocytoma cells, which are known to be NGF-responding cells. In addition, this protein promoted colony formation by human peripheral blood lymphocytes in soft agar culture.

Regional expression of the nerve growth factor gene family in rat brain during development.

The developmental expression patterns of three members (NGF, NGF-2/NT-3, and BDNF) of the NGF family in rat brain are different. NGF-2/NT-3 mRNA was the first detected during development followed by NGF and BDNF mRNAs. A substantial amount of NGF mRNA was found to be synthesized in the hippocampus and the cortex, and this regional expression pattern did not change during development. In contrast, NGF-2/NT-3 mRNA was detected in almost all the brain regions examined in the early developmental stage. In the late stage, the transcript was found in high concentration only in the hippocampus and the cerebellum. BDNF mRNA was widely distributed, and its level was augmented in the late developmental stage.

Tissue distribution and immunocytochemical localization of neurotrophin-3 in the brain and peripheral tissues of rats.

The tissue distribution of neurotrophin-3 (NT-3) was investigated in rats at 1 month of age using a newly established, sensitive two-site enzyme immunoassay system for NT-3, as well as the immunocytochemical localization of this protein. The immunoassay for NT-3 enabled us to quantify NT-3 at levels > 3 pg per assay. In the rat brain, NT-3 was detectable only in the olfactory bulb (0.54 ng/g wet weight), cerebellum (0.71 ng/g), septum (0.91 ng/g), and hippocampus (6.3 ng/g). By contrast, NT-3 was widely distributed in peripheral tissues. Appreciable levels of NT-3 were also found in the thymus (31 ng/g), heart (38 ng/g), diaphragm (21 ng/g), liver (45 ng/g), pancreas (892 ng/g), spleen (133 ng/g), kidney (40 ng/g), and adrenal gland (46 ng/g). An antibody specific for NT-3 bound to pyramidal cells in the CA2-CA4 regions of the hippocampus, to A cells in the islets of Langerhans in the pancreas, to unidentified cells in the red pulp of the spleen, to liver cells, and to muscle fibers in the diaphragm from rats at 1 month of age. Molecular masses of NT-3-immunoreactive proteins in the hippocampus and pancreas were 14 and 12 kDa, respectively. Thus, in rats, NT-3 was detected in restricted regions of the brain and in the visceral targets of the nodose ganglia at high concentrations. Our present results suggest that NT-3 not only functions as a classical target-derived neurotrophic factor but also can play other roles.

A specific host factor binds at a cis-acting transcriptionally silent locus required for stability control of yeast plasmid pSR1.

A cis-acting locus, Z, of plasmid pSR1 functions in stable maintenance of the plasmid in the native host, Zygosaccharomyces rouxii. The Z locus was shown to be located in a 482 bp sequence in the 5' upstream region of an open reading frame, P, by subcloning various DNA fragments in a plasmid replicating via the ARS1 sequence of the Saccharomyces cerevisiae chromosome. Northern analysis revealed that the Z region is not transcribed in either the native host Z. rouxii or the heterologous host S. cerevisiae. The Z region is protected from micrococcal nuclease attack in Z. rouxii but not in S. cerevisiae, its protection depending on the product of the S gene encoded by pSR1. Gel retardation assays suggested that a factor present in nuclear extracts of Z. rouxii cells, irrespective of the presence or absence of a resident pSR1 plasmid, binds to a 111 bp RsaI-SacII sequence in the Z region. These findings suggest that a host protein binds to the Z locus and that the S product interacts with this DNA-protein complex and stabilizes pSR1.