Effect of Zuogui Jiangtang Tongmai Prescription on SCFAs/GPR43/GLP-1/GLP-1R Signaling Pathway in Rats with Diabetes Mellitus Complicated with Cerebral Infarction / 中国实验方剂学杂志

ObjectiveTo explore the potential mechanism of Zuogui Jiangtang Tongmai prescription （ZJT） in the treatment of diabetes mellitus complicated with cerebral infarction （DM-CI） in rats based on the short-chain fatty acids （SCFAs）/G protein-coupled receptor 43 （GPR43）/glucagon-like peptide-1 （GLP-1）/GLP-1 receptor （GLP-1R） signaling pathway. MethodSixty SD rats were randomly divided into sham operation group， model group， low- and high-dose ZJT groups （12， 24 g·kg-1）， western medicine group （140 mg·kg-1 pioglitazone metformin tablets + 27 mg·kg-1 enteric-coated aspirin tablets）. Except for the sham operation group， all other groups were fed a high-sugar high-fat diet for 4 weeks and then subjected to intraperitoneal injection of 1% streptozotocin at 35 mg·kg-1 combined with middle cerebral artery occlusion （MCAO） to establish a DM-CI rat model. The corresponding interventions were performed with distilled water， low-dose ZJT， high-dose ZJT， pioglitazone metformin tablets， and enteric-coated aspirin tablets. After surgery， National Institutes of Health Stroke Scale （NIHSS） scoring and triphenyltetrazolium chloride （TTC） staining to measure the rat's cerebral infarct volume were carried out. Random blood glucose levels were measured， and hematoxylin-eosin （HE） staining was used to observe histopathological changes in rat brain tissues. Gas chromatography was employed to detect the content of SCFAs in the cecum contents. Enzyme-linked immunosorbent assay （ELISA） was adopted to measure serum GLP-1 level. Western blot was used to detect the protein expression of GPR43 in rat ileal tissues and GLP-1R in the ischemic brain tissues. ResultCompared with the sham operation group， the model group showed significantly increased NIHSS scores， random blood glucose levels， and cerebral infarct volumes （P<0.01）， and significantly decreased SCFAs content， GLP-1 levels， and GPR43 and GLP-1R protein expression （P<0.01）. Compared with the model group， the high-dose ZJT group and the western medicine group exhibited significantly reduced NIHSS scores， random blood glucose levels， and cerebral infarct volumes （P<0.05， P<0.01）， and significantly increased SCFAs content， GLP-1 levels， and GPR43 and GLP-1R protein expression （P<0.01）. ConclusionZJT can improve glucose metabolism disorder and reduce neurological damage in DM-CI rats， and its mechanism may be related to the increase in SCFAs content and the upregulation of the GPR43/GLP-1/GLP-1R signaling pathway.

Effect and mechanism of Zexie Decoction in promoting white adipose tissue browning/brown adipose tissue activation based on GLP-1R/cAMP/PKA/CREB pathway / 中国中药杂志

This study investigated the mechanism of Zexie Decoction(ZXD) in promoting white adipose tissue browning/brown adipose tissue activation based on the GLP-1R/cAMP/PKA/CREB pathway. A hyperlipidemia model was induced by a western diet(WD) in mice, and the mice were divided into a control group, a model group(WD), and low-, medium-, and high-dose ZXD groups. An adipogenesis model was induced in 3T3-L1 cells in vitro, and with forskolin(FSK) used as a positive control, low-, medium-, and high-dose ZXD groups were set up. Immunohistochemistry and immunofluorescence results showed that compared with the WD group, ZXD promoted the expression of UCP1 in white and brown adipose tissues, and also upregulated UCP1, CPT1β, PPARα, and other genes in the cells. Western blot analysis showed a dose-dependent increase in the protein expression of PGC-1α, UCP1, and PPARα with ZXD treatment, indicating that ZXD could promote the white adipose tissue browning/brown adipose tissue activation. Hematoxylin-eosin(HE) staining results showed that after ZXD treatment, white and brown adipocytes were significantly reduced in size, and the mRNA expression of ATGL, HSL, MGL, and PLIN1 was significantly upregulated as compared with the results in the WD group. Oil red O staining and biochemical assays indicated that ZXD improved lipid accumulation and promoted lipolysis. Immunohistochemistry and immunofluorescence staining for p-CREB revealed that ZXD reversed the decreased expression of p-CREB caused by WD. In vitro intervention with ZXD increased the protein expression of CREB, p-CREB, and p-PKA substrate, and increased the mRNA level of CREB. ELISA detected an increase in intracellular cAMP concentration with ZXD treatment. Molecular docking analysis showed that multiple active components in Alismatis Rhizoma and Atractylodis Macrocephalae Rhizoma could form stable hydrogen bond interactions with GLP-1R. In conclusion, ZXD promotes white adipose tissue browning/brown adipose tissue activation both in vivo and in vitro, and its mechanism of action may be related to the GLP-1R/cAMP/PKA/CREB pathway.

