ABSTRACT
Increased understanding of the role of incretin hormones in maintaining glucose homeostasis has enabled the development of pharmacotherapies that target deficient incretin activity in type 2 diabetes mellitus.lncretin-based therapies could be classified into 2 categories with different mechanisms of action:glucagon-like peptide 1 ( GLP-1 ) receptor agonists ( e.g.liraglutide,exenatide twice daily,and exenatide once weekly),and the dipeptidyl peptidase 4 ( DPP-4 ) inhibitors ( e.g.sitagliptin,linagliptin,saxagliptin,and vildagliptin),which inhibit the breakdown of endogenous GLP-1.Both categories share some specific characteristics,such as glucose-dependent insulin stimulation and low incidence of hypoglycemia.But there are also different profiles in efficacy between these 2 classes.This article reviews the pharmacokinetics and clinical aspects of efficacy and safety of these 2 classes of incretin-based therapies and elucidates their roles in treating type 2 diabetes mellitus.
ABSTRACT
Incretins ale gut hormones,which stimulate insulin secretion after meal.One of the incretin hormones,glucagon-like peptide-1(GLP-1),contributes to glucose homeostasis by stimulation of insulin secretion,suppression of glucagon secretion(both in a glucose dependent manner),slowing of gastric emptying,and induction of satiety.It could also improve beta cell function in type 2 diabetes.However,due to its short biological half-life,GLP-1 itself is not practical for type 2 diabetes therapy.Exenatide is a peptide found in the lizard Heloderma suspectum and has a high similarity to GLP-1.It has a much longer biological half life than GLP-1 and is a GLP-1 receptor agonist that Can be used for therapeutic purposes by twice daily injection.Clinical studies with exenatide have shown a significant reduction in HbA_(1C),fasting and postprandial glucose,and body weight in type 2 diabetic patients.Animal studies revealed an improvement of beta cell funotion and an increase in beta cell mass after exenatide treatment.Due to its special therapeutic efficacy,GLP-1 receptor agonist,such as exenatide,is now included in the recently published guidelines for the treatment of type 2 diabetes mellitus.
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BACKGROUND: Previous studies have confirmed that embryonic stem cells call be induced to differentiate into insulin-producing cells, but the induction process takes a long time. Most of the processes take about one month.OBJECTIVE: Activin A, all-trans retinoic acid (ATRA), basic fibroblast growth factor (bFGF) and nicotinamide were applied in vitro in combination to observe whether mouse embryonic stem cells could be induct to differentiate into insulin-producing cell in a relatively short time.DESIGN: Cell observation experiment.SETTING: Shanghai Institute of Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.MATERIALS: This study was performed at Shanghai Institute of Endocrine and Metabolic Diseases from October 2004 to February 2006. Two mice of clean grade and of 12.5-14.5 days of gestational age were provided by Shanghai SLAC Laboratory Animal Co., Ltd (Permission No. 2004A034). The protocol was performed in accordance with ethical guidelines for the use and care of animals. Mouse embryonic stem cell lines were supplied by Dr Changxian Zhang (CNRS UMR5641, France). Activin A was the product of the R & D Corporation. ATRA and nicotinamide were supplied by the Sigma Corporation, USA. BFGF was supplied by Gibco Corporaion. METHODS: Head and viscera were removed from embryos of the pregnant mouse. The remaining tissues were cut into pieces and digested with trypsin. Cell suspension was centrifuged and inoculated at 3×108L-1. The cells could be used as mouse feeder layer after 2-3 times of passage. The mouse embryonic stem cells (ESCs) were inoculated onto the feeder layer in knockout Dulbecco's modified Eagle medium (DMEM) supplemented with leukemia inhibitory factors (LIF). ESCs were passaged at 1:3-1:6 after 2-3 days of culture. Culture medium with serum was added into the culture dishes to terminate the digestion. Cell fluid was centrifuged and supernatant was discarded. The sediments were prepared into suspension and inoculated at 2.5×104 with LIF-free culture medium. After 24-48 hours, embryonic bodies (EBs) were collected and replated in 1% Matrigel-coated dishes. When began to adhere to the dishes, EBs were cultured in 10% FBS/DMEM supplemented with 100μg/L activin A for 24 hours. Then EBs were switched to 10% FBS/DMEM for 6-8 hours as an interval. After this interval. EBs differentiated were cultured in 10% FBS/DMEM with 10<-6mol/L RA for another 24 hours followed by culture in 10% FBS/DMEM supplemented with 10μg/L bFGFs for 3-5 days. Finally, EBs differentiated were cultured in DMEM/F12 supplemented with N2 supplement, B27 supplement, 1μg/L laminin, 10μg/L bFGFs, and 10mmol/L nicotinamide for 3-5 days. Dithizone (DTZ) staining, inununofluorescent staining and reverse transcription-polymerase chain reaction (RT-PCR) were applied to detect insulin expression in the differentiated cells.MAIN OUTCOME MEASURES: Induction of ESCs, DTZ staining and immunofluorescent staining as wel as RT-PCR detection.RESULTS: Mouse ESCs growing on a feeder layer formed many colonies with clear boundary and dense structure. However, there was no obvious outer limit between these ESCs. EBs began to adhere to the dishes, which were coated with matrigel, on the 2nd day. After activin A and ATRA interval induction, EBs spread, and most of the living cells were epithelial cell-like when cultured in 10% FBS/DMEM supplemented with 10μg/L bFGFs. After culturing in DMEM/F12 supplemented with N2, B27, nicotinamide, bFGFs and laminin, the cells formed small clusters. The insulin-producing cells were stained dark red with DTZ, and the cells stained with primary antibody to insulin were insulin-positive. After 2 weeks of induction of activin A, ATRA, bFGFs and nicotinamide, the insulin-producing cells expressed insulin 2, Pdxl, Nkx6.1, Nkx2.2, PP, IAPP, Glut2, Somastatin, Hnf3β and Neuro D mRNA but did not express insulin 1 mRNA.CONCLUSION: Mouse ESCs call be induced to differentiate into insulin-producing cells by activin A, ATRA, bFGFs and nicotinamide in vitro. Induction time call be shortened to 2 weeks.
ABSTRACT
Glucagon-like poptide-1 (GLP-1) is an incretin secreted by the enteroendocrine L cells of the gut. Upon the activation of GLP-1 receptor, adenylyl cyclase is activated and cAMP is generated, leading to the activation of protein kinase A and Epac signal pathway. GLP-1 could also activate calcium/calmodulin pathway as well as mitogen-activated protein kinases and phosphatidyl inositol-3 kinase pathway. GLP-1 not only stimulates the phase 1 and phase 2 insulin secretion, but also increases insulin synthesis. GLP-1 also stimulates proliferation and differentiation of islet β-cells, and protects β-cell from apoptosis and modulates endoplasmic reticulum stress response leading to promotion of β-cell adaptation and survival.
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Betacellulin (BTC) is one of "islets regeneration factors" which received more and more attention these years. BTCe is the C-terminal 50-residue region of mature BTC protein and can bind with erbB-1?erbB-4 receptor. It has the same mitogenic activity on cells as the whole section. BTC and BTCe can improve the level of glucose-stimulated insulin secretion (GSIS) during islets culture in vitro though they had no effect on the acute insulin secretion. BTCe also effectively ameliorated the hyperglycemia of STZ-induced diabetic rats by a single plasmid injection into muscle of rats. It is supposed that BTCe promote the proliferation of PDX-1 positive cells or repair some signal transduction pathway. Perhaps the latter is more important.
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Objective To study the association of glucagon like peptide 1 receptor (GLP1R) gene polymorphism with type 2 diabetes in Han population in Shanghai. Methods In the study, 360 type 2 diabetic patients and 313 normal control subjects were enrolled. Diabetic patients were further subdivided into insulin treated non obese patients (BMI28, 192 subjects). A single nucleotide polymorphism (SNP) rs 2268657 was genotyped in all the subjects enrolled in the study using allele specific real time PCR and its association with type 2 diabetes was examined. Results The frequencies of AA,AG, GG genotype incontrol group were0.086,0.447, 0.466 respectively, 0.155, 0.375, 0.470 in non obese diabetic patient group respectively, and 0.109, 0.500, 0.391 in obese diabetic patient group respectively. There was significant difference of the frequency of genotype AA between control group and non obese diabetic patient group (OR=1.939, P
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Objective To express the mouse peroxisome proliferator activated receptor ? 2 (mPPAR? 2) in NIH3T3 cells induced by the recombinant retrovirus vector. Methods mPPAR? 2 gene digested from the recombinant plasmid pcDNA3/mPPAR? 2 containing the target gene segment was subcloned into retrovirus vector pGCEN to generate the recombinant retrovirus pGCEN/mPPAR? 2. The recombinant retrovirus pGCEN/mPPAR? 2 and pGCEN were used to transfect PA317 cells with LipofectAMINE, and anti G418 clones of PA317 cells were selected and viral supernatants were harvested and used to infect NIH3T3 cells. The expressing products were identified with immune flurescence assay (IFA) and Western Blot. Results The recombinant retrovirus pGCEN/mPPAR? 2 was constructed, and 5?10 4 CFU/ml and 6?10 5 CFU/ml of pGCEN/mPPAR? 2 containing and pGCEN containing viral supernatants were obtained respectively. mPPAR? 2 was expressed in NIH3T3 cells mediated by the recombinant retrovirus vector. Conclusion This work is the basis for the foundation of adipocyte differentiation model in vitro and further researching on the molecular mechanism of adipocyte differentiation induced by PPAR? 2.
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Objective To study the association of single nucleotide polymorphism (SNP) of the protein tyrosine phosphatase-1B (PTP-1B) gene with type 2 diabetes mellitus and obesity in Chinese. Methods SNPs in the PTP-1B gene were detected by direct sequencing to PCR products, and the detected SNPs were genotyped in case-spouse samples with the technique of fluorescence real-time PCR. Results Totally 6 SNPs were found in PTP-1B gene. Three SNPs (I5/37 C→A,I6/82 A→G, I7/301 C→T) were in the intron regions and the other 3 (E8/45 C→T, E9/35 G→A, E10/372 G→A)in the exon regions. Among them, E9/35 G→A was a newly found mutation site. The A allele frequency of I5/37 C→A, T allele frequency of I7/301 C→T and G allele frequency of I6/82 A→G in type 2 diabetes were significantly higher than those in the normal spouse group (P