Role of the signal peptide in the synthesis and processing of the glucagon-like peptide-1 receptor

Background and purpose:

The glucagon-like peptide-1 receptor (GLP-1R) belongs to Family B of the G protein-coupled receptor superfamily and is a target for treatment of type 2 diabetes. Family B G protein-coupled receptors contain a putative N-terminal signal peptide, but its role in receptor synthesis and trafficking are unclear. Further, the signal peptide is not cleaved in at least one family member.

Experimental approach:

We examined receptor glycosylation and the role of the signal peptide in GLP-1R synthesis and trafficking using constructs containing epitope tags at the N- and/or C-terminus and in which the signal peptide sequence was either present or absent.

Key results:

The signal peptide was absolutely required for GLP-1R synthesis but could be substituted to some extent by increasing positive charge in the N-terminal region of the receptor flanking the signal peptide. The signal peptide is cleaved during synthesis and processing of the receptor. An enhanced GFP-epitope tag at the N-terminus of the receptor permitted synthesis of the receptor but blocked signal peptide cleavage and prevented trafficking to the plasma membrane. Cleavage site mutation allowed synthesis of a full-length receptor, blocked signal peptide cleavage and caused retention within the endoplasmic reticulum.

Conclusions and implications:

Signal peptide cleavage was not essential for receptor synthesis but was obligatory for processing and trafficking of receptors to the plasma membrane. Further, the GLP-1R is subject to N-linked glycosylation and only the mature, fully glycosylated form of the receptor is present in the plasma membrane. Inhibition of glycosylation prevents processing and cell surface expression of the GLP-1R.     

Associations between body mass index and substance use disorders differ by gender: results from the National Epidemiologic Survey on Alcohol and Related Conditions

Alcohol, illicit drugs, and nicotine can affect appetite and body weight, but few epidemiologic studies have examined relationships between body mass index (BMI) and substance use disorders. This study used logistic regression to examine effects of BMI and gender on risk for DSM-IV substance use disorders in a sample of 40 364 adults. Overweight and obesity were associated with increased risk for lifetime alcohol abuse and dependence in men but not women. Overweight and obesity were associated with decreased risk for past-year alcohol abuse in women. BMI was not associated with illicit drug use disorders. Overweight and obese men were at decreased risk for both lifetime and past-year nicotine dependence. Overweight women were at increased risk for lifetime nicotine dependence, and obese women were at decreased risk for past-year nicotine dependence. Further research is needed to identify reasons for observed gender differences in relationships between BMI and substance use disorders.     

The major determinant of exendin-4/glucagon-like peptide 1 differential affinity at the rat glucagon-like peptide 1 receptor N-terminal domain is a hydrogen bond from SER-32 of exendin-4

BACKGROUND AND PURPOSE

Exendin-4 (exenatide, Ex4) is a high-affinity peptide agonist at the glucagon-like peptide-1 receptor (GLP-1R), which has been approved as a treatment for type 2 diabetes. Part of the drug/hormone binding site was described in the crystal structures of both GLP-1 and Ex4 bound to the isolated N-terminal domain (NTD) of GLP-1R. However, these structures do not account for the large difference in affinity between GLP-1 and Ex4 at this isolated domain, or for the published role of the C-terminal extension of Ex4. Our aim was to clarify the pharmacology of GLP-1R in the context of these new structural data.

EXPERIMENTAL APPROACH

The affinities of GLP-1, Ex4 and various analogues were measured at human and rat GLP-1R (hGLP-1R and rGLP-1R, respectively) and various receptor variants. Molecular dynamics coupled with in silico mutagenesis were used to model and interpret the data.

KEY RESULTS

The membrane-tethered NTD of hGLP-1R displayed similar affinity for GLP-1 and Ex4 in sharp contrast to previous studies using the soluble isolated domain. The selectivity at rGLP-1R for Ex4(9–39) over Ex4(9–30) was due to Ser-32 in the ligand. While this selectivity was not observed at hGLP-1R, it was regained when Glu-68 of hGLP-1R was mutated to Asp.

CONCLUSIONS AND IMPLICATIONS

GLP-1 and Ex4 bind to the NTD of hGLP-1R with similar affinity. A hydrogen bond between Ser32 of Ex4 and Asp-68 of rGLP-1R, which is not formed with Glu-68 of hGLP-1R, is responsible for the improved affinity of Ex4 at the rat receptor.     