α/Sulfono-γ-AA peptide hybrids agonist of GLP-1R with prolonged action both in vitro and in vivo

Peptides are increasingly important resources for biological and therapeutic development, however, their intrinsic susceptibility to proteolytic degradation represents a big hurdle. As a natural agonist for GLP-1R, glucagon-like peptide 1 (GLP-1) is of significant clinical interest for the treatment of type-2 diabetes mellitus, but its in vivo instability and short half-life have largely prevented its therapeutic application. Here, we describe the rational design of a series of α/sulfono-γ-AA peptide hybrid analogues of GLP-1 as the GLP-1R agonists. Certain GLP-1 hybrid analogues exhibited enhanced stability (t 1/2 > 14 days) compared to t 1/2 (<1 day) of GLP-1 in the blood plasma and in vivo. These newly developed peptide hybrids may be viable alternative of semaglutide for type-2 diabetes treatment. Additionally, our findings suggest that sulfono-γ-AA residues could be adopted to substitute canonical amino acids residues to improve the pharmacological activity of peptide-based drugs.

Design of a highly potent GLP-1R and GCGR dual-agonist for recovering hepatic fibrosis

Currently, there is still no effective curative treatment for the development of late-stage liver fibrosis. Here, we have illustrated that TB001, a dual glucagon-like peptide-1 receptor/glucagon receptor (GLP-1R/GCGR) agonist with higher affinity towards GCGR, could retard the progression of liver fibrosis in various rodent models, with remarkable potency, selectivity, extended half-life and low toxicity. Four types of liver fibrosis animal models which were induced by CCl4, α-naphthyl-isothiocyanate (ANIT), bile duct ligation (BDL) and Schistosoma japonicum were used in our study. We found that TB001 treatment dose-dependently significantly attenuated liver injury and collagen accumulation in these animal models. In addition to decreased levels of extracellular matrix (ECM) accumulation during hepatic injury, activation of hepatic stellate cells was also inhibited via suppression of TGF-β expression as well as downstream Smad signaling pathways particularly in CCl4-and S. japonicum-induced liver fibrosis. Moreover, TB001 attenuated liver fibrosis through blocking downstream activation of pro-inflammatory nuclear factor kappa B/NF-kappa-B inhibitor alpha (NFκB/IKBα) pathways as well as c-Jun N-terminal kinase (JNK)-dependent induction of hepatocyte apoptosis. Furthermore, GLP-1R and/or GCGR knock-down results represented GCGR played an important role in ameliorating CCl4-induced hepatic fibrosis. Therefore, TB001 can be used as a promising therapeutic candidate for the treatment of multiple causes of hepatic fibrosis demonstrated by our extensive pre-clinical evaluation of TB001.

Glucagon-like peptide-1 and glucagon-like peptide-1 receptor agonists in the treatment of type 2 diabetes

The prevalence of type 2 diabetes (T2D) is increasing worldwide. Patients with T2D suffer from various diabetes-related complications. Since there are many patients with T2D that cannot be controlled by previously developed drugs, it has been necessary to develop new drugs, one of which is a glucagon-like peptide-1 (GLP-1) based therapy. GLP-1 has been shown to ameliorate diabetes-related conditions by augmenting pancreatic β-cell insulin secretion and having the low risk of causing hypoglycemia. Because of a very short half-life of GLP-1, many researches have been focused on the development of GLP-1 receptor (GLP-1R) agonists with long half-lives such as exenatide and dulaglutide. Now GLP-1R agonists have a variety of dosing-cycle forms to meet the needs of various patients. In this article, we review the physiological features of GLP-1, the effects of GLP-1 on T2D, the features of several GLP-1R agonists, and the therapeutic effect on T2D.

