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 beta 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 beta cells by activating GPR40. Our results indicate that GPR40 agonists and/or antagonists show potential for the development of new anti-diabetic drugs.

Galanin is a widely distributed neuropeptide with a variety of physiological functions. Three galanin receptor subtypes, GALR1, GALR2, and GALR3, have been reported. We isolated a novel galanin-like peptide (GALP) from porcine hypothalamus by observing its activity for increasing [35 S]GTP␥S binding to a membrane preparation of GALR2-transfected cells. The peptide had 60 amino acid residues and a non-amidated C terminus. The amino acid sequence of GALP-(9 -21) was completely identical to that of galanin-(1-13). A cloned porcine GALP cDNA indicated that GALP was processed from a 120-amino acid GALP precursor protein. The structures of rat and human GALP-(1-60) were deduced from cloned cDNA, which indicated that the amino acid sequences 1-24 and 41-53 were highly conserved between humans, rats, and pigs. Receptor binding studies revealed that porcine GALP-(1-60) had a high affinity for the GALR2 receptor (IC 50 ‫؍‬ 0.24 nM) and a lower affinity for the GALR1 receptor (IC 50 ‫؍‬ 4.3 nM). In contrast, galanin showed high affinity for the GALR1 (IC 50 ‫؍‬ 0.097 nM) and GALR2 receptors (IC 50 ‫؍‬ 0.48 nM). GALP is therefore an endogenous ligand that preferentially binds the GALR2 receptor, whereas galanin is relatively non-selective.Galanin, a C-terminally amidated peptide with 29 amino acid residues (non-amidated peptide with 30 residues in humans) was originally isolated from porcine intestine (1) and was later found to be ubiquitous in the central and peripheral nervous systems. It exerts diverse regulatory functions including central modulation of cognition, nociception, and feeding behavior, endocrine control of pituitary and pancreatic hormones, and regulation of gastrointestinal smooth muscle contractions (for review, see Refs. 2, 3).Structurally, galanin is unrelated to any known family of neuropeptides or regulatory peptides, suggesting the presence of unknown members of a galanin peptide family. Indeed, the existence of a galanin-like peptide(s) in mammalian tissues has been proposed for several reasons. First, molecular heterogeneity of galanin-like immunoreactivity has been reported by several groups. Rökaeus et al. (4) first reported that rat brain and ileum contained galanin-like peptides that cross-reacted with galanin antiserum but differed from galanin in chromatographic characterization. Nevertheless, some of these immunoreactivities may be the result of galanin precursors or galaninderived peptides, as described in the subsequent studies (5). Recently, Wang et al. (6) re-evaluated the existence of a novel galanin-like peptide in rat islets by showing the presence of galanin-like immunoreactivity that cross-reacted with antibody against porcine galanin but did not with antibody against rat galanin. Second, the physiological function of the three galanin receptor subtypes, GALR1 1 (7, 8), GALR2 (9, 10), and GALR3 (11,12), is unlikely to be solely the distribution of different signals to target cells. The low affinity of human galanin for human GALR3 (12) suggests the possibility that these receptor s...

Cells expressing oncogenic c-Myc are sensitized to TNF superfamily proteins. c-Myc also is an important factor in determining whether a cell is sensitive to TRAIL-induced apoptosis, and it is well established that the mitochondrial pathway is essential for apoptosis induced by c-Myc. We investigated whether c-Myc action on the mitochondria is required for TRAIL sensitivity and found that Myc sensitized cells with defective intrinsic signaling to TRAIL. TRAIL induced expression of antiapoptotic Mcl-1 and cIAP2 through activation of NF-kappaB. Both Myc and the multikinase inhibitor sorafenib block NF-kappaB. Combining sorafenib with TRAIL in vivo showed dramatic efficacy in TRAIL-resistant tumor xenografts. We propose the combination of TRAIL with sorafenib holds promise for further development.