2型糖尿病大鼠脂联素受体基因表达的相关性研究

目的研究2型糖尿病大鼠骨骼肌和肝脏脂联素受体的表达变化。方法将20只健康雄性SD大鼠随机分为糖尿病组(DM组)和对照组(NC组),每组10只,分别饲以高脂饲料和普通饲料。第13周末DM组大鼠一次性腹腔注射2%链脲佐菌素(STZ)溶液(STZ 25mg/kg,以0.1mol/L柠檬酸-柠檬酸钠缓冲液配制,pH=4.4),NC组腹腔注射等体积的无菌柠檬酸-柠檬酸钠缓冲液。第14周末进行葡萄糖耐量试验(OGTT)筛选成模大鼠,第25周末处死所有大鼠,取血检测空腹血糖、甘油三酯、胆固醇、脂联素、胰岛素水平,并计算胰岛素敏感指数(ISI),使用半定量反转录聚合酶链反应(RT-PCR)的方法分别检测2组大鼠骨骼肌和肝脏组织中脂联素受体(AR)mRNA表达水平。结果 DM组大鼠空腹血糖、甘油三酯、胆固醇、胰岛素水平高于NC组,脂联素水平和ISI低于NC组(P<0.05);DM组大鼠骨骼肌中AR1mRNA和肝脏组织中AR2mRNA的表达水平低于NC组(0.64±0.25与1.36±0.30,0.93±0.18与1.52±0.21;P<0.05),AR1和AR2表达水平分别与空腹血糖、甘油三酯、胆固醇、胰岛素、脂联素、ISI相关。结论 2型糖尿病大鼠AR mRNA的表达水平显著降低,与胰岛素敏感性密切相关。     

胶质瘤促血管生成素2基因表达的临床意义（附52例分析）

目的　探讨胶质瘤促血管生成素2 (Ang2) 基因在脑胶质瘤的表达及其临床意义。方法　采用逆转录聚合酶链反应(RT PCR) 和链酶亲和素过氧化物酶复合物SABC法检测52例人脑胶质瘤和8例正常脑组织中Ang2基因表达。结果　50 例脑胶质瘤和8 例正常脑组织均可表达Ang2 mRNA片段。高恶度胶质瘤Ang2 mRNA及蛋白表达显著高于低恶度胶质瘤(P<0 01); 低恶度胶质瘤Ang2 mRNA及蛋白表达显著高于正常脑组织(P<0 05)。免疫组化染色显示, Ang2蛋白主要分布在高恶度胶质瘤组织的瘤细胞和内皮细胞, 低恶度胶质瘤呈低水平表达, 正常脑组织不表达。结论　Ang2的表达与胶质瘤的分级密切相关, 可能对胶质瘤的恶性演进起促进作用。     

可卡因对性成熟期大鼠睾丸谷胱甘肽过氧化物酶基因表达的影响

目的：研究可卡因对大鼠睾丸谷胱甘肽过氧化物酶（glutathione peroxidase GSH-PX）基因表达的影响。方法：性成熟期健康SD雄性大鼠皮下注射可卡因制造吸毒动物模型。化学比色法检测睾丸氧化应激指标;半定量RT-PCR方法检测睾丸组织的GSH-PXmRNA表达。结果：可卡因处理第7天,实验组GSH-PX活性、MDA含量与对照组比较无显著性差异（P〉0．05）;而GSH-PXmRNA水平显著高于对照组（P〈0．05）。可卡因处理第14天,实验组GSH-PX活性显著低于对照组（P〈0．05）,MDA含量显著高于对照组（P〈0．05）,而GSH-PXmRNA水平显著高于对照组（P〈0．05）。可卡因处理第21天,实验组GSH-PX活性及mRNA表达水平显著低于对照组（P〈0．05）,MDA含量显著高于对照组（P〈0．05）。可卡因处理第28天,实验组GSH-PX活性及mRNA表达水平显著低于对照组（P〈0．05）,MDA含量显著高于对照组（P〈0．05）。     

The value of short- and long-acting glucagon-like peptide-1 agonists in the management of type 2 diabetes mellitus: experience with exenatide

Background:

Only about half of patients with type 2 diabetes treated with antihyperglycemic drugs achieve glycemic control (HbA1c <7%), most commonly due to poor treatment adherence. Glucagon-like peptide-1 (GLP-1) receptor agonists act on multiple targets involved in glucose homeostasis and have a low risk of causing hypoglycemia. While GLP-1 receptor (GLP-1R) agonists share the same mechanism of action, clinical profiles of individual agents differ, particularly between short- and long-acting agents. In this article, recent findings regarding the pharmacology of GLP-1 agonists are reviewed, and the clinical effects of short- versus long-acting agents are compared.

