Angiopoietin-related growth factor antagonizes obesity and insulin resistance.

Angiopoietin-related growth factor (AGF), a member of the angiopoietin-like protein (Angptl) family, is secreted predominantly from the liver into the systemic circulation. Here, we show that most (>80%) of the AGF-deficient mice die at about embryonic day 13, whereas the surviving AGF-deficient mice develop marked obesity, lipid accumulation in skeletal muscle and liver, and insulin resistance accompanied by reduced energy expenditure relative to controls. In parallel, mice with targeted activation of AGF show leanness and increased insulin sensitivity resulting from increased energy expenditure. They are also protected from high-fat diet-induced obesity, insulin resistance and nonadipose tissue steatosis. Hepatic overexpression of AGF by adenoviral transduction, which leads to an approximately 2.5-fold increase in serum AGF concentrations, results in a significant (P < 0.01) body weight loss and increases insulin sensitivity in mice fed a high-fat diet. This study establishes AGF as a new hepatocyte-derived circulating factor that counteracts obesity and related insulin resistance.

Identification and relative quantitation of an orphan G-protein coupled receptor SREB2 (GPR85) protein in tissue using a linear ion trap mass spectrometer.

SREB2 (GPR85) is an orphan G-protein coupled receptor (GPCR) whose function is unknown. We previously prepared a SREB2-overexpressing transgenic mouse for functional analysis but were unable to confirm SREB2 protein expression level by immunochemical or biochemical methods. In this article, we report mass spectrometric identification and relative quantitative analysis of SREB2 in the forebrains of transgenic and wild type mice using nanoliquid chromatography coupled with a linear ion-trap mass spectrometer. By analyzing Chinese hamster ovary (CHO) cells overexpressing the SREB2 gene, we identified a proteotypic SREB2 peptide, GPTPPTLLGIR. Using a stable isotope-labeled analog as an authentic peptide for protein identification and as an internal control for relative quantitation, SREB2 was directly identified from the membrane fraction of forebrains from wild type and SREB2 transgenic mice. SREB2 protein expression level in the transgenic mouse was estimated to be 3-fold higher than that in the wild type littermate.

The evolutionarily conserved G protein-coupled receptor SREB2/GPR85 influences brain size, behavior, and vulnerability to schizophrenia.

The G protein-coupled receptor (GPCR) family is highly diversified and involved in many forms of information processing. SREB2 (GPR85) is the most conserved GPCR throughout vertebrate evolution and is expressed abundantly in brain structures exhibiting high levels of plasticity, e.g., the hippocampal dentate gyrus. Here, we show that SREB2 is involved in determining brain size, modulating diverse behaviors, and potentially in vulnerability to schizophrenia. Mild overexpression of SREB2 caused significant brain weight reduction and ventricular enlargement in transgenic (Tg) mice as well as behavioral abnormalities mirroring psychiatric disorders, e.g., decreased social interaction, abnormal sensorimotor gating, and impaired memory. SREB2 KO mice showed a reciprocal phenotype, a significant increase in brain weight accompanying a trend toward enhanced memory without apparent other behavioral abnormalities. In both Tg and KO mice, no gross malformation of brain structures was observed. Because of phenotypic overlap between SREB2 Tg mice and schizophrenia, we sought a possible link between the two. Minor alleles of two SREB2 SNPs, located in intron 2 and in the 3' UTR, were overtransmitted to schizophrenia patients in a family-based sample and showed an allele load association with reduced hippocampal gray matter volume in patients. Our data implicate SREB2 as a potential risk factor for psychiatric disorders and its pathway as a target for psychiatric therapy.

Disruption of the ether-a-go-go K+ channel gene BEC1/KCNH3 enhances cognitive function.

The K+ channel, one of the determinants for neuronal excitability, is genetically heterogeneous, and various K+ channel genes are expressed in the CNS. The therapeutic potential of K+ channel blockers for cognitive enhancement has been discussed, but the contribution each K+ channel gene makes to cognitive function remains obscure. BEC1 (KCNH3) is a member of the K+ channel superfamily that shows forebrain-preferential distribution. Here, we show the critical involvement of BEC1 in cognitive function. BEC1 knock-out mice performed behavioral tasks related to working memory, reference memory, and attention better than their wild-type littermates. Enhanced performance was also observed in heterozygous mutants. The knock-out mice had neither the seizures nor the motor dysfunction that are often observed in K+ channel-deficient mice. In contrast to when it is disrupted, overexpression of BEC1 in the forebrain caused the impaired performance of those tasks. It was also found that altering BEC1 expression could change hippocampal neuronal excitability and synaptic plasticity. The results indicate that BEC1 may represent the first K+ channel that contributes preferentially and bidirectionally to cognitive function.

