The effect of Exenatide Therapy on inflammation, insulin requirement and weight in Obese Type 2 diabetes Mellitus pat

1. The effect of Exenatide Therapy on inflammation, insulin requirement and weight in Obese Type 2 diabetesMellitus patients on Insulin ShahidMukhtarBanday MBBS Mentors DR PARESH DANDONA MBBS., Ph.D., F.R.C.P, F.A.C.P, F.A.C.C DR AJAY CHOUDHARY MBBS. MRCP

2. Diabetes Mellitus along with its complications costs 174 billion Dollars(Economic Costs of Diabetes in the U.S. in 2007 Diabetes Care March 2008 ) • GLP1 mimetic/analogues/DPP4 inhibitors – • New and effective adjunctive treatment strategy to manage Type 2 Diabetes Mellitus

3. STIMULATES INSULIN RELEASE GLP1 INCRETIN LOWERING BLOOD SUGAR INHIBITS GLUCAGON RELEASE DPP-4 DPP-4 INHIBITORS BLOCKS DPP4 ENZYME TO DECREASE GLUCOSE

4. Insulinotropic effect,8,9 INSULIN RELEASE IN PRESENCE OF HYPERGLYCEMIA insulin biosynthesis and gene expression5,10 The transcription of glucokinase and the GLUT 2 transporter genes .11 GLP1 • L cells of jejunum/ileum • GLUCAGONOSTIC EFFECT:VIA8,18,19 • GLP1 R ON ALPHA CELLS • INSULIN • SOMATOSTATIN GLP-1 receptor activation directly stimulates beta-cell replication and neogenesis14-16 (RODENT STUDIES) ANIMAL STUDIES INHIBITS BETA CELL APOPTOSIS animal models of obesity and hyperglycemia.17

5. Exenatide exhibits actions that are similar to those of GLP-1 • Stimulation of insulin secretion only when blood glucose concentrations are elevated • Suppression of postprandial glucagon secretion. BUT DOES Not impair normal glucagon response to hypoglycemia • Restoration of First-phase of insulin response • Slowing of gastric emptying and promotes satiety(5, 6) EXENATIDE Synthetic exendin-4

6. Obesity IL6 /CRP/TNF ALPHA ⁵⁷ Insulin Resistance • Pro inflimmatory • High oxidative state ⁴⁸⁻⁵⁶ Diabetes Mellitus Insulin IV

7. DIET INDUCED WEIGHT LOSS • TNF ALPHA • IL6 • CRP52,76-81 CALORIE RESTRICTION • OXIDATIVE STRESS • INFLAMMATORY • MEDIATORS49,52,82

8. NFkB • Glucose • and mixed meal63-65 Total cellular IB Two cardinal indices of inflammation at the cellular level

9. RATIONALE • Obesity and Diabetes • States of increased inflammation • Exenatide is expected to lead to decreased inflammation by virtue of better glycemic control and weight loss. • Exenatide results in better control of T2 Diabetes Mellitus • Long term use of exenatide reduces insulin requirement.

10. AIMS • Exenatide has anti inflammatory effect • Single dose of Exenatide causes anti inflammatory effect • Effect of exenatide over 12 weeks v/s placebo on • HbA1c • Fasting blood glucose • Body weight • Total Insulin Requirement

11. Single-center, randomized, placebo-control, single blinded (patient) prospective study. N=24 subjects Type2DM On insulin EXENATIDE N=12 PLACEBO N=12

12. Inclusion criteria • Males or females 20-75 years of age inclusive. • Type 2 diabetes • On insulin therapy • HbA1c 7.5% and ≤ 10.0% • BMI ≥ 30 kg/m2 • Subjects on statins, ACE inhibitors, metformin and will be allowed as long as they are on stable doses of these compounds and the dosage in not changed during the study. 

13. Exclusion criteria • Coronary event or procedure (myocardial infarction, unstable angina, coronary artery bypass surgery or coronary angioplasty) in the previous four weeks • Pregnancy • Hepatic disease (abnormal LFT’s) • Use of DPP4 inhibitors. • Renal impairment (serum creatinine > 1.5) • Participation in any other concurrent clinical trial • Any other life-threatening, non-cardiac disease • Uncontrolled hypertension (BP > 160/100 mm of Hg) • Use of an investigational agent or therapeutic regimen within 30 days of study

14. Dietitian/Certified Diabetes Educator meeting on Day 0 • Dietary recommendations - American Diabetes Association guidelines • They were randomized to receive either • Exenatide 10micg or Placebo 30 min before breakfast and dinner for 12wk. • The dose of Exenatide was started at 5micg twice daily for 1 wk to ensure tolerability.