Data sources:

Relevant articles were identified through a search of PubMed using the keywords glucagon-like peptide-1, GLP-1, glucagon-like peptide-1 receptor agonist, GLP-1R agonist, and exenatide for publications up to 22 May 2015. Supporting data were obtained from additional searches for albiglutide, dulaglutide, liraglutide and lixisenatide as well as from the bibliographies of key articles.

Findings:

Short-acting GLP-1R agonists produce greater reductions in postprandial glucose levels by slowing gastric emptying, whereas long-acting GLP-1R agonists produce greater reductions in fasting blood glucose by stimulating insulin secretion from the pancreas. These characteristics can be exploited to provide individualized treatment to patients. A large body of evidence supports the benefits of short- and long-acting exenatide as add-on therapy in patients with inadequate glycemic control despite maximum tolerated doses of metformin and/or sulfonylurea. Exenatide is generally well tolerated and no new safety concerns were identified during long-term follow-up of up to 5 years. A limitation of this review of short-and long-acting GLP-1 receptor agonists is that it focuses on exenatide rather than all the drugs in this class. However, the focus on a single molecule helps to avoid any confusion that may be introduced as a result of differences in molecular structure and size.

Conclusions:

Short-acting GLP-1R agonists including exenatide are well suited to patients with type 2 diabetes with exaggerated postprandial glucose excursions and for co-administration with basal insulin therapy. Long-acting GLP-1R agonists including once weekly exenatide offer greater convenience and are well suited to patients who require specific control of fasting hyperglycemia.     

Inhibitory effect of trilinolein on endothelin-1-induced c-fos gene expression in cultured neonatal rat cardiomyocytes

Trilinolein, isolated from the traditional Chinese herb Sanchi (Panax notoginseng), has been shown to have myocardial protective effects via its antioxidant ability. However, the cellular and molecular mechanisms of the protective effect of trilinolein in the heart remain to be elucidated. Oxidative mechanisms have been implicated in neonatal cardiomyocyte hypertrophy. We previously reported that ET-1 induces ROS generation via the ET_A receptor and ROS modulates c-fos gene expression. We have therefore examined whether trilinolein attenuates ROS production and ET-1-induced c-fos gene expression in cardiomyocytes. Cultured neonatal rat cardiomyocytes were stimulated with ET-1 (10 nM), and c-fos gene expression was examined. Trilinolein (1 and 10 μM) inhibited ET-1-induced c-fos gene expression in cardiomyocytes. We also examined the effects of trilinolein on ET-1- increased NADPH oxidase activity and superoxide formation. Trilinolein inhibited ET-1-increased NADPH oxidase activity and superoxide formation in a concentration-dependent manner. This increase in superoxide production by ET-1 was significantly inhibited by trilinolein, diphenyleneiodonium, or N-acetylcysteine. Trilinolein also decreased ET-1- or H₂O₂-induced extracellular signal-regulated kinase (ERK) phosphorylation, c-Jun NH₂-terminal kinase (JNK) phosphorylation, and activator protein-1 activation. These data indicate that trilinolein inhibits ET-1-induced ERK phosphorylation, JNK phosphorylation, and c-fos gene expression via attenuating superoxide production in cardiomyocytes. Hung-Yu Yang and Ju-Chi Liu equally contributed to this work     

Hypothalamic insulin and glucagon-like peptide-1 levels in an animal model of depression and their effect on corticotropin-releasing hormone promoter gene activity in a hypothalamic cell line

Background

In depression, excessive glucocorticoid action may cause maladaptive brain changes, including in the pathways controlling energy metabolism. Insulin and glucagon-like peptide-1 (GLP-1), besides regulation of glucose homeostasis, also possess neurotrophic properties. Current study was aimed at investigating the influence of prenatal stress (PS) on insulin, GLP-1 and their receptor (IR and GLP-1R) levels in the hypothalamus. GLP-1 and GLP-1R were assayed also in the hippocampus and frontal cortex – brain regions mainly affected in depression. The second objective was to determine the influence of exendin-4 and insulin on CRH promoter gene activity in in vitro conditions.

The effect of adjunctive mood stabilizers on antipsychotic utilization pattern and health resource utilization for Medicaid enrollees with schizophrenia

ABSTRACT

Background: Prescribing adjunctive mood stabilizers to manage schizophrenia is prevalent, despite the lack of substantial evidence to support the long-term use of this treatment regimen.

