The worldwide prevalence of obesity is steadily increasing, nearly doubling between 1980 and 2008. Obesity is often associated with insulin resistance, a major risk factor for type 2 diabetes mellitus (T2DM): a costly chronic disease and serious public health problem. The underlying cause of T2DM is a failure of the beta cells of the pancreas to continue to produce enough insulin to counteract insulin resistance. Most current T2DM therapeutics do not prevent continued loss of insulin secretion capacity, and those that do have the potential to preserve beta cell mass and function are not effective in all patients. Therefore, developing new methods for preventing and treating obesity and T2DM is very timely and of great significance. There is now considerable literature demonstrating a link between inhibitory guanine nucleotide-binding protein (G protein) and G protein-coupled receptor (GPCR) signaling in insulin-responsive tissues and the pathogenesis of obesity and T2DM. These studies are suggesting new and emerging therapeutic targets for these conditions. In this review, we will discuss inhibitory G proteins and GPCRs that have primary actions in the beta cell and other peripheral sites as therapeutic targets for obesity and T2DM, improving satiety, insulin resistance and/or beta cell biology.
Animals ; Diabetes Mellitus, Type 2/drug therapy/*metabolism ; GTP-Binding Protein alpha Subunits/genetics/*metabolism ; Humans ; Insulin-Secreting Cells/metabolism ; Obesity/drug therapy/*metabolism ; Receptor, Melatonin, MT2/genetics/*metabolism ; Receptors, Adrenergic, alpha-1/genetics/*metabolism ; Receptors, Prostaglandin/genetics/*metabolism

OBJECTIVETo investigate the expression and distribution of alpha1-adrenoceptor subtypes in rabbit submandibular gland and the effect of phenylephrine on salivary secretion.

METHODSThe expressions of alpha1A-, alpha1B-, and alpha1D-adrenoceptor mRNA and protein were investigated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot in rabbit submandibular gland. Immunohistochemical assay was applied to detect the distribution of alpha1A-, alpha1B-, and alpha1D-adrenoceptor and localization of aquaporin 5 in rabbit submandibular gland. Different concentrations of phenylephrine (1 x 10(-8))-(1 x 10(-6)) mol/L were administrated through a polyethylene tube, which was intubated into Wharton's duct of submandibular gland. Heart rate and blood pressure of rabbits were observed during phenylephrine administration. Salivary flow was measured by the length of moist filter paper (35 mm x 5 mm) within 5 min.

RESULTSAlpha1A-, alpha1B-, and alpha1D-adrenoceptor mRNA and protein were expressed in rabbit submandibular gland. Three alpha1-adrenoceptor subtypes were widely distributed in the membrane and cytoplasma of both acinar and ductal cells. Phenylephrine (1 x 10(-7) mol/L, 100 microl) stimulated effectively salivary secretion without inducing significant alteration of blood pressure and heart rate in rabbit. Immunohistochemical assay showed that aquaporin 5 was mainly localized in the apical and lateral plasma membrane in both acinar and ductal cells in unstimulated condition, while the expression of aquaporin 5 was increased after administration of phenylephrine.

CONCLUSIONSExpression of alpha1A-, alpha1B-, and alpha1D-adrenoceptor mRNA and protein was existed in rabbit submandibular gland. Phenylephrine safely and effectively promoted salivary secretion when it was administrated through Wharton's duct of submandibular gland. The mechanism of phenylephrine on salivary secretion may involve in the increase of expression of aquaporin 5 in the apical and lateral plasma membrane in rabbit submandibular gland. This study will hopefully lead to a novel strategy for clinical treatment of dysfunction of submandibular gland.

