Yangpresentationdiabetesandobesity.ppt

《Yangpresentationdiabetesandobesity.ppt》由会员分享，可在线阅读，更多相关《Yangpresentationdiabetesandobesity.ppt（39页珍藏版）》请在淘文阁 - 分享文档赚钱的网站上搜索。

1、ATAXIA-TELANGIECTASIA:Clinical Phenotype Cerebellar Ataxia Telangiectasias Cancer Predisposition Premature Aging Growth Retardation Insulin Resistance Glucose IntoleranceATAXIA-TELANGIECTASIA:Cellular Phenotype Genetic Instability Hypersensitivity to Ionizing Radiation G1, S, G2 Cell Cycle Checkpoin

2、t Defects Early senescence Cytoskeletal defects Poor Growth in Culture - higher requirements for serum growth factorsATM: a member of the PI-3 kinase superfamilyPI-3 K domainRad3domainATRS. pombeRad3PI-3 kinaseMec1S. cerevisiaeTel1S. cerevisiaeTOR1/2S. cerevisiaeFRAP/mTORmammalianDNA PKmammalianMei-

3、41mammalianATMmammalian D. melanogasterPI-3 K domainFKBP-12rapamycinbinding domainIRATM p53chk2mdm2nbs1G1 Checkpoint S-phase Checkpoint 4E-BP1S15S343S20Rad50Mre11Brca1InsulinTranslation Initiation -adaptinTranslocation of CytoplasmicProteinsNuclear ATM ProteinCytoplasmic ATM ProteinAnti-ATM32P-4EBP-

4、14EBP-1 staining Insulin - + - + A. HEK-293 B. 3T3-L1 1.0 2.0 1.0 3.0 Yang et. al., Nature Cell Biology, 2000Type 2 diabetes and A-T A high incidence (59%) of Type 2 diabetes in A-T patients has been reported. A-T patients with type 2 diabetes exhibit symptoms of glucose intolerance and insulin resi

5、stance. Insulin resistance is caused by defective glucose uptake mediated by glucose transporter 4 (GLUT4). GLUT-4 translocation is regulated by Insulin through PI-3K/AKT pathway.Hypothesis ATM may participate in GLUT4 translocation in response to insulin signalingType 1 and type 2 diabetes:IRS-IGlu

6、coseTransporter 4GLUCOSE ENERGY (MUSCLE)STORAGE (FAT)PI 3-Kinase ? PKB/AKTInsulinPP?IRS-IPI 3-KinasePDK1 PKB/AKT ATMInsulinCell survivalPPProtein translationGlucoseuptakeCell membraneSer473Thr308Viniegra et al., J. Biol. Chem., 2005; Halaby et al., Cell Signalling, 2008 A38 (A-T) A29 (NL) + - + -Ins

7、ulin-ATM -Akt-S473-P -Akt-Total -Rats on a high-fat diet Control rats p valuesATM- -Tubulin- rats on a high-fat dietcontrol ratsA.AKT-S473-P-Total AKT-Insulin - + - +Rats on a high-fat dietControl ratsB.Insulin - + - +KU-55933 - - + +The ATM inhibitor KU-55933 inhibits Akt phosphorylation at both Se

8、r473 and Thr308 in L6 myoblastsATM-AKT-S473-P-Total AKT-AKT-T308-P-Treatment with the ATM inhibitor KU-55933 leads to a significant decrease in glucose uptake in L6 myoblasts *A.C.D.B.Indirect immunofluorescense- insulin + insulin Insulin PI3-kinaseAktThr308Ser473PDK1ATMGLUT4 translocation and Gluco

9、se uptakeSynip?, VAMP2?ChloroquineCaffeineKU-55933Role of ATM in insulin-mediated Akt phosphorylation and glucose uptake p-Akt473- Control Insulin Chloro. Ins. + Chloro. t-Akt-*A.B.2-DG uptake (pmol/mg/min)P-Akt -T- Akt - Chloroquine + + - - - -Insulin - + - + - +Fat-fed Chow-fedConclusions Our resu

10、lts further demonstrated that ATM mediates the Ser-473 phosphorylation and the full activation of AKT in response to insulin In muscle tissues, ATM may regulate insulin-mediated GLUT4 translocation process through regulation of AKT activity Chloroquine is a promising drug candidate that could be use

11、d to treat type 2 diabetes, possibly through up-regulation of Akt-mediated glucose uptake Future Directions Characterize the upstream kinase (PI 3-kinase, or others) that regulates ATM activity in response to insulin Identify and characterize downstream substrates of ATM (mTOC2, PDK1, ATR) that may

12、directly phosphorylate Ser473 of Akt in response to insulin Further determine the mechanism used by ATM to regulate the insulin-mediated glucose uptake process (ATM/mTORC2/Akt/AS-160/GLUT4, or ATM/VAMP2/GLUT4) Examine how ATM regulate Akt-mediated glycogen synthesis process in response to insulin AT

13、M and beta cell functionAtm-/- mice develop diabetes with age as their blood glucose increased and their plasma insulin levels decreased. C-Peptide levels, which are indicative of insulin secretion, were significantly decreased in aging A-T mice (27 weeks or older), as compared to wild-type mice.It

14、was hypothesized that Atm-/- mice may exhibit increased cell apoptosis and reduced cell mass as they age (Miles et al., Am J Physiol Endocrinol Metab 2007). Current model of type 2 diabetes mellitus is defined by an initial symptom of insulin resistance/reduced glucose uptake and a subsequent decrea

15、se in insulin secretion due to reduced pancreatic -cell mass or function. IRS-IPI 3-KinasePDK1 PKB/AKT ATMInsulinCell survivalPPCellProliferationGlucoseuptakeCell membraneSer473Thr308Viniegra et al., J. Biol. Chem., 2005; Halaby et al., Cell Signalling, 2008ATMElghazi et al., Regulation of -cell mas

16、s and function by the Akt/protein kinase B signalling pathway. Diabetes, obesity, and metabolism, 2007ChloroquineHypothesis Chloroquine, by activating ATM and Akt, may play a positive role in promoting beta-cell mass and function Con KU chlo Con KU chloInsulinINS-1E (rat cells)p-Akt (Thr308)Total-Ak

17、tbeta-actinp-Akt (Ser473)Chloroquine, a promising drug candidate for cell regeneration INS-1E cells were seeded in a 48 well plate and treated with different chemicals for 48 hours under 0.5 % FBS. MTT assay was performed (four duplicates each treatment). Components of cytokines (cyto): IFN-gamma +

18、IL-1-beta. Chloroquine: chlo; GLP-1: GLP;Absorbance (570 nm)MTT assay in cultured INS1E cellsINS-1E cells were seeded in a 24 well plate and treated with different chemicals for 48 hours. Cell death ELISA was performed (four duplicates each treatment). *MTT assay in isolated primary mouse islet cell

19、sDissociated islets cells from ATM+/+, ATM+/- and ATM-/- mice were pretreated with chloroquine or GLP-1 for 1 hr and then treated with cytokines (IL-1, IFN-) for 24 hrs. The cell proliferation rate in each well was determined by MTT assay. *, P0.005.*Cell death ELISA assay in isolated primary mouse

20、islet cellsIslets from ATM+/+ or ATM+/- mice were isolated following a video protocol by Szot GL, et al. JoVE. 2007. Islets were dissociated to single cells with trypsin. Islet cells were then pretreated with chloroquine (chlo) or GLP-1 (GLP) for 1 hour before the addition of cytokines (cyto, IL-1 p

21、lus IFN-) for another 24 hours. A cell death ELISA assay (measuring DNA fragmentation) was then performed. * p 0.05, * p 0.001 from four duplicates of the experiments. ATMElghazi et al., Regulation of -cell mass and function by the Akt/protein kinase B signalling pathway. Diabetes, obesity, and meta

22、bolism, 2007ChloroquineConclusions Results from my group and others have demonstrated that ATM is an activator of Akt, a key regulator of beta cell mass and function We have shown that chloroquine, a known activator of ATM, stimulates activity of Akt and its downstream substrates in INS1E cells We a

23、lso observed that chloroquine can prevent cytokine-mediated cell death of INS-1E and primary mouse islet cells in an ATM-dependent manner Our results suggest that choloroquine may be a promising drug candidate for beta cell regeneration and functionEvidence supporting the clinical usage of choloroqu

24、ine Chloroquine has been tested in mouse and rat models of type 2 diabetes and in multiple pilot-scale human clinical trials for type 2 diabetic patients showing significant improvement of insulin sensitivity and glucose tolerance Chloroquine has long been used as a drug for treating malaria and it

25、is in general safe for human use As an anti-rheumatic drug, chloroquine can also inhibit the release of cytokines, such as IL-1beta and TNF-alpha. Our preliminary data using NOD mice and STZ-induced mice showed that chloroquine may increase beta cell mass Therefore, in addition to type 2 diabetes, c

26、hloroquine may be a promising therapeutic agent for type 1 diabetes as well Other related studies Chloroquine has also been shown to play a role in body weight reduction Our recent results suggest this may be related to ATMs role in regulating lipid metabolism Under serum starvation or energy defici

27、ent conditions, ATM also regulates LKB1 and AMPK pathway activity We have recently start to collaborate with Mayo Clinic Diabetes Center to further explore a metabolomics related project to determine ATMs role in the AMPK pathway in response to metformin and resveratrolOther related studies Overacti

28、vation of Akt leads to various types of cancer Highly expression of GLUT1 and elevated glucose uptake are common features of malignant cancers KU-55933s role in inhibiting ATM and glucose uptake has been further explored for its anti-tumor function Abnormal cancer glucose metabolism and the end prod

29、ucts have been explored as potential biomarkers of cancer Dr. Yosef Shiloh Dr. Michael B. Kastan Dr. Margot ClearyDr. Amira Klip Dr. Keith Miskimins Dr. Adrian HegemanDr. Adrian Vella Dr. Carston Wagner Dr. Douglas YeeAmerican Diabetes Association Breast Cancer Research Program, Department of Defense ATCP (A-T Childrens Project)NIEHS, NIH; and NCI, NIH; GIA; ACS-IRG; and Prostate Cancer TWG; UMN