15. 24 obese Type 2 diabetes HbA1c(7.5 --9%) • All patients were on Insulin • Stable doses of antidiabetic medications • Stable weight over prior 4 weeks • All were on 1 -2g of Metformin • Statins/ACEI DOSES were not changed during the study • No patient was on Thiazolidinediones, Antioxidants,NSAIDS . • 14/24:sulfonylureas glyburide/ glipizide 5–10 mg/d). • 10/24: long acting insulin(glargine/detemir) • 12/24:long acting + pre meal bolus • 2/24: Novolog 70/30 BID

16. 0 day 3wk 6 wk 12 wk 12 WEEK study 0hr 5micg exenitide 2hr 4hr placebo 6hr Single dose study

17. Insulin titration wk3 wk6 • Target fasting blood glucose 100 • 2 hour post prandial 160

18. Mono Nuclear Cells collection- Na EDTA- washed Hanks salt solution- yeild 95% • Reactive Oxygen Species generation:measured by chemiluminiscence • Nuclear NFkBand Oct-1 DNA-binding activity was measured by EMSA (electrophoretic mobility shift assay) • Nuclear extracts -salt extraction method from MNC

19. Quantification of JNK-1, TLR-2, TLR-4, TNF alpha Real Time-PCR. • The mRNA expression of JNK-1, TLR-2, TLR-4, TNFalpha,SOCS-3, IL-1B, and IL-10

20. Statistical analysis Statistical analysis was conducted using Sigma Stat software (SPSS Inc., Chicago, IL) • All data are represented as mean ± SE.Baseline measurements were normalized to 100%, and changes from baseline were calculated as percent change from baseline. • Statistical analysis was carried out using one way repeated measures ANOVA (RMANOVA) with Holm-Sidak post hoc test. • Two-factor RMANOVA followed by Dunnett’s post hoc was used for multiple comparisons between different treatments. • Paired t test and Student’s t test were used where appropriate. • Multivariate analysis of changes in inflammatory mediators from baseline with changes in free fatty acids (FFA), insulin, glucose, percent HbA1c, body mass index, Systolic and diastolic blood pressure was performed using multiple linear regression.

22. Data represented as mean ± Standard Error(SE) • Â - P value <0.05(paired t test compared with baseline)

23. Fasting blood glucose fell from 139 ±17 to 110 ±9 mg/dl(P<0.05) • HbA1c from 8.6% ± 0.4% to 7.4%±0.5%(P<0.05) • Data are presented as mean +/- SE; n =12 each. * and • **, P < 0.05 by RMANOVA (compared with baseline) in exenatide and placebo groups, respectively; # P < 0.05 by two-way RMANOVA compared with control groups.

24. Insulin increased (P <0.05 ) in the Exenatide group whereas it did not change significantly in the placebo group • Data are presented as mean ±SE; n =12 each. * and • **, P # 0.05 by RMANOVA (compared with baseline) in exenatide and placebo groups, respectively; # P < 0.05 by two-way RMANOVA compared with control groups.

25. Percent change in FFA(C) after placebo and exenatide 10 mic g twice daily for 12 wk • Percentage change in FFA (D) after a single dose of 5 mic g exenatide or placebo in type 2 diabetic subjects. • Percentage FFA decreased by 21.5% from baseline (P<0.05)with exenatide • Data are presented as mean +/- SE; n =12 each. * and • **, P # 0.05 by RMANOVA (compared with baseline) in exenatide and placebo groups, respectively; # P < 0.05 by two-way RMANOVA compared with control groups.

26. Percent change in ROS generation by MNC after • Placebo and exenatide 10 micg bid for 12 wk • Single dose of placebo or exenatide (5 micg) and after 6 h

27. Time (weeks) Time (hours) • Change in NFkB/Oct-1 DNA-binding activity (B and C) • Data are presented as mean +/-SE; n = 12 each. *, P < 0.05 by RMANOVA (compared with baseline); #, P <0.05 by two-way RMANOVA • compared with control groups..

28. Change of mRNA expression of TNFalpha (D and E) • IL-1B (F and G) from baseline (100%) after placebo and exenatide 10 micg twice-daily treatment for 12 wk and after 6 h of a single dose of placebo or exenatide (5 micg) in type 2 diabetic subjects.

29. Percent change in JNK-1 (B), TLR-2 (C), and SOCS-3 (D) proteins in MNC after placebo and exenatide 10 micg twice-daily treatment for 12 wk (W) in type 2 diabetic subjects. • Data are presented as mean +/-SE; n =12 each. *, P <0.05 by RMANOVA (compared with baseline); #, P <0.05 by two-way RMANOVA compared with control

30. Discussion • Data shows clearly that exenatidesuppresses several indices of inflammation when given over a period of 12wk. • They include ROS generation by MNC, • IntranuclearNFkB binding, • Expression of TNF alpha, JNK-1, TLR-2, IL-1B, and SOCS-3 in MNC • There was Fall in the plasma concentrations of MCP-1,SAA, IL-6, and MMP-9 • All these changes were independent of weight loss

31. Effect more impressive as all patients were on insulin which has its own anti-inflammatory effect • An explanation for the lack of exenatide induced weight loss could be the short duration of our study in subjects on relatively large doses of insulin. • HbA1c was also reduced significantly from 8.6 to 7.4%, and the reduction in calorie loss from glycosuria could have neutralized the effect of exenatide on weight loss.

