Signal transduction pathways of muscarinic receptors in circular smooth muscle from the rabbit caecum

The effects of muscarinic acetylcholine receptor stimulation on phosphoinositides breakdown and adenylate cyclase activity were examined in the circular smooth muscle of the rabbit caecum. InMyo-[³H]inositol-labeled circular smooth muscle cells, carbachol caused a concentration-dependent increase in [³H]inositol phosphates ([³H]IPs) accumulation (EC₅₀ of 3±1 M). The M₁-selective antagonist pirenzepine (PRZ), the M₂-selective AF-DX 116 (11-2[[2-[(diethyl-amino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6Hpyrido[2,3-b][1,4]benzodiazepin-6-one) and the M₃-selective para-fluoro-hexahydrosiladifenidol (p-F-HHSiD) inhibited the carbachol-induced [³]inositol phosphates accumulation with the following order of potency: p-F-HHSiD>PRZ>AF-DX 116. In saponin-permeabilized circular smooth muscle cells, carbachol and GTP[S] elicited a concentration-dependent increase in [³H]inositol phosphates accumulation. The concentration-response curve for GTP[S] was shifted to the left when cells were incubated with 1 M carbachol. The [³H]inositol phosphates accumulation elicited by simultaneous addition of 0.1 M GTP[S] and 1 M carbachol to permeabilized cells was significantly decreased (78.28±18.23% inhibition) when cells were preincubated for 5 min with 0.1 mM GDP[S]. In nonpermeabilized cells, pertussis toxin did not alter the carbachol-induced increase in [³H]inositol phosphates accumulation. On the other hand, the 0.1 mM carbachol-induced inhibition of forskolin-stimulated adenylate cyclase activity in circular smooth muscle homogenates was significantly reversed by atropine and AF-DX 116, whereas PRZ and p-F-HHSiD were ineffective (muscarinic antagonists were used at 1 M final concentration). Moreover, the carbachol-induced inhibition of the cyclic AMP accumulation elicited by 10 M isoproterenol was abolished by pertussis toxin pretreatment of isolated circular smooth muscle cells. In conclusion, our data suggest that in circular smooth muscle of rabbit caecum, the muscarinic receptor stimulation of [³H]inositol phsophates accumulation is mediated by M₃ subtype receptors coupled to a pertussis toxin-insensitive G protein, whereas inhibition of adenylate cyclase activity is mediated by M₂ subtype receptors coupled to a pertussis toxin-sensitive GTP-binding protein G_i.     

Peptide fragment of the m3 muscarinic acetylcholine receptor activates G(q) but not G(i2).

G(q), a heterotrimeric guanine nucleotide-binding protein, plays important roles such as the regulation of calcium mobilization and cell proliferation. This protein is considered as a promising drug target for the treatment of cardiac hypertrophy. Selective activation of G(q) would be quite useful for analyzing the role of G(q) in signaling pathways. We synthesized m3i3c-a peptide with 16 amino acid residues that corresponds to the junction between the C-terminus of the third intracellular loop and the sixth transmembrane helix (TM-VI) of human m3 muscarinic acetylcholine receptor, which couples to G(q) but not G(i2). At micromolar concentrations, this peptide was found to activate G(q) but not G(i2). This peptide is the first small compound that selectively activates G(q) but not G(i2). Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Altered clock gene expression and vascular smooth muscle diurnal contractile variations in type 2 diabetic db/db mice