Inhibition of angiogenesis and vascular leakiness by angiopoietin-related protein 4.

Angiopoietins and angiopoietin-related proteins (ARPs) have been shown to regulate angiogenesis, a process essential for various neovascular diseases including tumors. Here, we identify ARP4/fasting-induced adipose factor/peroxisome proliferator-activated receptor gamma angiopoietin-related as a novel antiangiogenic modulatory factor. We hypothesized that ARP4 may regulate angiogenesis. In vitro experiments using purified recombinant ARP4 protein revealed that ARP4 markedly inhibited the proliferation, chemotaxis, and tubule formation of endothelial cells. Moreover, using corneal neovascularization and Miles permeability assays, we found that both vascular endothelial growth factor-induced in vivo angiogenesis and vascular leakiness were significantly inhibited by the addition of ARP4. Finally, we found remarkable suppression of tumor growth within the dermal layer associated with decreased numbers of invading blood vessels in transgenic mice that express ARP4 in the skin driven by the keratinocyte promoter. These findings demonstrate that ARP4 functions as a novel antiangiogenic modulatory factor and indicate a potential therapeutic effect of ARP4 in neoplastic diseases.

cDNA cloning and characterization of porcine histamine H4 receptor.

The cDNA encoding histamine H4 receptor was cloned from the porcine spleen cDNA library. Porcine H4 receptor, which shares 72% homology with its human counterpart, bound to histamine in receptor-expressing mammalian cells. Isolation of the porcine H4 receptor, which is important for understanding of the pharmacology, will aid in better interpretation of physiological role of this subtype of histamine receptor.

Molecular cloning and characterization of prokineticin receptors.

Recent studies have identified two novel biofunctional proteins, termed prokineticin 1/EG-VEGF and prokineticin 2, which were mammalian homologues of mamba MIT1 and frog Bv8. Prokineticins have been demonstrated to exert their physiological functions through G-protein coupled receptors (GPCRs). In this study, we report the molecular identification of two endogenous prokineticin receptors, designated PK-R1 and PK-R2, through a search of the human genomic DNA database. PK-R1, locating in chromosome 2, and PK-R2, locating in chromosome 20p13, shared 87% homology, which was an extremely high value among known GPCRs. In functional assays, mammalian cells expressing PK-Rs responded to prokineticins in a concentration-dependent manner. Tissue distribution analysis revealed that expression of PK-R1 was observed in the testis, medulla oblongata, skeletal muscle and skin, while that of PK-R2 showed preferential expression in the central nervous system. The tissue distribution of PK-Rs reported in this paper suggests that the prokineticins play multifunctional roles in vivo.

Cyclopropane-based conformational restriction of histamine. (1S,2S)-2-(2-aminoethyl)-1-(1H-imidazol-4-yl)cyclopropane, a highly selective agonist for the histamine H3 receptor, having a cis-cyclopropane structure.

A series of cyclopropane-based conformationally restricted analogues of histamine, the "folded" cis-analogues, i.e., (1S,2R)-2-(aminomethyl)-1-(1H-imidazol-4-yl)cyclopropane (11), (1S,2S)-2-(2-aminoethyl)-1-(1H-imidazol-4-yl)cyclopropane (13), and their enantiomers ent-11 and ent-13, and the "extended" trans-analogues, i.e., (1R,2R)-2-(aminomethyl)-1-(1H-imidazol-4-yl)cyclopropane (12) and its enantiomer ent-12, were designed as histamine H(3) receptor agonists. These target compounds were synthesized from the versatile chiral cyclopropane units, (1S,2R)- and (1R,2R)-2-(tert-butyldiphenylsilyloxy)methyl-1-formylcyclopropane (14 and 15, respectively) or their enantiomers ent-14 and ent-15. Among the conformationally restricted analogues, the "folded" analogue 13 (AEIC) having the cis-cyclopropane structure was identified as a potent H(3) receptor agonist, which showed a significant binding affinity (K(i) = 1.31 +/- 0.16 nM) and had an agonist effect (EC(50) value of 10 +/- 3 nM) on the receptor. This compound owes its importance to being the first highly selective H(3) receptor agonist to have virtually no effect on the H(4) subtype receptor. These studies showed that the cis-cyclopropane structure is very effective in the conformational restriction of histamine to improve the specific binding to the histamine H(3) receptor.

RMI1 deficiency in mice protects from diet and genetic-induced obesity.

The aim of this study is to discover and characterize novel energy homeostasis-related molecules. We screened stock mouse embryonic stem cells established using the exchangeable gene trap method, and examined the effects of deficiency of the target gene on diet and genetic-induced obesity. The mutant strain 0283, which has an insertion at the recQ-mediated genome instability 1 (RMI1) locus, possesses a number of striking features that allow it to resist metabolic abnormalities. Reduced RMI1 expression, lower fasting-blood glucose and a reduced body weight (normal diet) were observed in the mutant mice. When fed a high-fat diet, the mutant mice were resistant to obesity, and also showed improved glucose intolerance and reduced abdominal fat tissue mass and food intake. In addition, the mutants were also resistant to obesity induced by the lethal yellow agouti (A(y)) gene. Endogenous RMI1 genes were found to be up-regulated in the liver and adipose tissue of KK-A(y) mice. RMI1 is a component of the Bloom's syndrome gene helicase complex that maintains genome integrity and activates cell-cycle checkpoint machinery. Interestingly, diet-induced expression of E2F8 mRNA, which is an important cell cycle-related molecule, was suppressed in the mutant mice. These results suggest that the regulation of energy balance by RMI1 is attributable to the regulation of food intake and E2F8 expression in adipose tissue. Taken together, these findings demonstrate that RMI1 is a novel molecule that regulates energy homeostasis.

Distinct localization of prokineticin 2 and prokineticin receptor 2 mRNAs in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the master circadian clock that regulates physiological and behavioral circadian rhythms in mammals. Prokineticin 2 (PK2) is highly expressed in the SCN, and its involvement in the generation of circadian locomotor activity has been reported previously. In the present study, using in situ hybridization methods, we investigated the localization of PK2 and prokineticin receptor 2 (PKR2), a specific receptor for PK2, in the rat SCN. In steady light : dark (L : D = 12 : 12 h) and constant dark conditions, rPK2 mRNA displayed a robust circadian oscillation with a peak occurring during the day. Moreover, during peak expression, the rPK2 mRNA-positive neurons were scattered in both the dorsomedial and ventrolateral SCN, which are two functionally and morphologically distinct subregions. Furthermore, double-labeling in situ hybridization experiments revealed that greater than 50% of the rPK2 mRNA-containing neurons co-expressed either vasoactive intestinal peptide (VIP), gastrin-releasing peptide (GRP) or arginine vasopressin (AVP) in the SCN. In contrast, the rPKR2 mRNA levels did not show significant diurnal alterations. rPKR2 mRNA-containing neurons were also clustered in the dorsolateral part of the SCN, which shows negligible labeling of either rAVP, rVIP, rGRP or rPK2 transcripts. In addition, this region exhibited a delayed cycling of the rPer1 gene. These results suggest an intrinsic PK2 neurotransmission and functionally distinct roles for PKR2-expressing neurons in the SCN.

The brain-specific carnitine palmitoyltransferase-1c regulates energy homeostasis.

Fatty acid synthesis in the central nervous system is implicated in the control of food intake and energy expenditure. An intermediate in this pathway, malonyl-CoA, mediates these effects. Malonyl-CoA is an established inhibitor of carnitine palmitoyltransferase-1 (CPT1), an outer mitochondrial membrane enzyme that controls entry of fatty acids into mitochondria and, thereby, fatty acid oxidation. CPT1c, a brain-specific enzyme with high sequence similarity to CPT1a (liver) and CPT1b (muscle) was recently discovered. All three CPTs bind malonyl-CoA, and CPT1a and CPT1b catalyze acyl transfer from various fatty acyl-CoAs to carnitine, whereas CPT1c does not. These findings suggest that CPT1c has a unique function or activation mechanism. We produced a targeted mouse knockout (KO) of CPT1c to investigate its role in energy homeostasis. CPT1c KO mice have lower body weight and food intake, which is consistent with a role as an energy-sensing malonyl-CoA target. Paradoxically, CPT1c KO mice fed a high-fat diet are more susceptible to obesity, suggesting that CPT1c is protective against the effects of fat feeding. CPT1c KO mice also exhibit decreased rates of fatty acid oxidation, which may contribute to their increased susceptibility to diet-induced obesity. These findings indicate that CPT1c is necessary for the regulation of energy homeostasis.

Abnormal development of the olfactory bulb and reproductive system in mice lacking prokineticin receptor PKR2.

Prokineticins, multifunctional secreted proteins, activate two endogenous G protein-coupled receptors PKR1 and PKR2. From in situ analysis of the mouse brain, we discovered that PKR2 is predominantly expressed in the olfactory bulb (OB). To examine the role of PKR2 in the OB, we created PKR1- and PKR2-gene-disrupted mice (Pkr1(-/-) and Pkr2(-/-), respectively). Phenotypic analysis indicated that not Pkr1(-/-)but Pkr2(-/-)mice exhibited hypoplasia of the OB. This abnormality was observed in the early developmental stages of fetal OB in the Pkr2(-/-) mice. In addition, the Pkr2(-/-) mice showed severe atrophy of the reproductive system, including the testis, ovary, uterus, vagina, and mammary gland. In the Pkr2(-/-) mice, the plasma levels of testosterone and follicle-stimulating hormone were decreased, and the mRNA transcription levels of gonadotropin-releasing hormone in the hypothalamus and luteinizing hormone and follicle-stimulating hormone in the pituitary were also significantly reduced. Immunohistochemical analysis revealed that gonadotropin-releasing hormone neurons were absent in the hypothalamus in the Pkr2(-/-) mice. The phenotype of the Pkr2(-/-) mice showed similarity to the clinical features of Kallmann syndrome, a human disease characterized by association of hypogonadotropic hypogonadism and anosmia. Our current findings demonstrated that physiological activation of PKR2 is essential for normal development of the OB and sexual maturation.

Molecular cloning of monkey histamine H4 receptor.

Histamine H(4) receptor is considered as a novel therapeutic target for allergic diseases. To enhance the knowledge about species difference, which is essential for drug discovery research, monkey H(4) receptor was identified. Monkey H(4) receptor was characterized to have comparable similarity with its human counterpart. Discovery of monkey H(4) receptor will contribute to a better interpretation of effective drug discovery.

Identification of MrgX2 as a human G-protein-coupled receptor for proadrenomedullin N-terminal peptides.

Proadrenomedullin N-terminal 20 peptide (PAMP[1-20]/PAMP-20) and its truncated analog, PAMP[9-20]/PAMP-12, are endogenous peptides that elicit hypotension through inhibiting catecholamine secretion from sympathetic nerve endings and adrenal chromaffin cells. Although the binding sites for PAMP are widely distributed, the nature of its receptor has been elusive. In an effort to identify potential PAMP receptor(s), we found that a human G-protein-coupled receptor, MrgX2, was specifically activated by PAMP. Although a previous study revealed that MrgX2 was a receptor for cortistatin, a neuropeptide involved in sleep regulation and locomotor activity, our present data indicated that the rank order of the agonistic effect against MrgX2 was "PAMP-12> or =cortistatin>PAMP-20". These activities were confirmed by the inhibition of the forskolin-elevated cAMP accumulation, Ca(2+) mobilization, and [(35)S]guanosine 5'-(gamma-thio)triphosphate binding assays. These findings suggest that MrgX2 couples with not only G(alpha q) but also G(alpha i), consistent with previous reports on the pharmacological profile of PAMP signaling. Furthermore, by immunostaining, we found that MrgX2 was expressed in the adrenal chromaffin cells as well as the dorsal root ganglia. From these results, we concluded that MrgX2 is a potential human PAMP-12 receptor that regulates catecholamine secretion from adrenal glands. The present discovery will eventually lead to a better understanding of the pathophysiological role of proadrenomedullin peptides.