Adrenergic alpha-Agonists ; pharmacology ; Animals ; Aquaporin 5 ; metabolism ; Phenylephrine ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Receptors, Adrenergic, alpha-1 ; genetics ; metabolism ; Salivation ; drug effects ; Submandibular Gland ; drug effects ; secretion

Using matchmaker yeast two-hybrid system, it has been demonstrated that there exists an interaction between the cellular C terminal of alpha(1A)-adrenergic receptor (alpha(1A)-AR) and a segment of bone morphogenetic protein-1 (BMP-1). In the present study binding between the two proteins was further determined in human embryonic cell 293 (HEK293), a mammalian expression system. Mammalian expression vector PCP3HA was constructed by PCR and consisted of segments of BMP-1 cDNA, and vector PDT-alpha(1A) consisted of the full-length cDNA of human alpha(1A)-AR. They were transfected to HEK293 cells and examined by Western blot. alpha(1A)-AR and the segment of BMP-1 could be detected in the lysis of transfected cells. Then binding between alpha(1A)-AR and the segment of BMP-1 in HEK293 cell was determined by enzyme-linked immunosorbent assays (ELISA) and co-immunoprecipitation. In ELISA experiment, the ELISA microwell plate was first coated with anti-FLAG M2 antibody, which recognizes the FLAG-tagged alpha(1A)-AR, then the cell lysis, anti-HA rabbit polyclonal antibody and HRP conjugated anti-rabbit antibody were added in turn. The OD(490) values among the control group, PDT-alpha(1A) transfection group and PCP3HA transfection group, exhibited no significant difference (0.034+/-0.027, 0.042+/-0.019, 0.030+/-0.0096), but the OD(490) values of PDT-alpha(1A) and PCP3HA co-transfection group (0.57+/-0.12) were significantly higher than those of the other groups (P<0.001, respectively). In co-immunoprecipitation experiments, HEK293 cells expressing alpha(1A)-AR or/and segment of BMP-1 were lysed and incubated with anti-FLAG M2 antibody, then the immunoprecipitation pellet was immunoblotted with either the HRP conjugated anti-FLAG antibody or the anti-HA antibody, which recognizes the HA-tagged segment of BMP-1. Segment of BMP-1 was present in the pellet immunoprecipitation of PDT-alpha(1A) and PCP3HA co-transfected group. In conclusion, the results indicate that alpha(1A)-AR and the segment of BMP-1 are present in the same complex in HEK293 cells.
Adrenergic alpha-Agonists ; pharmacology ; Bone Morphogenetic Protein 1 ; Bone Morphogenetic Proteins ; genetics ; metabolism ; Cells, Cultured ; Embryo, Mammalian ; Enzyme-Linked Immunosorbent Assay ; Humans ; Kidney ; cytology ; metabolism ; Metalloendopeptidases ; genetics ; metabolism ; Protein Binding ; Receptors, Adrenergic, alpha-1 ; genetics ; metabolism ; Transfection ; Two-Hybrid System Techniques

OBJECTIVEDuring the development of hypoxic pulmonary hypertension, the quantity of the protein and mRNA of alpha1-adrenergic receptor (alpha1-adrenergic receptor,alpha1-AR) in the lung tissue increased, while no particular reports were found about the change of the alpha1-AR subtypes during that course. The study aimed to understand the quantity and location of alpha1-AR subtypes mRNA expression in control and hypoxia rats,and the effect of phentolamine in hypoxic pulmonary hypertension.

METHODSFifty-five male Wistar rats were divided randomly into 5 groups: control group (n = 10), hypoxia 2 weeks (n = 13), hypoxia 4 weeks (n = 10), saline control group (n = 10) and hypoxia 4 weeks plus phentolamine group (n = 12). Semi-quantitative reverse transcription and polymerase chain reaction (RT-PCR) were used to examine the mRNA expression of alpha1-AR subtypes in each group. In situ hybridization was also used to detect the location of the alpha1-AR in lungs of normal rats.

RESULTS(1) The pulmonary artery pressure increased with the extension of hypoxia. (2) The results of RT-PCR showed that the mRNA expression of alpha1A-AR was the most,alpha1B-AR the second and alphaZD-AR was the least in all of the groups. (3) The expression of alpha1A-AR and alpha1B-AR mRNA increased with the extension of hypoxia, and the expressions among groups showed difference. (4) The expression of alpha1D-AR also increased with the extension of hypoxia but no difference was found among groups. (5) No difference was found in mRNA quantity of all three subtypes between phentolamine group and hypoxia saline group. (6) In situ hybridization showed that mRNA of the three alpha1-AR subtypes located mainly in the artery and venous smooth muscle cells and endothelial cells.