This study was designed to determine whether the 24-h rhythms of clock gene expression and vascular smooth muscle (VSM) contractile responses are altered in type 2 diabetic db/db mice. Control and db/db mice were euthanized at 6-h intervals throughout the day. The aorta, mesenteric arteries, heart, kidney, and brain were isolated. Clock and target gene mRNA levels were determined by either real-time PCR or in situ hybridization. Isometric contractions were measured in isolated aortic helical strips, and pressor responses to an intravenous injection of vasoconstrictors were determined in vivo using radiotelemetry. We found that the 24-h mRNA rhythms of the following genes were suppressed in db/db mice compared with control mice: the clock genes period homolog 1/2 (Per1/2) and cryptochrome 1/2 (Cry1/2) and their target genes D site albumin promoter-binding protein (Dbp) and peroxisome proliferator-activated receptor-γ (Pparg) in the aorta and mesenteric arteries; Dbp in the heart; Per1, nuclear receptor subfamily 1, group D, member 1 (Rev-erba), and Dbp in the kidney; and Per1 in the suprachiasmatic nucleus. The 24-h contractile variations in response to phenylephrine (α(1)-agonist), ANG II, and high K(+) were significantly altered in the aortas from db/db mice compared with control mice. The diurnal variations of the in vivo pressor responses to phenylephrine and ANG II were lost in db/db mice. Moreover, the 24-h mRNA rhythms of the contraction-related proteins Rho kinase 1/2, PKC-potentiated phosphatase inhibitory protein of 17 kDa, calponin-3, tropomyosin-1/2, and smooth muscle protein 22-α were suppressed in db/db mice compared with control mice. Together, our data demonstrated that the 24-h rhythms of clock gene mRNA, mRNA levels of several contraction-related proteins, and VSM contraction were disrupted in db/db mice, which may contribute to the disruption of their blood pressure circadian rhythm.     

驼乳源益生菌对db/db鼠肠道菌群的调节作用

目的 探究驼乳来源复合益生菌对db/db鼠血糖及肠道菌群的调节作用。方法 将60只db/db鼠随机分为5组,每组12只,分别为模型组、二甲双胍组、利拉鲁肽组、低剂量复合益生菌组和高剂量复合益生菌组,12只C57BL/Ks小鼠为正常对照组。阳性药组分别灌胃0.3 mg/g二甲双胍及皮下注射0.2 μg/g利拉鲁肽,低剂量复合益生菌组灌胃乳酸菌1.0×108 CFU/d和酵母菌1.0×106 CFU/d,高剂量复合益生菌组灌胃乳酸菌1.0×1010 CFU/d和酵母菌1.0×108 CFU/d,其他组灌胃生理盐水。血糖仪检测空腹血糖（FBG）及不同时间段血糖水平。ELISA法检测糖化血红蛋白（HbA1c）含量。实时荧光定量PCR检测粪便菌群含量。结果 与模型组相比,低、高剂量复合益生菌组FBG水平明显减少,改善葡萄糖耐受（OGTT）能力,降低HbA1c含量。与模型组相比,低、高剂量复合益生菌组显著降低厚壁菌门、放线菌门和大肠埃希菌属水平,增加拟杆菌门、乳杆菌属和双歧杆菌属水平,但对总菌与梭杆菌属无影响。结论 驼乳来源复合益生菌可通过调节肠道菌群降低db/db鼠血糖含量。     

M3 receptor modulates extracellular matrix synthesis via ERK1/2 signaling pathway in human bladder smooth muscle cells

Extracellular matrix (ECM) accumulation plays a key role in the progression of bladder outlet obstruction (BOO). Muscarinic receptors have been widely reported to serve as pivotal regulators in lung tissue remodeling. However, the influence of them on human bladder smooth muscle cells (HBSMCs) and the underlying molecular mechanisms have not yet been evaluated. The purposes of the present study are to investigate the effect of muscarinic receptors on the synthesis of ECM in HBSMCs and the involvement of intracellular signal transducers. The results indicated that M₁-M₅ muscarinic receptors were all encoded in HBSMCs. The expression rank order was M₂ > M₁ > M₅ > M₃ > M₄. The gene and protein expression of collagen I (COL1), TIMP-1, and TIMP-2 was carbachol (CCH) concentration-dependently enhanced. The synthesis of COL1 in the supernatant of cell culture medium was significantly elevated by exposure to CCH. The CCH-induced protein expression of COL1, TIMP-1, and TIMP-2, however, was obviously reduced by the pretreatment of muscarinic receptor antagonists, atropine, and M₃-preferring antagonist (1,1-dimethyl-4-diphenyl-acetoxypiperidinium iodide [4-DAMP]). Furthermore, ERK1/2 was activated by 100 µM CCH when compared with the control group and the pretreatment of ERK1/2 inhibitor significantly suppressed the synthesis of COL1 induced by 100 µM CCH. Besides, CCH-induced phosphorylation of ERK1/2 was remarkably restrained by the pretreatment of 4-DAMP. All in all, these findings demonstrated that M₃ receptor can modulate extracellular matrix synthesis via the ERK1/2 signaling pathway, which may provide potential novel therapeutic targets for BOO.     