Molecular cloning and characterization of a novel Gq-coupled orphan receptor GPRg1 exclusively expressed in the central nervous system.

G-protein-coupled receptors (GPCRs) are important mediators of signal transduction and are therefore potential targets for pharmacological therapeutics. Here, we report the identification and characterization of an orphan GPCR, termed GPRg1, which was found in the GenBank database following searches with GPCR query sequences. Quantitative PCR analysis revealed that GPRg1 transcripts are expressed almost exclusively in the brain. Moreover, in situ hybridization experiments in brain demonstrated that GPRg1 is abundantly expressed in the ventrolateral region of caudate putamen, the habenular nucleus, the zona incerta, and the medial mammillary nucleus. In addition, overexpression of GPRg1 in 293-EBNA cells activates serum response factor mediated transcription, which was completely inhibited by the Gq/11 selective inhibitor YM-254890, indicating the coupling of GPRg1 with Gq/11. These findings suggest that GPRg1 is a candidate receptor for novel physiologically bioactive substrates and that it plays important roles in the central nervous system.

Lysophosphatidylcholine enhances glucose-dependent insulin secretion via an orphan G-protein-coupled receptor.

A lysophospholipid series, such as lysophosphatidic acid, lysophosphatidylserine, and lysophosphatidylcholine (LPC), is a bioactive lipid mediator with diverse physiological and pathological functions. LPC has been reported to induce insulin secretion from pancreatic beta-cells, however, the precise mechanism has remained elusive to date. Here we show that an orphan G-protein-coupled receptor GPR119 plays a pivotal role in this event. LPC potently enhances insulin secretion in response to high concentrations of glucose in the perfused rat pancreas via stimulation of adenylate cyclase, and dose-dependently induces intracellular cAMP accumulation and insulin secretion in a mouse pancreatic beta-cell line, NIT-1 cells. The Gs-protein-coupled receptor for LPC was identified as GPR119, which is predominantly expressed in the pancreas. GPR119-specific siRNA significantly blocked LPC-induced insulin secretion from NIT-1 cells. Our findings suggest that GPR119, which is a novel endogenous receptor for LPC, is involved in insulin secretion from beta-cells, and is a potential target for anti-diabetic drug development.

Molecular identification of nicotinic acid receptor.

Nicotinic acid and its derivative, Acipimox, have been widely used in the treatment of hyperlipidemia. Pharmacological studies have demonstrated that they exert the beneficial effect through the activation of a Gi-protein-coupled receptor on adipocyte, which has remained elusive to date. Here we show that a novel GPCR, designated HM74b because of its high similarity to HM74, is a receptor for nicotinic acid. HM74b mRNA is found in human, murine, and rat adipose tissues. Nicotinic acid and Acipimox inhibit forskolin-stimulated intracellular cAMP accumulation in human HM74b-expressing cells and activate GTP gamma S binding in a dose-dependent manner. [3H]Nicotinic acid specifically binds to HM74b-expressing membrane and its binding is replaced by Acipimox. This finding will open a new phase of research on the physiological role of nicotinic acid and will be a clue to develop novel antihyperlipidemic drugs.

Angiopoietin-related growth factor (AGF) promotes epidermal proliferation, remodeling, and regeneration.

We report here the identification of an angiopoietin-related growth factor (AGF). To examine the biological function of AGF in vivo, we created transgenic mice expressing AGF in epidermal keratinocytes (K14-AGF). K14-AGF mice exhibited swollen and reddish ears, nose and eyelids. Histological analyses of K14-AGF mice revealed significantly thickened epidermis and a marked increase in proliferating epidermal cells as well as vascular cells in the skin compared with nontransgenic controls. In addition, we found rapid wound closure in the healing process and an unusual closure of holes punched in the ears of K14-AGF mice. Furthermore, we observed that AGF is expressed in platelets and mast cells, and detected at wounded skin, whereas there was no expression of AGF detected in normal skin tissues, suggesting that AGF derived from these infiltrated cells affects epidermal proliferation and thereby plays a role in the wound healing process. These findings demonstrate that biological functions of AGF in epidermal keratinocytes could lead to novel therapeutic strategies for wound care and epidermal regenerative medicine.