32. Potent but transient Rapid Anti inflammatory effect: single injection of 5micg exenatide: peak effect at 2hrs : coincides with time of peak plasma concentration.

33. Possible mechanisms for the anti inflammatory/antioxidant effects of exenatide include the • suppression of FFA ₤ • enhancement of the anti inflammatory action of insulin ₤ • Suppression of Glucagon(not studied)

34. Limitations of study • Small sample size • Diet recommendations were made at baseline and dietary history was not collected at the end of the study. • Likely that subjects did not make any substan-tial dietary changes during the course of the study.

35. Conclusion Exenatide when administered for 12 wk • comprehensive ROS suppressive and anti inflammatory effect in presence of insulin • Single dose of 5micg: rapid but transient anti inflammatory effect.

36. Future studies • Whether exenatidemight be used in acute inflammatory settings in the intensive care unit or following heart attacks and strokes, where a rapid anti-inflammatory effect is required and such drugs may be of potential use.

37. THANK YOU

38. Acknowledgement • Faculty Diabetic Research Center of WNY

39. Reference • 5. Drucker DJ, Philippe J, Mojsov S, Chick WL, Habener JF. Glucagon-like peptide I stimulates insulin gene expression and increases cyclic AMP levels in a rat islet cell line. Proc NatlAcadSci U S A 1987;84(10):3434-8 • 7. Kreymann B, Williams G, Ghatei MA, Bloom SR. Glucagon-like peptide-1 7-36: a physiological incretin in man. Lancet 1987;2(8571):1300-4. • 8. Weir GC, Mojsov S, Hendrick GK, Habener JF. Glucagonlike peptide I (7-37) actions on endocrine pancreas. Diabetes 1989;38(3):338-42. • 9. Nauck MA, Kleine N, Orskov C, Holst JJ, Willms B, Creutzfeldt W. Normalization of fasting hyperglycaemia by exogenous glucagon-like peptide 1 (7-36 amide) in type 2 (non-insulin-dependent) diabetic patients. Diabetologia 1993;36(8):741-4. • 10. Fehmann HC, Habener JF. Insulinotropic hormone glucagon-like peptide-I(7-37) stimulation of proinsulin gene expression and proinsulin biosynthesis in insulinoma beta TC-1 cells. Endocrinology 1992;130(1):159-66. • 14. Xu G, Stoffers DA, Habener JF, Bonner-Weir S. Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats. Diabetes 1999;48(12):2270-6. • 15. Zhou J, Wang X, Pineyro MA, Egan JM. Glucagon-like peptide 1 and exendin-4 convert pancreatic AR42J cells into glucagon- and insulin-producing cells. Diabetes 1999;48(12):2358-66. • 16. Hui SL, Epstein S, Johnston CC, Jr. A prospective study of bone mass in patients with type I diabetes. J ClinEndocrinolMetab 1985;60(1):74-80. 17. Wang Q, Brubaker PL. Glucagon-like peptide-1 treatment delays the onset of diabetes in 8 week-old db/db mice. Diabetologia 2002;45(9):1263-73. • 18. Nauck MA, Heimesaat MM, Behle K, et al. Effects of glucagon-like peptide 1 on counterregulatory hormone responses, cognitive functions, and insulin secretion during hyperinsulinemic, stepped hypoglycemic clamp experiments in healthy volunteers. J ClinEndocrinolMetab 2002;87(3):1239-46. • 19. Heller RS, Kieffer TJ, Habener JF. Insulinotropic glucagon-like peptide I receptor expression in glucagon-producing alpha-cells of the rat endocrine pancreas. Diabetes 1997;46(5):785-91.