Hyperosmolality-induced abnormal patterns of calcium mobilization in smooth muscle cells from non-diabetic and diabetic rats

Hyperglycemia and/or hyperosmolality may disturb calcium homeostasis in vascular smooth muscle cells (SMCs), leading to altered vascular contractility in diabetes. To test this hypothesis, the KCl induced increases in [Ca²⁺]_i in primarily cultured vascular SMCs exposed to different concentrations of glucose were examined. With glucose concentration in solutions kept at 5.5 mM, KCl induced a fast increase in [Ca²⁺]_i which then slowly declined (type 1 response) in 83% of SMCs from non-diabetic rats. In 9% of non-diabetic SMCs KCl induced a slow increase in [Ca²⁺]_i (type 2 response). Interestingly, under the same culture conditions KCl induced type 1 and type 2 responses in 47 and 35% of SMCs from diabetic rats. When SMCs from non-diabetic or diabetic rats were cultured in 36 mM glucose, KCl induced a fast increase in [Ca²⁺]_i which, however, maintained at a high level (type 3 response). The sustained level of [Ca²⁺]_i in the presence of KCl was significantly higher in cells cultured with 36 mM glucose than that in non-diabetic cells cultured with 5.5 mM glucose. Furthermore, the hyperglycemia-induced alterations in calcium mobilization were similarly observed in cells cultured in high concentration of mannitol (30.5 mM) or L-glucose, indicating that hyperosmolality was mainly responsible for the abnormal calcium mobilization in diabetic SMCs.     

Dissociation of the metabolic from the contractile response to muscarinic stimulation in the rabbit urinary bladder

The calcium dependence of contraction and NADH flurorescence was investigated in rabbit bladder stimulated with bethanechol or KCl. The absence of calcium in the bathing solution induced a rightward shift in the dose response to bethanechol for both contraction and NADH flurorescence. The contractile response was shifted to a greater degree than the fluorescence response and the maximal response to bethanechol was reduced by 80% for contraction but only 20% for NADH fluorescence. This rightward shift was also induced by the benzothiazepine calcium antagonist diltiazem (200 M) and again the contractile response was shifted significantly more than the fluorescence response. The combination of zero calcium and 200 M diltiazem virtually abolished contractions but only inhibited the NADH fluorescence by 65% at maximally effective bethanechol concentrations. Unlike the effect of diltiazem on the response to bethanechol, diltiazem (200 M) shifted both the contraction and fluorescence curves to the right equally in response to KCl stimulation. These results indicate that a metabolic response to muscarinic stimulation (decreased NADH) can occur in the absence of any observable contractile response. This metabolic response may be due to post receptor signal processing events. For KCl stimulation, the NADH response is probably secondary to and a result of the contractile response.Abbreviations ATP Adenosine Triphosphate - KCl Potassium Chloride - HPLC High Performance Liquid Chromatography - NADH reduced nicotinamide Adenine Dinucleotide - NAD Oxidized Nicotinamide Adenine Dinucleotide     

Characterization of contractile function and expression of muscarinic receptors,G proteins and adenylate cyclase in cultured tracheal smooth muscle of Swine