Development and evaluation of a cell model targeted on GLP-1 receptor / 中国药理学通报

Aim To establish a cell model targeting on GLP-1 R,and evaluate its function by the cAMP assay,for screening the new class of GLP-1 analogues as anti-diabetic candidates. Methods An eukaryotic expression vector pEGFP-GLP-1 R was constructed and transfected into HEK293A cells.After selecting with G41 8,a cell line stably expressing GLP-1 R-GFP was estab-lished.The expression and the cellular distribution of GLP-1 R-GFP fusion protein were investigated by Western blot and fluo-rescence microscopy.Then,the activity of GLP-1 analogue Lira-glutide was evaluated by monitoring the content of cAMP via HTRF using this cell model.Results GLP-1 R-GFP-293A cell line was successfully established.GLP-1 R-GFP fusion proteins were mainly distributed in the cell membrane.The dose-respon-sive relationship experiments revealed that cAMP could be effec-tively stimulated by Liraglutide using this cell model.Conclu-sion This cell model could be used to detect the bioactivity of GLP-1 analogues in vitro,which lays a foundation for the screen-ing of GLP-1 analogues and small GLP-1 R agonists.

Application of High Resolution Melting Method in the Detection of GLP1R Gene Polymorphism Site rs3765467 / 中国药房

OBJECTIVE:To establish the method for the detection of the known glucagon-like peptide 1 receptor (GLP1R) gene mutation site rs3765467(NT_007592.16,position:39065819),and to evaluate its accuracy and practicability. METHODS:Peripheral venous blood samples of 72 healthy subjects were collected in medical examination center of our hospital during Oct. 2015-Feb. 2016. The whole blood DNA was extracted by column extraction method. After amplified by touch down PCR,high reso-lution melting(HRM)method was adopted to analyze amplified product. Sequencing verification by double stranded chain termina-tion method(Sanger sequencing method)was performed for 38 test samples. The results of 2 methods were compared. RESULTS:The results of mutation scanning showed that there were 39065817 and 39065819 polymorphism sites in amplified segments. Four types of mutations were detected by HRM method [GCG/GCG,GCA/GCG or ACG/GCG,GCA/GCA or ACG/ACG,A(G) CA(G)],but 6 types of mutations was detected by Sanger sequencing method [GCG/GCG,ACG/GCG,ACG/ACG,A(G)CA (G),GCA/GCG,GCA/GCA]. CONCLUSIONS:HRM method can identify GCG/GCG and A(G)CA(G)genotype,but can not identify GCA/GCG and ACG/GCG heterozygous mutation,GCA/GCA and ACG/ACG homozygous mutation. The method is not suitable for the detection of single nucleotide polymorphism for multiple neighboring sites. In the detection of single nucleotide mu-tation,economical and simple method should be selected after comprehensive analysis of sequence.

Research Progress in the Relationship Between GLP-1,GLP-2 and Colorectal Cancer / 医学研究生学报

Glucagon-like peptide (GLP-1, GLP-2) is a recently discovered intestinal epithelium-specific growth factor.This paper reviews the relationship between the origin, the secretion and the degradation of GLP-1 and GLP-2 and colorectal cancer.It provides basic data for the treatment of colorectal cancer and it′s useful to trade-offs the potential carcinogenesis between the applications of GLP-1, GLP-2 analogues with the inhibitors of degradation of the enzyme treatment.Developing the treatment of applying GLP-1 and GLP-2 into clinical practice has already become the new subject to carry out, which depends on the relevant basic and clinical experiments.

Effects of exenatide on the cell proliferation, invasion and apoptosis of human tongue squamous cell carcinoma SCC-25 / 天津医药

Objective To detect the effects of exenatide on the related indicators of proliferation, invasion and apoptosis of cell line SCC-25. Methods SCC-25 cells were cultured in vitro. The expression level of glucagon like peptide 1 receptor (GLP-1R) was determined by Western blot assay in SCC-25 cells. SCC-25 cells were divided into four groups:control group and exenatide group (1,10 and 100 nmol/L). The ability of cell proliferation was detected using MTT assay after 24 h, 48 h and 72 h of culture. The ability of invasion was measured with Transwell assays. The expression levels of MMP-2, Caspase-3 and Phospho-p38 MAPK were measured by Western blot assay. Results GLP-1 receptor expression was found in SCC-25 cells. Compared with control group, the cell survival rate, invasion rate and the expression of MMP-2 were significantly decreased in SCC-25 group (P＜0.05). The expression of Caspase-3 were significantly increased (P＜0.05).Changes were in a concentration-dependent and time-dependent manner (P＜0.05). The expression of Phospho-p38 MAPK was significantly increased at 24 h in 10 nmol/L exenatide group (P＜0.05). Conclusion Exenatide can inhibit the cell proliferation and invasion, which may contribute the apoptosis by promoting expressions of Phospho-p38 MAPK and Caspase-3 of SCC-25 cells.