1. بنام خالق هستی Glucose Transporters (GLUT or SLC2A family) دکتر گائینی مجید قلی پور

2. Insulin u انسولین هورمونی است که از سلول های بتای لوزالمعده ترشح می شود. u در حضور آن، بافت چربی، عضلات و کبد می توانند گلوکز خون را برداشت کرده یا به مصرف برسانند و یا به صورت گلیکوژن ذخیره نمایند. u هرگونه نارسایی و نقص در سنتز و یا عمل آن باعث بیماری قند (diabetes) شده که در نتیجه گلوکز از جریان خون برداشت نمی شود.

3. Definition of Diabetes u Diabetes is any disorder characterized by excessive urine excretion. u 1- Diabetes mellitus: A metabolic disorder in which there is an inability to oxidize carbohydrate due to disturbances in insulin function. u 2- Diabetes insipidus: Is the result of a deficiency of antidiuretic hormone. u 3- Brittle diabetes: Is a form that is very difficult to control. It is characterized by unexplained oscillations between hypoglycemia and acidosis

4. Types of Diabetes Mellitus u Diabetes mellitus is a heterogeneous clinical disorder with numerous causes. Two main classifications of diabetes mellitus are Idiopathic and Secondary u Idiopathic: Which is divided into two main types. 1- (IDDM) Insulin Dependent Diabetes Mellitus (type 1 diabetes) which most often manifests in childhood and is the result of an autoimmune destruction of the β-cells of the pancreas. 2- (NIDDM) Non-Insulin-Dependent Diabetes Mellitus (type 2 diabetes) is characterized by persistent hyperglycemia but rarely leads to ketoacidosis. Type 2 diabetes generally manifests after age 40. u There are two main forms of type 2 diabetes: 1. Late onset associated with obesity. 2. Late onset not associated with obesity.

5. Types of Diabetes Mellitus u Secondary: or other specific types of diabetes mellitus are the result of many causes including: 1-Maturity onset type diabetes of the young (MODY)1-6 2-Pancreatic disease. 3-Endocrine disease. Counter-regulatory hormones are glucagon, epinephrine, growth hormone and cortisol. 4-Drug-induced diabetes. 5-Anti-insulin receptor autoantibodies (Type B insulin resistance). 6-Mutations in the insulin gene.

6. Types of Diabetes Mellitus 7-Mutations in insulin receptor gene. u a. Leprachaunism u b. Rabson-Mendenhall syndrome u c. Type A insulin resistance 8-Gestational diabetes. 9-Many other genetic syndromes include: Wolfram syndrome, Down syndrome, Huntington disease, Werner syndrome, …

7. Glucose transporter Glucose : 1- Glucose is an essential substrate for the metabolism of most cells 2- Glucose is a polar molecule u Thus transport through biological membranes requires specific transport proteins. Glucose transporter (GLUT)

9. Glucose transporter u Transport of glucose through the membrane of intestinal and kidney epithelial cells depends on the presence of secondary active Na+/glucose symporters, SGLT-1 and SGLT-2 which concentrate glucose inside the cells, using the energy provided by cotransport of Na+ ions down their electrochemical gradient. intestinalkidney

11. Glucose transporter u GLUTs are integral membrane proteins which contain 12 membrane-spanning helices with both the C and N terminal exposed on the cytoplasmic side of the plasma membrane. u GLUT proteins transport glucose and related hexoses according to a model of alternate conformation. u Binding of glucose to one site provokes a conformational change associated with transport, and releases glucose to the other side of the membrane. u glucose-binding sites are probably located in transmembrane segments 9, 10, 11. u the QLS motif located in the seventh transmembrane segment could be involved in the selection and affinity of transported substrate.

12. Glucose transporter

14. u برای انتقال دهنده گلوکز از دو نشان و علامت استفاده می شود. u 1- protein symbol: GLUT (Glucose transporter). u 2- gene symbol: SLC2 (Solute Carrier Family 2). u برای تعیین نوع آن به ترتیب از شماره و یا حرف A + شماره، استفاده می شود. u بعنوان مثال GLUT 1 یا SLC2 A1.

15. GLUTs Types u Most mammalian cells transport glucose through a family of membrane proteins known as glucose transporters. u Each glucose transporter isoform plays a specific role in glucose metabolism determined by : 1- pattern of tissue expression. 2- substrate specificity. 3- transport kinetics. 4- regulated expression in different physiological conditions.