Smooth muscle cells lose their contractile function and phenotype very rapidly when placed in culture. During organ culture of smooth muscle strips, phenotype is lost more slowly. In the present studies, we established an organ culture model to study contractile function and expression of muscarinic receptors, G proteins and adenylyl cyclase in different serum concentrations in tracheal smooth muscle from swine. The results show that contractile function and the amounts of M(3) receptors, G proteins and adenylyl cyclase were maintained for up to 5 days in culture. The expression of M(2) receptors was significantly decreased in culture when compared to freshly isolated muscles. Maximal isometric tension was significantly increased in cultured muscles compared with freshly isolated muscles. Different serum concentrations did not significantly affect contractile function and expression of muscarinic receptors, G proteins and adenylyl cyclase. In conclusion, our studies suggest that cultured smooth muscle might be used as a model to study the regulation of contractile function of smooth muscle by various signal transduction pathways.     

Antidiabetic effect of enterolactone in cultured muscle cells and in type 2 diabetic model db/db mice

Enterolactone (ENL) is formed by the conversion of dietary precursors like strawberry lignans via the gut microbiota. Urinary concentrations of lignan metabolites are reported to be significantly associated with a lower risk of Type 2 diabetes (T2D). In the present study, antidiabetic effect of ENL and its modes of action were studied in vitro and in vivo employing a rat skeletal muscle-derived cell line, L6 myocytes in culture, and T2D model db/db mice. ENL dose-dependently increased glucose uptake in L6 myotubes under insulin absent condition. This increase by ENL was canceled by compound C, an inhibitor of 5′-adenosine monophosphate-activated protein kinase (APMK). Activation (=phosphorylation) of AMPK and translocation of glucose transporter 4 (GLUT4) to plasma membrane in L6 myotubes were demonstrated by Western blotting analyses. Promotion by ENL of GLUT4 translocation to plasma membrane was also visually demonstrated by immunocytochemistry in L6 myoblasts that were transfected with glut4 cDNA-coding vector. T2D model db/db mice were fed the basal 20 % casein diet (20C) or 20C supplemented with ENL (0.001 or 0.01 %) for 6 weeks. Fasting blood glucose (FBG) levels were measured every week and intraperitoneal glucose tolerance test (IPGTT) was conducted. ENL at a higher dose (0.01 % in 20C) suppressed the increases in FBG levels. ENL was also demonstrated to improve the index of insulin resistance (HOMA-IR) and glucose intolerance by IPGTT in db/db mice. From these results, ENL is suggested to be an antidiabetic chemical entity converted from dietary lignans by gut microbiota.     

Impairment of contractility associated with muscarinic supersensitivity in trachea isolated from diabetic rats: lack of correlation with ultrastructural changes or quinuclidinyl benzylate binding to lung membranes

Evolution of cholinergic response of rat isolated trachea was determined after various durations of diabetes (17, 40, 90, 150 and 210 days). Long-term diabetes was associated with both impairment of contractility and supersensitivity to cholinergic stimulation. However, the mechanism of these alterations remains to be determined, as response to field stimulation was not specifically altered while electron microscopy studies could not detect any significant change in the aspect of nerves, smooth muscle or epithelium. As well, binding studies of lung cholinergic receptors using the antagonist ligand [³H]-quinuclidinyl benzylate and the agonist carbachol did not detect any change in diabetic animals.     