CONCLUSIONSThis study suggested that alpha1-AR subtypes worked in the development of hypoxia pulmonary hypertension. More research on alpha1-AR subtypes may help the clinical treatment of pulmonary hypertension.

Adrenergic alpha-Antagonists ; pharmacology ; Animals ; Disease Models, Animal ; Hypertension, Pulmonary ; drug therapy ; etiology ; genetics ; Hypoxia ; complications ; In Situ Hybridization ; Lung ; metabolism ; pathology ; Male ; Phentolamine ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Receptors, Adrenergic, alpha-1 ; classification ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUNDChronic heart failure (CHF) had been characterized as an activated sympathetic system leading to the alteration of adrenergic receptor (AR) levels in the heart. Thus far, not much research has been done with regard to traditional Chinese medical treatment for CHF. We investigated the effect of Shexiangbaoxin pills (SXBXP) on the function of the heart and the expression of a(1)-AR and b-AR subtypes in the messenger RNA (mRNA) levels and protein levels of non-infarction left ventricular tissue from rats with CHF induced by myocardial infarction.

METHODSModels of CHF were established by left anterior descending coronary artery ligature. Fifty-four Wistar rats were randomly divided into five groups: normal control group (group A), sham operation group (group B), CHF model group (group C), positive medicine control group (group D), and small-dose SXBXP group (group E) and large-dose SXBXP group (group F), deployed intragastrically. Cardiac function was examined by echocardiography before and after therapy; mRNA expressed levels were measured by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) for b(1)-AR, b(2)-AR, b(3)-AR, a(1A)-AR, a(1B)-AR, and a(1D)-AR; protein levels were measured by Western blotting analysis for b(1)-AR, b(2)-AR, a(1A)-AR, a(1B)-AR, and a(1D)-AR in non-infarction left ventricular tissue.

RESULTSThere was no significant difference in the left ventricular ejection fraction (LVEF) between groups A and B. Compared to group B, LVEF of groups C, D, E, and F were significantly decreased (P < 0.01) before therapy. After therapy, compared to group C, LVEF of group F was significantly improved (P < 0.05). Compared to group B, b(1)-AR and a(1B)-AR expressed levels were markedly decreased (P < 0.05), a(1A)-AR and b(3)-AR were significantly increased (P < 0.01) in group C, and in both mRNA and protein expressed levels b(2)-AR had no significant difference between groups B and C (P > 0.05). a(1D)-AR mRNA levels were unchanged in each group (P > 0.05), but a(1D)-AR protein level was significantly decreased in group C (P < 0.05). After treatment, compared to group C, mRNA levels of b(1)-AR and a(1B)-AR were significantly increased (P < 0.05 and P < 0.01), and a(1A)-AR was markedly decreased in groups D, E, and F (P < 0.05). b(3)-AR level significantly declined in both groups D and F (P < 0.01), but b(2)-AR and a(1D)-AR expressed levels remained unchanged in each group (P > 0.05). Protein levels, compared to group C, b(1)-AR was significantly increased (P < 0.01, P < 0.05, and P < 0.01) and a(1A)-AR was markedly decreased in groups D, E, and F (P < 0.05, P < 0.01, and P < 0.01). b(2)-AR expressed level was significantly increased in group F (P < 0.05). a(1B)-AR expressed level was significantly increased in both groups E and F (P < 0.05), and a(1D)-AR was remarkably increased in both groups D and F (P < 0.05).

CONCLUSIONSAfter SXBXP treatment, LVEF was increased and cardiac function was significantly ameliorated in rats with CHF. The therapeutic effect of SXBXP may be related to better blood supply for myocardium and up-regulation of b(1)-AR and a(1B)-AR, and down-regulation of a(1A)-AR and b(3)-AR. The results show that SXBXP can be used in treatment of CHF and the therapeutic effect of large-dose SXBXP is superior to small-dose SXBXP.

Animals ; Blotting, Western ; Drugs, Chinese Herbal ; therapeutic use ; Echocardiography ; Heart Failure ; diagnostic imaging ; drug therapy ; Male ; Myocardial Infarction ; diagnostic imaging ; drug therapy ; Radiography ; Rats ; Rats, Wistar ; Receptors, Adrenergic, alpha-1 ; genetics ; metabolism ; Receptors, Adrenergic, beta ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction