Ryo Yoshimoto

Effects of adrenomedullin and calcitonin gene-related peptide on contractions of the rat aorta and porcine coronary artery

Effects of adrenomedullin and α‐calcitonin gene‐related peptide (CGRP) on the contractions and cytosolic Ca2+ concentrations ([Ca2+]i) of the rat aorta and porcine coronary artery were investigated. Characteristics of the receptors mediating the effects of adrenomedullin and α‐CGRP were also investigated. Adrenomedullin and α‐CGRP caused a concentration‐dependent relaxation in the rat aorta contracted with noradrenaline. The IC50 values for adrenomedullin and α‐CGRP were 2.4 nM and 4.0 nM, respectively. The relaxant effects of these peptides were abolished by removal of the endothelium and significantly attenuated by an inhibitor of nitric oxide synthase, NG‐monomethyl‐L‐arginine (L‐NMMA, 100 μM), but not by a cyclo‐oxygenase inhibitor, indomethacin (10 μM). Adrenomedullin and α‐CGRP increased the endothelial [Ca2+]i in the rat aorta with endothelium, whereas they did not change [Ca2+]i in the smooth muscle. An antagonist of the CGRP1 receptor, CGRP (8–37), antagonized the relaxant effects of α‐CGRP and the β‐isoform of CGRP (β‐CGRP) but not those of adrenomedullin in the rat aorta. In the porcine coronary artery contracted with U46619, adrenomedullin and α‐CGRP caused a concentration‐dependent relaxation with an IC50 of 27.6 and 4.1 nM, respectively. Removal of the endothelium altered neither the IC50 values nor the maximal relaxations induced by adrenomedullin or α‐CGRP. When the artery was contracted with high K+ solution (72.7 mM), these peptides caused a small relaxation. Adrenomedullin and α‐CGRP increased cyclic AMP content and decreased the smooth muscle [Ca2+]i in the porcine coronary artery. CGRP (8–37) significantly antagonized the relaxant effects of adrenomedullin and α‐CGRP in the porcine coronary artery. However, it had little effect on the relaxations induced by the β‐isoform of CGRP (β‐CGRP). These results suggest that in the rat aorta, adrenomedullin and α‐CGRP increase the endothelial [Ca2+]i, activate nitric oxide synthase and release nitric oxide, without a direct inhibitory action on smooth muscle. In the porcine coronary artery, in contrast, adrenomedullin and α‐CGRP directly act on smooth muscle, increase cyclic AMP content, decrease the smooth muscle [Ca2+]i and inhibit contraction. The rat aortic endothelium seems to express the CGRP receptor which is sensitive to α‐CGRP, β‐CGRP and CGRP (8–37) and the adrenomedullin specific receptor. The porcine coronary smooth muscle, in contrast, seems to express two types of CGRP receptor; one of which is sensitive to α‐CGRP, CGRP (8–37) and adrenomedullin and the other is sensitive only to β‐CGRP.

Role of MGAT2 and DGAT1 in the release of gut peptides after triglyceride ingestion

Triglyceride ingestion releases gut peptides from enteroendocrine cells located in the intestinal epithelia and provides feedback regulations of gastrointestinal function. The precise mechanisms sensing lipids in the intestinal wall, however, are not well characterized. In the current study, we investigated the release of gut peptides following oral triglyceride loading in mice deficient for monoacylglycerol acyltransferase 2 (MGAT2KO) and diacylglycerol acyltransferase 1 (DGAT1KO), enzymes that sequentially re-synthesize triglyceride to secrete as chylomicron at the small intestine. In wild-type (Wt) mice, oral triglyceride loading resulted in hypertriglycemia. In addition, plasma glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) were significantly increased 30 min after triglyceride loading, before decaying in 2h. In MGAT2KO and DGAT1KO mice, oral triglyceride loading did not result in hypertriglycemia and the increase in GIP was significantly suppressed in both KO mouse strains. In contrast, the increases in plasma GLP-1 and PYY in both KO mouse strains were comparable to Wt mice 30 min after triglyceride loading, however, they remained elevated in DGAT1KO mice even 2h after triglyceride loading. In parallel to the changes in GLP-1 and PYY, gastric emptying was delayed after oral triglyceride loading in MGAT2KO mice comparably to Wt type mice and was further delayed in DGAT1KO mice. STC-1 and GLUTag, GLP-1-producing intestinal endocrine L-cell lines, displayed a significant level of DGAT1 activity but not MGAT activity. These findings suggest that synthesis and/or secretion of triglyceride-rich lipoproteins play an important role in the release of GIP. Moreover, DGAT1 may directly regulate the release of GLP-1 and PYY in L-cells.

Chronic effect of doxorubicin on vascular endothelium assessed by organ culture study.

We have attempted to determine the chronic effects of doxorubicin, a commonly used anticancer agent, on vascular endothelium using an organ culture system. In rabbit mesenteric arteries treated with 0.3 microM doxorubicin for 7 days, rounding and concentrated nuclei and TUNEL-positive staining were observed in endothelial cells, indicating DNA damage and the induction of apoptosis. However, the endothelium-dependent relaxation induced by substance P and the expression of mRNA encoding endothelial NO synthase (eNOS) did not differ from those in control arteries. In arteries treated with a higher concentration (1 microM) of doxorubicin, apoptosis and damage to nuclei occurred in the endothelial cells at the third day of treatment, and the detachment and excoriation of endothelium from the tunica interna of the vascular wall were also observed. The impairment of endothelium-dependent relaxation was observed at the fifth day of the treatment with 1 microM doxorubicin. Additionally, apoptotic change in the smooth muscle layer was observed at this concentration of doxorubicin. Apoptotic phenomena were further confirmed by DNA fragmentation using isolated bovine aortic endothelial cells (BAECs) and A7r5 vascular smooth muscle cells, and it was revealed that BAECs are more sensitive than A7r5 to the apoptotic effect of doxorubicin. These results suggest that chronic treatment with doxorubicin at therapeutic concentrations induces apoptosis and excoriation of endothelial cells, which diminishes endothelium-dependent relaxation.

Identification of a stable chemerin analog with potent activity toward ChemR23.

Chemerin is a novel peptide that was identified as a natural ligand for ChemR23. As it has been reported to be involved in the regulation of immune responses and adipogenesis, chemerin may have a variety of physiological functions. Chemerin is synthesized as a precursor (prochemerin) and is proteolytically activated and inactivated in sequential steps, which control its physiological roles in a coordinated manner. Chemerin-9 (chemerin148-156) was previously identified as the smallest peptide with low nanomolar potency. However, like mature chemerin, chemerin-9 is rapidly degraded and inactivated in plasma, which has limited the use of chemerin-9 in in vivo experiments. In order to identify stable chemerin analogs that facilitate in vivo studies, we synthesized a series of chemerin-9 analogs and examined intrinsic activity and metabolic stability. We identified an agonistic and metabolically stable chemerin-9 analog (d-Tyr(147)-[d-Ser(151), d-Ala(154), Tic(155)]chemerin148-156) that shows enhanced plasma exposure with prolonged half-life in mice upon intraperitoneal administration. Improvement of metabolic stability resulted in a reduction in the plasma free fatty acid levels in fasted mice, which cannot be accomplished by unstable-mouse chemerin-9. This reduction in plasma free fatty acids reflects the anti-lipolysis activity of chemerin-9 and analogs in mouse primary adipocytes. The discovery of a metabolically stable chemerin analog will facilitate investigation of the pharmacological roles of chemerin in vivo. Moreover, this stable chemerin analog might provide new therapeutic approaches to inflammatory diseases such as asthma and metabolic disorders such as obesity and diabetes where ChemR23 activation may be of benefit.

海洋由来天然生理活性物質，ペクテノトキシン-2のアクチン重合阻害作用

Pectenotoxin-2 (PCTX-2), which is one of Diarrhetic Shellfish Poisoning (DSP), is a family of cyclic polyether macrolide toxin isolated from scallop Patinopecten yessoensis. Although PCTX-2 has a potent cytotoxic activities against several cancer cell lines, the biochemical activity of PCTX-2 has not been determined yet. To clarify the biochemical activity of PCTX-2 is the aime in this study. PCTX-2 inhibited the contractions elicited by 72.7 mM KCl or 1 microM phenyrephrine in a concentration dependent manner in the isolated rat aorta. In A10 cells, actin stressfiber in center but not in periphery of the cell was disrupted by PCTX-2 without any visible shape change. By monitoring fluorescent intensity of pyrenyl-actin, PCTX-2 was found to inhibit the velocity and the degree of actin polymerization in a concentration dependent manner. In addition, PCTX-2 decreased viscosity of F-actin measured with falling ball viscometry. Stoichiometric analysis indicated that PCTX-2 forms 1:4 complex with G-actin. These results suggest that PCTX-2 is a potent natural actin depolymerizing compound with unique mode of action.

Pharmacological inhibition of diacylglycerol acyltransferase 1 reduces body weight and modulates gut peptide release—Potential insight into mechanism of action

Investigation was conducted to understand the mechanism of action of diacylglycerol acyltransferase 1 (DGAT1) using small molecules DGAT1 inhibitors, compounds K and L.

Discovery and characterization of a novel potent, selective and orally active inhibitor for mammalian ELOVL6

The elongase of long chain fatty acids family 6 (ELOVL6) is a rate-limiting enzyme for the elongation of saturated and monounsaturated long chain fatty acids. ELOVL6 is abundantly expressed in lipogenic tissues such as liver, and its mRNA expression is up-regulated in obese model animals. ELOVL6 deficient mice are protected from high-fat-diet-induced insulin resistance, suggesting that ELOVL6 might be a new therapeutic target for diabetes. We previously identified an indoledione compound, Compound A, as the first inhibitor for mammalian ELOVL6. In this study, we discovered a novel compound, Compound B, and characterized its biochemical and pharmacological properties. Compound B has a more appropriate profile for use as a pharmacological tool compared to Compound A. Chronic treatment with Compound B in model animals, diet-induced obesity (DIO) and KKAy mice, showed significant reduction in hepatic fatty acid composition, suggesting that it effectively inhibits ELOVL6 activity in the liver. However, no improvement in insulin resistance by ELOVL6 inhibition was found in these model animals. Further studies need to address the impact of ELOVL6 inhibition on pharmacological abnormalities in several model animals. This is the first report on pharmacology data from chronic studies using a selective ELOVL6 inhibitor. Compound B appears to be a useful tool to further understand the physiological roles of ELOVL6 and to evaluate the therapeutic potential of ELOVL6 inhibitors.

Synthesis and evaluation of a novel indoledione class of long chain fatty acid elongase 6 (ELOVL6) inhibitors.

Novel indoledione derivatives were synthesized and evaluated as long chain fatty acid elongase 6 (ELOVL6) inhibitors. Systematic optimization of an indole class of lead 1 led to the identification of potent ELOVL6 selective inhibitors. Representative inhibitor 37 showed sustained plasma exposure and good liver penetrability in mice. After oral administration, 37 potently inhibited ELOVL6 activity in the liver in mice.

In vivo imaging of obesity-induced inflammation in adipose tissue.

Obesity is associated with low-grade inflammation in adipose tissue, which contributes to the development of obesity-related diseases such as insulin resistance, hypertension and arteriosclerosis. Here we developed an animal model that non-invasively monitors inflammation in adipose tissue using in vivo bioluminescent imaging (BLI) technique. In vitro, stimulation with TNFalpha or co-culture with RAW264 macrophages increased bioluminescence in 3T3-L1 adipocytes expressing NF-kappaB-mediated luciferase gene (3T3-L1/NF-kappaB-re-luc2P). In vivo, lipopolysaccharide increased bioluminescence in mice transplanted with 3T3-L1/NF-kappaB-re-luc2P cells. Moreover, light emission derived from implanted cells was significantly higher in diet-induced obese mice transplanted with 3T3-L1/NF-kappaB-re-luc2P than in lean mice. Our results showed that BLI technique and 3T3-L1/NF-kappaB-re-luc2P cells provide a useful approach to non-invasively monitor obesity-induced inflammation in adipose tissue in in vivo.

Synthesis and Biological Evaluation of a Novel 3-Sulfonyl-8-azabicyclo[3.2.1]octane Class of Long Chain Fatty Acid Elongase 6 (ELOVL6) Inhibitors

Long chain fatty acid elongase 6 (ELOVL6) catalyzes the elongation of long chain fatty acyl-CoAs and is a potential target for the treatment of metabolic disorders. The ultrahigh throughput screen of our corporate chemical collections resulted in the identification of a novel 3-sulfonyl-8-azabicyclo[3.2.1]octane class of ELOVL6 inhibitor 1a. Optimization of lead 1a led to the identification of the potent, selective, and orally available ELOVL6 inhibitor 1w.