Estradiol restores diabetes-induced reductions in sex steroid receptor expression and distribution in the vagina of db/db mouse model

Sex steroid hormones and receptors play an important role in maintaining vaginal physiology. Disruptions in steroid receptor signaling adversely impact vaginal function. Limited studies are available investigating the effects of diabetic complications on steroid receptor expression and distribution in the vagina. The goals of this study were to investigate type 2 diabetes-induced changes in expression, localization and distribution of estrogen (ER), progesterone (PR) and androgen receptors (AR) in the vagina and to determine if estradiol treatment ameliorates these changes. Eight-week-old female diabetic (db/db) mice (strain BKS.Cg-m+/+ Lepr^db/J) were divided into two subgroups: untreated diabetic and diabetic animals treated with pellets containing estradiol. Control normoglycemic littermates were subcutaneously implanted with pellets devoid of estradiol. At 16 weeks of age, animals were sacrificed, vaginal tissues excised and analyzed by Western blot and immunohistochemical methods. Diabetes produced marked reductions in protein expression of ER, PR, and AR. Diabetes also resulted in marked differences in the distribution, staining intensity and proportion of immunoreactive cells containing these steroid receptors in the epithelium, lamina propria and muscularis. Treatment of diabetic animals with estradiol restored receptor protein expression and distribution similar to those levels observed in control animals. This study demonstrates that type 2 diabetes markedly reduces steroid receptor protein expression and distribution in the vagina. Estradiol treatment of diabetic animals ameliorates these diabetes-induced changes.     

Whole-cell K+ current activation in response to voltages and carbachol in gastric parietal cells isolated from guinea pig

Summary Patch-clamp studies of whole-cell ionic currents were carried out in parietal cells obtained by collagenase digestion of the gastric fundus of the guinea pig stomach. Applications of positive command pulses induced outward currents. The conductance became progressively augmented with increasing command voltages, exhibiting an outwardly rectifying current-voltage relation. The current displayed a slow time course for activation. In contrast, inward currents were activated upon hyperpolarizing voltage applications at more negative potentials than the equilibrium potential to K⁺ (E _K). The inward currents showed time-dependent inactivation and an inwardly rectifying current-voltage relation. Tail currents elicited by voltage steps which had activated either outward or inward currents reversed at nearE _K, indicating that both time-dependent and voltagegated currents were due to K⁺ conductances. Both outward and inward K⁺ currents were suppressed by extracellular application of Ba²⁺, but little affected by quinine. Tetraethylammonium inhibited the outward current without impairing the inward current, whereas Cs⁺ blocked the inward current but not the outward current. The conductance of inward K⁺ currents, but not outward K⁺ currents, became larger with increasing extracellular K⁺ concentration. A Ca²⁺-mobilizing acid secretagogue, carbachol, and a Ca²⁺ ionophore, ionomycin, brought about activation of another type of outward K⁺ currents and voltage-independent cation currents. Both currents were abolished by cytosolic Ca²⁺ chelation. Quinine preferentially inhibited this K⁺ current. It is concluded that resting parietal cells of the guinea pig have two distinct types of voltage-dependent K⁺ channels, inward rectifier and outward rectifier, and that the cells have Ca²⁺-activated K⁺ channels which might be involved in acid secretion under stimulation by Ca²⁺-mobilizing secretagogues.     

Coincident signalling between the Gi/Go-coupled delta-opioid receptor and the Gq-coupled m3 muscarinic receptor at the level of intracellular free calcium in SH-SY5Y cells

In SH-SY5Y cells, activation of delta-opioid receptors with [D-Pen(2,5)]-enkephalin (DPDPE; 1 microM) did not alter the intracellular free Ca(2+) concentration [Ca(2+)](i). However, when DPDPE was applied during concomitant Gq-coupled m3 muscarinic receptor stimulation by carbachol or oxotremorine-M, it produced an elevation of [Ca(2+)](i). The DPDPE-evoked increase in [Ca(2+)](i) was abolished when the carbachol-sensitive intracellular Ca(2+) store was emptied. There was a marked difference between the concentration-response relationship for the elevation of [Ca(2+)](i) by carbachol (EC(50) 13 microM, Hill slope 1) and the concentration-response relationship for carbachol's permissive action in revealing the delta-opioid receptor-mediated elevation of [Ca(2+)] (EC(50) 0.7 mM; Hill slope 1.8). Sequestration of free G protein beta gamma dimers by transient transfection of cells with a beta gamma binding protein (residues 495-689 of the C terminal tail of G protein-coupled receptor kinase 2) reduced the ability of delta opioid receptor activation to elevate [Ca(2+)](i). However, DPDPE did not elevate either basal or oxotremorine-M-evoked inositol phosphate production indicating that delta-opioid receptor activation did not stimulate phospholipase C. Furthermore, delta-opioid receptor activation did not result in the reversal of muscarinic receptor desensitization, membrane hyperpolarization or stimulation of sphingosine kinase. There was no coincident signalling between the delta-opioid receptor and the lysophosphatidic acid receptor which couples to elevation of [Ca(2+)](i) in SH-SY5Y cells by a PLC-independent mechanism. In SH-SY5Y cells the coincident signalling between the endogenously expressed delta-opioid and m3 muscarinic receptors appears to occur in the receptor activation-Ca(2+) release signalling pathway at a step after the activation of phospholipase C.     

Characterization of ion movements and their relationship to muscarinic receptor binding and excitation-contraction coupling in guinea pig ileal longitudinal smooth muscle

The relationship between ion movements (sodium uptake and potassium release) and agonist-induced contractile responses or muscarinic receptor binding was investigated in the guinea pig ileal longitudinal muscle (GPLM). Sodium uptake and potassium release were agonist-dependent, concentration-dependent, and stereoselective, with the following rank order of maximum ion movement: muscarinic agonists greater than histamine greater than substance P = serotonin. Potassium depolarization did not initiate sodium uptake or potassium release. Sodium uptake was rapid and monophasic, preceding potassium release which was biphasic in nature. Full muscarinic agonists produced equal maximal increases in sodium uptake, while maximal potassium release varied for all muscarinic agonists and in addition differed from sodium uptake in the following ways: time course, stereoselectivity, sensitivity to calcium antagonists, modulation by the guanylyl nucleotide derivative, 5'-guanylylimidodiphosphate (Gpp(NH)p), and inhibition by muscarinic receptor blockade with benzilylcholine mustard. The calcium ionophores A23187 and ionomycin (SQ23377) did not produce any sodium uptake; A23187 but not ionomycin produced potassium release comparable to that evoked by muscarinic agonists. Ion movement in response to combinations of agonists were not additive. Muscarinic agonist binding as measured by competition for [3H]quinuclidinyl benzilate ([3H]QNB) binding, was best described by multiple sites and was regulated by Gpp(NH)p. Excellent correlations were observed between the dissociation constants for binding and sodium uptake, potassium release, and contraction. The best correlations were those between the pharmacologic responses and the high affinity binding site in the absence, and the low affinity site in the presence, of Gpp(NH)p, respectively. Furthermore, the potencies of muscarinic agonists to evoke ion movements and to inhibit [3H]QNB binding were similar, and from one to two orders of magnitude less than those for contraction. It is suggested that contraction and potassium release were mediated by the high affinity, and sodium uptake by the low and average affinity muscarinic agonist binding sites, respectively. These findings suggest an agonist-activated receptor-effector coupling model in GPLM that leads to the activation of sodium uptake, potassium release, and subsequently, contraction.     

Features of insulin receptor interaction in placenta from normal,overt and poorly controlled gestational diabetic patients