Objective: The objective of this study was to assess the impact of using adjunctive mood stabilizers on antipsychotic utilization, total health expenditures, inpatient hospital­izations, long-term care stays, and emergency room (ER) visits for patients with schizophrenia.

Methods: Georgia Medicaid claims from 1999 through 2001 were analyzed to identify recipients diagnosed with schizophrenia (International Classification of Diseases, Ninth Revision, Clinical Modification [ICD?9?CM]: 295.XX). The treatment groups consisted of subjects who received combination therapy of mood stabilizers and anti­psychotics (including both atypical and typical medica­tions), while the comparison group consisted of subjects who were on antipsychotic medications without exposure to the mood stabilizers under investigation. Four treatment groups (valproate, lithium, carbamazepine, and combina­tion mood stabilizer therapy) were formed based on the mood stabilizers patient received. Differences in annual health care use and expenditures were estimated between propensity score matched treatment and comparison groups controlling for comorbidity, prior utilization, demographic, and health provider specialty.

Results: During the 1?year observation period, subjects in treatment groups filled an average of 200-days supply of adjunctive mood stabilizers. These adjunctive mood stabilizer recipients had significantly longer antipsychotic treatment durations than the subjects who did not have exposure to mood stabilizers (valproate + antipsychotic vs. antipsychotic only, net difference: 56.47 days, p < 0.0001; lithium + antipsychotic vs. antipsychotic only, net difference: 90.25 days, p < 0.0001; carbamazepine + antipsychotic vs. antipsychotic only, net difference: 41.27 days, p = 0.0439; multiple mood stabilizers + anti­psychotic vs. antipsychotic only, net difference: 83.14 days, p < 0.0001). The intensive pharmacotherapy associated with treatment groups resulted in $900–$1300 higher pharmacy costs than the comparison groups (valproate + antipsychotic vs. antipsychotic only, net difference: $1218.43, p < 0.0001; lithium + antipsychotic vs. antipsychotic only, net difference: $985.79, p = 0.0015; carbamazepine + antipsychotic vs. anti­psychotic only, net difference: $911.63, p = 0.0497; multiple mood stabilizers + antipsychotic vs. antipsychotic only, net difference: $1281.91, p < 0.0047). However, there were no statistically significant differences for total health expenditures, hospitalizations, emergency room visits, and nursing home admissions between propensity-matched treatment and control groups.

Conclusions: There were no differences in health care costs or utilization of ER, long-term care, and inpatient services between schizophrenia patients who did and did not receive adjunctive mood stabilizer; however, longer anti­psychotic treatment durations were observed in patients receiving adjunctive mood stabilizers. Interpretation of these results is limited by the unknown selection bias between the treatment and the comparison groups and the relatively small number of patients in some treatment groups. The development of a better-controlled study to further evaluate this treatment regimen is warranted.     

氟哌啶醇联合用药治疗儿童抽动障碍及精神分裂症的研究进展

氟哌啶醇为一种重要的抗精神分裂药,临床上用于治疗精神疾病引起的行为冲动和焦虑等症状,但单独使用该药物治疗的总有效率较低,与其他类型药物联合用于精神疾病的治疗往往具有更好的疗效和安全性。本文就近年来关于氟哌啶醇联合用药治疗儿童抽动障碍及精神分裂症的情况进行综述,探讨氟哌啶醇联合用药用于儿童抽动障碍及精神分裂症方面的研究进展,为临床用药提供参考依据。     

利拉鲁肽对超重及肥胖2型糖尿病患者体质量和胰岛素抵抗的影响

目的 观察利拉鲁肽对超重及肥胖2型糖尿病患者体质量和胰岛素抵抗的影响。方法 选取安徽医科大学第二附属医院2012年6月至2014年1月住院治疗的2型糖尿病患者,将口服药物血糖控制不佳且体质量指数（BMI）≥24 kg/m²的90例2型糖尿病患者按系统随机化法分成治疗组（45例）和对照组（45例）,对照组在原方案上加用基础胰岛素,治疗组加用利拉鲁肽,治疗组根据BMI进一步分为超重亚组（BMI<28 kg/m²）24例,和肥胖亚组（BMI≥28 kg/m²）21例,连续治疗12周。比较治疗后各项指标（BMI、收缩压、空腹血糖、餐后血糖等）变化。结果 治疗组BMI及稳态模型胰岛素抵抗指数（HOMA-IR）治疗后较治疗前显著降低（P<0.05）,对照组治疗后增加（P<0.05）;收缩压（SBP）、空腹血糖（FBG）、餐后2 h血糖（2 h PG）、糖基化血红蛋白（HbA1C）、三酰甘油（TG）、总胆固醇（TCH）在两组中均较前下降（P<0.05）,治疗组SBP、TG、TCH下降优于对照组（P<0.05）,FBG、2 h PG、HbA1C下降,组间差异无统计学意义（P>0.05）;治疗组中肥胖亚组BMI下降程度优于超重亚组（P<0.05）;治疗过程中两组均未发生严重低血糖。结论 利拉鲁肽在降糖的同时也可以降低患者体质量并改善患者胰岛素抵抗;随着BMI增加,利拉鲁肽降低体质量及改善胰岛素抵抗作用越强。     

黄连有效成分盐酸小檗碱、盐酸药根碱、黄连碱及其混合物对NCI-H716细胞分泌胰高血糖素样肽1(GLP-1)的影响

目的:研究黄连有效成分对NCI-H716细胞分泌胰高血糖素样肽1(GLP-1)的影响。方法:分别设置空白对照组、盐酸药根碱(JAT)组、盐酸小檗碱(BER)组、黄连碱(COP)组、盐酸巴马汀(BM)及BER十COP十JAT高剂量组和BER+COP+JAT低剂量组,同时以BER组为阳性对照组。采用CCK-8法检测各组药物不同浓度下对细胞增殖的抑制作用,确定药物浓度范围;采用酶联免疫吸附法(ELISA)检测NCI-H716细胞分泌GLP-1的量;采用聚合酶链式反应(PCR)检测NCI-H716细胞表达GLP-1RNA的量。结果:COP、JAT,以及高、低浓度BER+COP+JAT均能促进NCI-H716细胞分泌GLP-1(P<0.05),其中,BER+COP+JAT高浓度组及BER+COP+JAT低浓度组促GLP-1分泌作用均优于阳性对照组(P<0.05);COP、JAT组与阳性对照组相比无显著性差异(P>0.05)。结论:JAT、COP对NCI-H716细胞分泌GLP-1均有促进作用,且BER+COP+JAT混合组效果优于其单体组。     

Rupatadine effectively prevents the histamine-induced up regulation of histamine H1R and bradykinin B2R receptor gene expression in the rat paw

BackgroundActivation of histamine H₁ receptor (H₁R) is a well-known hallmark of allergic and inflammatory pathology. Both types of bradykinin receptors (B₁R and B₂R) are also known to contribute significantly to the latter and some sort of functional interaction between them and H₁R has been alluded to in the past. Here we use an experimental model of rat paw oedema formation to examine the effect of exogenously added histamine on the gene expression of H₁R and bradykinin receptors B₁R and B₂R, alone or in combination to rupatadine, a second generation antihistamine agent.MethodsHistamine-induced oedema formation was monitored with a plethysmometer. The gene expression of H₁R, B₁R and B₂R was analyzed with both conventional and real-time PCR. Rupatadine fumarate was used in pure form and administered intraperitoneally, prior to histamine injection into the paw. Microscopy of haematoxylin and eosin-stained sections of paw tissue was used to examine effects on tissue architecture.ResultsHistamine injection into the paw resulted in significant up regulation of H₁R and B₂R without inducing significant cellular infiltration, but appears to affect less the expression of B₁R. Rupatadine was, under the conditions used in this study, very effective in preventing this effect and in suppressing oedema formation through its antihistamine action.ConclusionRupatadine has a suppressing effect on H₁R and B₂R gene expression which could add to its efficacy towards allergy and allergy-like conditions.     

抑癌基因PTEN和胰岛素样生长因子-1受体在子宫内膜癌中的表达及临床意义

目的研究抑癌基因PTEN和胰岛素样生长因子1受体(IGF1R)与子宫内膜癌临床及病理特征的关系。方法应用免疫组织化学方法(SP法)检测45例子宫内膜癌组织中PTEN基因和胰岛素样生长因子1受体的表达。结果45例子宫内膜癌组织中,PTEN阳性表达率为28.89%;Ⅰ期患者和Ⅱ～Ⅲ期患者阳性表达率分别为44.00%和10.00%(P<0.05);在高、中、低分化子宫内膜癌中的阳性表达率分别为60.00%、15.00%和10.00%(P<0.05);无、有淋巴结转移者的阳性表达率分别为38.71%和7.14%(P<0.05);IGF1R阳性表达率为86.67%。结论PTEN基因抑制子宫内膜癌的生长、浸润及淋巴结转移;IGF1R在子宫内膜癌的发生中发挥着重要作用。