16. GLUTs Types GLUT1 : Is widely distributed in fetal tissues. In adult, mediates glucose transport into red cells, and throughout the blood brain barrier. GLUT2 : Is expressed by renal tubular and small intestinal epithelial cells. It provides glucose to liver and pancreatic β cells. GLUT3 : Is the main transporter in neurons. GLUT4 : Is the insulin-regulated glucose transporter found in adipose tissues and striated muscle (skeletal and cardiac) that is responsible for insulin- regulated glucose disposal.

17. GLUTs Types GLUT5 : Transports fructose in intestine and testis. GLUT6 : Name was previously assigned to a pseudoegene. It is highly expressed in brain, spleen, and leukocytes. GLUT7 : Expressed in liver and other gluconeogenic tissues, mediates glucose flux across endoplasmic reticulum membrane.

18. GLUTs Types GLUT8 : High levels are found in adult testis and placenta. GLUT9 : It is expressed in kidney and liver. It is also detected in placenta, lung, blood leukocytes, heart, and skeletal muscle. GLUT10 : Has been identified as a candidate gene for NIDDM susceptibility. It is widely expressed with highest levels in liver and pancreas.

19. GLUTs Types GLUT11 : It is expressed in heart and skeletal muscle. GLUT12 : It is expressed in skeletal muscle, adipose tissue, and small intestine. GLUT13 : (H+ myo-inositol transporter - HMIT) It is predominantly expressed in brain.

20. GLUTs Types u 13 نوع GLUT/SLC2 شناسایی شده، بر اساس شباهت و همگنی، به 3 دسته تقسیم می شوند. u دسته اول : GLUT4 - GLUT3 - GLUT2 - GLUT1 u دسته دوم : GLUT11 - GLUT9 - GLUT7 - GLUT5 u دسته سوم :GLUT12 - GLUT10 - GLUT8 - GLUT6 - GLUT 13 (H+/myoinositol transporter HMIT) Most members of classes II and III have been identified recently in homology searches.

21. New Nomenclature (HUGO) of Gluts Protein Other Names Gene Chromos. Accession MajorTissue Major Isoform Name Locuse Numbers Experssion GLUT1 GTR1, Hu: 492aa SLC2A1 1p35-31.3 AC023331 Erythrocytes, brain GLUT2 GTR2, Hu: 524 aa SLC2A2 3q26.2-27 AC068853 Liver, islets GLUT3 GTR3, Hu: 496 aa SLC2A3 12p13.3 AC007536 Brain (neuronal) GLUT4 GTR4, Hu: 509 aa SLC2A4 17p13 AC003688 Muscle, fat, heart GLUT5 GTR5, Hu: 501 aa SLC2A5 1p36.2 AC041046 Intestine, testis, kidney GLUT6 GTR6, Hu: 507 aa SLC2A6 9q34 AC002355 Spleen, leukocytes, brain GLUT7 GTR7, Rt: 528 aa SLC2A7 1p36.2 AL356306 Liver GLUT8 GTR8, Hu: 477 aa SLC2A8 9 AL445222 Testis, blastocyst, brain GLUT9 GTR9, Hu: 511/540aa SLC2A9 4p15.3-16 AC005674 Liver, kidney GLUT10 GTR10, Hu: 541 aa SLC2A10 20q12-13.1 AC031055 Liver, pancreas GLUT11 GTR11, Hu: 496 aa SLC2A11 22q11.2 AP000350 Heart, muscle GLUT12 GTR12, Hu: 617 aa SLC2A12 6q23.2 AL449363 Heart, prostate GLUT13 HMIT, Hu: 618/629 aa SLC2A13 AJ315644 Brain

22. Regulation of insulin secretion Na + K+K+ K+K+ K+K+ K+K+ VmVm GLUT2 Ca 2+ Voltage-gated Ca 2+ channel KIR Pancreatic ß cell Insulin granules Ca 2+ -

23. Basal insulin secretion Na + K+K+ K+K+ K+K+ K+K+ GLUT2 Ca 2+ Voltage-gated Ca 2+ channel KIR Pancreatic ß cell Pacemaker ß cells Signal Insulin granules Ca 2+ VmVm

24. Insulin granules Glucose-stimulated insulin secretion Na + K+K+ K+K+ K+K+ K+K+ ATP Na + K+K+ - K+K+ Glucose GLUT2 Ca 2+ Voltage-gated Ca 2+ channel KIR VmVm Pancreatic ß cell IP 3 cAMP Glucokinase K m = 7-9 mM ß cell integrates input from various metabolites, hormones and neurotransmitters

25. Na + K+K+ K+K+ K+K+ K+K+ GLUT2 Ca 2+ Voltage-gated Ca 2+ channel KIR Pancreatic ß cell Insulin granules Ca 2+ - Sulfonylureas - VmVm Sulfonylureas: Mechanism of Action

26. Insulin : mechanism of action Cell-surface receptors:  subunits contain insulin binding sites  subunits have tyrosine kinase activity plasma membrane

27. Insulin receptor signaling PO 4  IRS-1 + ATP IRS-1  PO 4 Insulin binding to  subunit regulates  subunit activityInsulin autophosphorylation of  subunit GLUT4 phosphorylation of other substrates  tyr kinase activity activation of phospho- inositide 3-kinase Glucose transporter translocation to plasma membrane

28. PO 4  IRS-1 + ATP IRS-1  PO 4 Insulin GLUT4 phosphorylation of MAP kinase MAPK + ATP MAPK  PO 4 Transcriptional regulation Protein synthesis, proliferation & differentiation Insulin binding to  subunit regulates  subunit activity autophosphorylation of  subunit phosphorylation of other substrates  tyr kinase activity Insulin receptor signaling e.g.  GLUT expression

29. PO 4  IRS-1 + ATP IRS-1  PO 4 Insulin GLUT4 glycogen synthase protein phosphatase-1 phosphorylase kinase phosphorylase  + + -  - Glycogen deposition Insulin binding to  subunit regulates  subunit activity autophosphorylation of  subunit phosphorylation of other substrates  tyr kinase activity phosphorylation of MAP kinase Insulin receptor signaling

30. Normal kinetics of intestinal glucose absorption in the absence of GLUT2 u در این تحقیق از موش های پرورش یافته فاقد GLUT2-/- ( گروه تجربی) استفاده شد که موش های سالم (گروه کنترل)، گلوکز را همراه با Phlorizin مصرف کردند. u نمونه خون سیاهرگی از دم آن ها تهیه شد. u کل RNA بافت های مختلف (acid guanidinium thiocyanate technique) بدست آمد. u برای سنجش مقادیر GLUT1, 2, 3, 5, 8, and 9 mRNA از روش Northern blot analysis استفاده شد. u گلوکز خون در گروه ها بعد از مصرف گلوکز خوراکی افزایش یافت. u به طور مشابه در افرادی با Fanconi-Bickel syndrome که به علت غیر فعال شدن هر دو ژن GLUT2 بوجود می آید، هضم و جذب غیر طبیعی در کربوهیدرات وجود ندارد.

31. Normal kinetics of intestinal glucose absorption in the absence of GLUT2

32. u mRNA مربوط به GLUT1 در هیچ بافتی یافت نشد. u mRNA مربوط به GLUT2 فقط در گروه کنترل یافت شد. u mRNA مربوط به GLUT3 در هیچ بافتی یافت نشد. u سطح GLUT8 مربوط به روده هر دو گروه کنترل و فاقد GLUT2 در سطح پائینی قرار داشت. u سطح GLUT5 در گروه کنترل پائین بود لیکن در گروه فاقدGLUT2 افزایش یافت. u سطح G6Pase و G6PTase در تمام گروه ها یکسان بود.

33. Normal kinetics of intestinal glucose absorption in the absence of GLUT2

34. u در موشهای فاقد GLUT2 برداشت گلوکز تقریباً مسدود شده بود لیکن خروجی گلوکز بدون تغییر ماند که می توان نتیجه گرفت مسیر دیگری برای انتقال وجود دارد که احتمالاً گلوکز از شبکه آندو پلاسمی مستقیماً به غشاء خارجی هدایت می شود و بنابر مدارک موجود کبد هم دارای این مکانیزم می باشد. Membrane Traffic-based Pathway u این مسیر در سلول های طبیعی روده وجود دارد زیرا : u 1- با مهار SGLT-1 تقریباً 15% کاهش در جذب گلوکز بوجود آمد. u 2- مسیر (MTP) تقریباً فقدان GLUT2 را جبران کرد.

36. glucose transporter 4 u In most insulin-resistant states, such as obesity and NIDDM, GLUT4 gene expression is reduced in adipose cells, in skeletal muscle GLUT4 expression is normal. u Thus, the decreased glucose uptake in response to insulin (insulin resistance) in skeletal muscle results from alterations in the translocation, docking, or fusion of glucose transporters at the plasma membrane or T tubules, or potentially from changes in the specific activity of the transporters (moles of glucose transported/transporter/unit of time)

37. glucose transporter 4 u Exercise and insulin utilize distinct signaling pathways that lead to the activation of glucose transport in skeletal muscle. u In insulin-resistant, defective activation of PI3- kinase, results impaired intracellular signaling. u However, the activation of PI 3-kinase is not important for the effects of exercise to stimulate glucose transport. u Some of the beneficial effects of exercise training on insulin action may be indirect, resulting from weight loss or changes in body composition (increased lean body mass).