Features of insulin binding to trophoblast plasma membranes were studied in six normal pregnant women (NP), six overt diabetes (ODP) and six poorly controlled glycemic gestational patients (PCDP) i.e. women who did not strictly follow the management of diabetes mellitus during pregnancy. A decreased maximum specific insulin receptor binding per 0.1 mg membrane protein in placenta from PCDP (12%) was found comparing with that from ODP or NP (17.5% and 36.2%, respectively, P<0.01), The insulin binding in PCDP declined at a faster rate until it reached minimum when studied at a higher temperature (25–37°C). The binding equilibrium was likewise attained faster at this temperature than that at lower temperature of 4°C for all studied groups.The insulin receptor binding in all studied groups was pH dependent. The maximum binding in ODP and PCDP groups was attained at pH 7.8 while for NP maximum binding was at pH 7.4. The competitive dinding assay was carried out with 14 concentrations of unlabelled insulin and the half maximal displacement of¹²⁵I-insulin was at 8×10^–9 M, 6×10^–9 M and 4×10^–9 M for NP, ODP and PCDP, respectively (P<0.05) suggesting the differences in the effect of glycemic control on the insulin binding. Furthermore the binding yielded curvilinear Scatchard plots with the apparent affinity of the receptors being affected in the ODP and PCDP groups.The molecular characteristics of the receptors in the diabetic patients as revealed by the cross-linking technique used in this study did not reveal any changes in the subunit structures when compared with normals except that the¹²⁵I-insulin bound as shown by the band intensity was much less in PCDP. These findings indicate that control of hyperglycemia could optimize the outcome of insulin receptor function during diabetic pregnancy.     

Basic fibroblast growth factor regulates extracellular matrix and contractile protein expression independent of proliferation in vascular smooth muscle cells

Summary Basic fibroblast growth factor (bFGF) can influence proliferation and differentiation in vascular smooth muscle cells. Basic FGF promotes some features of the synthetic phenotype (proliferation) but is known to inhibit others (collagen synthesis). Whether bFGF availability influences smooth muscle cell phenotype independent of proliferation is not known. The purpose of this study was to determine if the effects of bFGF on extracellular matrix and contractile protein expression are dependent on changes in proliferation. Basic FGF availability was manipulated by adding bFGF to cultured cells or by inhibiting bFGF expression using antisense RNA, and adjusting culture conditions such that proliferation was held constant. Compared to cells cultured in serum alone, smooth muscle α-actin and myosin heavy chain expression was markedly reduced by added bFGF, but was not influenced by antisense inhibition of bFGF expression. Under the same conditions, collagen synthesis was inhibited by added bFGF, and was stimulated by reduced bFGF expression. These consequences of altering bFGF availability were not associated with changes in FGF receptor expression. These findings demonstrate that alterations in bFGF availability can regulate smooth muscle cell phenotype independent of proliferation, which may be related to the regulation of smooth muscle cell phenotype in vivo.     

Novel GPR119 agonist AS1535907 contributes to first-phase insulin secretion in rat perfused pancreas and diabetic db/db mice

G protein-coupled receptor (GPR) 119 is highly expressed in pancreatic β-cells and enhances the effect of glucose-stimulated insulin secretion (GSIS) on activation. The development of an oral GPR119 agonist that specifically targets the first phase of GSIS represents a promising strategy for the treatment of type 2 diabetes. In the present study, we evaluated the therapeutic potential of a novel small molecule GPR119 agonist, AS1535907, which was modified from the previously identified 2,4,6-tri-substituted pyrimidine core agonist AS1269574. AS1535907 displayed an EC₅₀ value of 4.8 μM in HEK293 cells stably expressing human GPR119 and stimulated insulin secretion in rat islets only under high-glucose (16.8 mM) conditions. In isolated perfused pancreata from normal rats, AS1535907 enhanced the first phase of insulin secretion at 16.8 mM glucose, but had no effect at 2.8 mM glucose. In contrast, the sulfonylurea glibenclamide predominantly induced insulin release in the second phase at 16.8 mM glucose and also markedly stimulated insulin secretion at 2.8 mM glucose. In in vivo studies, a single 10 μM administration of AS1535907 to diabetic db/db mice reduced blood glucose levels due to the rapid secretion of insulin secretion following oral glucose loading. These results demonstrate that GPR119 agonist AS1535907 has the ability to stimulate the first phase of GSIS, which is important for preventing the development of postprandial hypoglycemia. In conclusion, the GPR119 agonist AS1535907 induces a more rapid and physiological pattern of insulin release than glibenclamide, and represents a novel strategy for the treatment of type 2 diabetes.