Cell-cycle checkpoints controlling the orderly progression through mitosis are frequently disrupted in human cancers. One such checkpoint, entry into metaphase, is regulated by the CHFR gene encoding a protein possessing forkhead-associated and RING finger domains as well as ubiquitin-ligase activity. Although defects in this checkpoint have been described, the molecular basis and prevalence of CHFR inactivation in human tumors are still not fully understood. To address this question, we analyzed the pattern of CHFR expression in a number of human cancer cell lines and primary tumors. We found CpG methylation-dependent silencing of CHFR expression in 45% of cancer cell lines, 40% of primary colorectal cancers, 53% of colorectal adenomas, and 30% of primary head and neck cancers. Expression of CHFR was precisely correlated with both CpG methylation and deacetylation of histones H3 and H4 in the CpG-rich regulatory region. Moreover, CpG methylation and thus silencing of CHFR depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3b, as their genetic inactivation restored CHFR expression. Finally, cells with CHFR methylation had an intrinsically high mitotic index when treated with microtubule inhibitor. This means that cells in which CHFR was epigenetically inactivated constitute loss-of-function alleles for mitotic checkpoint control. Taken together, these findings shed light on a pathway by which mitotic checkpoint is bypassed in cancer cells and suggest that inactivation of checkpoint genes is much more widespread than previously suspected.DNA methylation ͉ DNA methyltransferase ͉ mitotic checkpoint ͉ histone deacetylation

We isolated a cDNA encoding an orphan G proteincoupled receptor, TGR7, which has been recently reported to correspond to MrgD. To search for ligands for TGR7, we screened a series of small molecule compounds by detecting the Ca 2؉ influx in Chinese hamster ovary cells expressing TGR7. Through this screening, we found that ␤-alanine at micromolar doses specifically evoked Ca 2؉ influx in cells expressing human, rat, or mouse TGR7. A structural analogue, ␥-aminobutyric acid, weakly stimulated cells expressing human or rat TGR7, but another analogue, glycine, did not. In addition, ␤-alanine decreased forskolin-stimulated cAMP production in cells expressing TGR7, suggesting that TGR7 couples with G proteins G q and G i . In guanosine 5-O-3-thiotriphosphate binding assays conducted using a membrane fraction of cells expressing TGR7, ␤-alanine specifically increased the binding of guanosine 5-O-3-thiotriphosphate. When a fusion protein composed of TGR7 and green fluorescent protein was expressed in cells, it localized at the plasma membrane but internalized into the cytoplasm after treatment with ␤-alanine. In addition, we found that ␤-[3 H]alanine more efficiently bound to TGR7-expressing cells than to control cells. From these results, we concluded that TGR7 functioned as a specific membrane receptor for ␤-alanine. Quantitative PCR analysis revealed that TGR7 mRNA was predominantly expressed in the dorsal root ganglia in rats. By in situ hybridization and immunostaining, we confirmed that TGR7 mRNA was co-expressed in the small diameter neurons with P2X 3 and VR1, both in rat and monkey dorsal root ganglia. Our results suggest that TGR7 participates in the modulation of neuropathic pain.

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OBJECTIVE-GPR40 is a G protein-coupled receptor regulating free fatty acid-induced insulin secretion. We generated transgenic mice overexpressing the hGPR40 gene under control of the mouse insulin II promoter and used them to examine the role of GPR40 in the regulation of insulin secretion and glucose homeostasis.RESEARCH DESIGN AND METHODS-Normal (C57BL/6J) and diabetic (KK) mice overexpressing the hGPR40 gene under control of the insulin II promoter were generated, and their glucose metabolism and islet function were analyzed.RESULTS-In comparison with nontransgenic littermates, hGPR40 transgenic mice exhibited improved oral glucose tolerance with an increase in insulin secretion. Although islet morphologic analysis showed no obvious differences between hGPR40 transgenic and nontransgenic mice, isolated islets from hGPR40 transgenic mice had enhanced insulin secretion in response to high glucose (16 mmol/l) compared with those from nontransgenic mice, and they both had similar low glucose (3 mmol/l)-stimulated insulin secretion. In addition, hGPR40 transgenic islets significantly increased insulin secretion against a naturally occurring agonist palmitate in the presence of 11 mmol/l glucose. hGPR40 transgenic mice were also found to be resistant to high-fat diet-induced glucose intolerance, and hGPR40 transgenic mice harboring KK background showed augmented insulin secretion and improved oral glucose tolerance compared with nontransgenic littermates. CONCLUSIONS-Our results suggest that GPR40 may have a role in regulating glucose-stimulated insulin secretion and plasma glucose levels in vivo and that pharmacological activation of GPR40 may provide a novel insulin secretagogue beneficial for the treatment of type 2 diabetes.

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.