38. glucose transporter 4 u Training cessation for 14 days in endurance- trained (runners) or strength-trained (weight lifters) subjects was associated with a decrement in insulin sensitivity but no reduction in GLUT4 expression in muscle, suggesting that multiple factors are involved in the changes in insulin sensitivity with detraining.

40. Dynamic traffic of GLUT4 u In unstimulated fat or muscle cells (i.e., the basal state), ~3–10% of GLUT4 is located at the cell surface and >90% is in intracellular compartments. u Insulin shifts the steady-state distribution of GLUT4 toward the PM by elevating the exocytic rate of GLUT4 and minimally reducing its endocytic rate. u The excursion of GLUT4-containing vesicles to and from the PM, the dynamic traffic of GLUT4 between multiple intracellular compartments, and vesicle-membrane fusion are exquisitely regulated.

41. regulated recycling

42. Insulin-stimulated glucose uptake u Two signaling pathways downstream of insulin receptor appear to control the movement of GLUT4 to the membrane: 1- The phosphatidylinositol 3-kinase (PI3-kinase) pathway. 2- The Rho-family GTPase TC10 pathway.

43. The phosphatidylinositol 3-kinase (PI3-kinase) pathway

44. The Rho-family GTPase TC10 pathway

45. u The caveolin gene family has three members in vertebrates: CAV1, CAV2, and CAV3, coding for the proteins caveolin-1, caveolin-2 and caveolin-3, respectively. All three members are membrane proteins with similar structure. u Caveolin forms oligomers and associates with cholesterol and sphingolipids in certain areas of the cell membrane, leading to the formation of caveolae.oligomers cell membrane caveolae u Oligomers of caveolin form the coat of a domains. Many functions are ascribed to these domains, ranging from endocytosis and transcytosis to signal transduction. Oligomersendocytosistranscytosissignal transduction

46. The Rho-family GTPase TC10 pathway u Caveolin-1 is most prominently expressed in endothelial, fibrous and adipose tissue. endothelialfibrousadipose u The expression pattern of caveolin-2 is similar to that of caveolin-1; it seems to be co-expressed with caveolin-1.co-expressed u The expression of caveolin-3 is restricted to skeletal muscle. skeletal muscle

47. Insulin-stimulated glucose uptake

49. Exercise-stimulated glucose uptake

50. Exercise-stimulated glucose uptake (Ca2+ concentration) In rat’s muscle, raising intracellular Ca2+ by treatment with caffeine in vitro also increases glucose transport. In contrast, no effect of Ca2+ ionophores on basal skeletal muscle glucose transport, with an inhibition of insulin-stimulated glucose transport. The discrepancies between these findings may relate to the magnitude and duration of the increase in cytosolic Ca2+.

51. Exercise-stimulated glucose uptake (Calmodulin) CaM inhibitors show reductions in contraction- stimulated glucose transport, perhaps because CaM inhibition leads to reduced tension development. Specific inhibition by KN62/93 that are activated by Ca2+-bound CaM (i.e., CaMKI, -II, -IV) leads to partial reduction in contraction-stimulated glucose transport without affecting tension development. CaMKII activity is increased in contracting skeletal muscle of humans during exercise/contraction and KN62/93 impairs the activation of CaMKII.

52. Exercise-stimulated glucose uptake (Nitric oxide synthase) Nitric oxide synthase (NOS) is also activated by Ca2+-bound CaM. NOS activity and it’s production are increased during exercise in skeletal muscle cells. Inhibition of NOS leads to reduced glucose uptake without affecting total blood flow across the working limb of humans. The effect of NOS inhibition on glucose uptake in humans might be explained by regulation of nutritive flow rather than membrane transport, although this remains to be established.

53. Exercise-stimulated glucose uptake (AMPK) Energy-sensing signaling system is likely to be involved in stimulating glucose transport with contractions. 5'-AMP-activated protein kinase (AMPK) may be the enzyme that fulfils this role as reflected by the ratios of AMP/ATP and creatine/phosphocreatine. Because activation of AMPK in resting muscle by the drug 5'-aminoimidazole-4-carboxyamide- ribonucleoside (AICAR) increases glucose transport/uptake, it would seem logical to ascribe a role for AMPK in contraction-stimulated glucose transport.

54. Exercise-stimulated glucose uptake (AMPK) u Thus, although AMPK is an attractive candidate for signaling in contraction- stimulated muscle glucose uptake, the available evidence at this time does not lead to a clear conclusion regarding its role.

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