40. 17. Wang Q, Brubaker PL. Glucagon-like peptide-1 treatment delays the onset of diabetes in 8 week-old db/db mice. Diabetologia 2002;45(9):1263-73. • 18. Nauck MA, Heimesaat MM, Behle K, et al. Effects of glucagon-like peptide 1 on counterregulatory hormone responses, cognitive functions, and insulin secretion during hyperinsulinemic, stepped hypoglycemic clamp experiments in healthy volunteers. J ClinEndocrinolMetab 2002;87(3):1239-46. • 19. Heller RS, Kieffer TJ, Habener JF. Insulinotropic glucagon-like peptide I receptor expression in glucagon-producing alpha-cells of the rat endocrine pancreas. Diabetes 1997;46(5):785-91. • 48. Dandona P, Thusu K, Cook S, et al. Oxidative damage to DNA in diabetes mellitus. Lancet 1996;347(8999):444-5. • 49. Dandona P, Mohanty P, Ghanim H, et al. The suppressive effect of dietary restriction and weight loss in the obese on the generation of reactive oxygen species by leukocytes, lipid peroxidation, and protein carbonylation. J ClinEndocrinolMetab 2001;86(1):355-62. • 50. Higdon JV, Frei B. Obesity and oxidative stress: a direct link to CVD? ArteriosclerThrombVascBiol 2003;23(3):365-7. • 51. Vincent HK, Powers SK, Stewart DJ, Shanely RA, Demirel H, Naito H. Obesity is associated with increased myocardial oxidative stress. Int J ObesRelatMetabDisord 1999;23(1):67-74. • 52. Dandona P, Weinstock R, Thusu K, Abdel-Rahman E, Aljada A, Wadden T. Tumor necrosis factor-alpha in sera of obese patients: fall with weight loss. J ClinEndocrinolMetab 1998;83(8):2907-10. • 53. Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 1993;259(5091):87-91. • 54. Katsuki A, Sumida Y, Murashima S, et al. Serum levels of tumor necrosis factor-alpha are increased in obese patients with noninsulin-dependent diabetes mellitus. J ClinEndocrinolMetab 1998;83(3):859-62. • 55. Madrid LV, Mayo MW, Reuther JY, Baldwin AS, Jr. Akt stimulates the transactivation potential of the RelA/p65 Subunit of NF-kappa B through utilization of the Ikappa B kinase and activation of the mitogen-activated protein kinase p38. J BiolChem 2001;276(22):18934-40. • 56. Davi G, Guagnano MT, Ciabattoni G, et al. Platelet activation in obese women: role of inflammation and oxidant stress. Jama 2002;288(16):2008-14.

41. 63. Aljada A, Mohanty P, Ghanim H, et al. Increase in intranuclear nuclear factor kappaB and decrease in inhibitor kappaB in mononuclear cells after a mixed meal: evidence for a proinflammatory effect. Am J ClinNutr 2004;79(4):682-90. • 64. Mohanty P, Ghanim H, Hamouda W, Aljada A, Garg R, Dandona P. Both lipid and protein intakes stimulate increased generation of reactive oxygen species by polymorphonuclear leukocytes and mononuclear cells. Am J ClinNutr 2002;75(4):767-72. • 65. Aljada A, Ghanim H, Mohanty P, Assian E, Dandona P. Glucose intake stimulates intranuclearNFkB and p47phox in mononuclear cells. ENDO '2000, the 82nd Annual Meeting of the Endocrine Society 2000 • 76. Ziccardi P, Nappo F, Giugliano G, et al. Reduction of inflammatory cytokine concentrations and improvement of endothelial functions in obese women after weight loss over one year. Circulation 2002;105(7):804-9. • 77. Zahorska-Markiewicz B, Janowska J, Olszanecka-Glinianowicz M, Zurakowski A. Serum concentrations of TNF-alpha and soluble TNF-alpha receptors in obesity. Int J ObesRelatMetabDisord 2000;24(11):1392-5. • 78. Heilbronn LK, Noakes M, Clifton PM. Energy restriction and weight loss on very-low-fat diets reduce C-reactive protein concentrations in obese, healthy women. ArteriosclerThrombVascBiol 2001;21(6):968-70. • 79. Bastard JP, Jardel C, Bruckert E, et al. Elevated levels of interleukin 6 are reduced in serum and subcutaneous adipose tissue of obese women after weight loss. J ClinEndocrinolMetab 2000;85(9):3338-42. • 80. Abad LW, Schmitz HR, Parker R, Roubenoff R. Cytokine responses differ by compartment and wasting status in patients with HIV infection and healthy controls. Cytokine 2002;18(5):286-93. • 81. Nicklas BJ, Ambrosius W, Messier SP, et al. Diet-induced weight loss, exercise, and chronic inflammation in older, obese adults: a randomized controlled clinical trial. Am J ClinNutr 2004;79(4):544-51. • 82. Dandona P, Mohanty P, Hamouda W, et al. Inhibitory effect of a two day fast on reactive oxygen species (ROS) generation by leucocytes and plasma ortho-tyrosine and meta-tyrosine concentrations. J ClinEndocrinolMetab 2001;86(6):2899-902.

42. 3. MacDonald PE, El-Kholy W, Riedel MJ, Salapatek AM, Light PE,Wheeler MB 2002 The multiple actions of GLP-1 on the process of • glucose-stimulated insulin secretion. Diabetes 51(Suppl 3):S434–S442 • 4. Holz GG, Chepurny OG 2003 Glucagon-like peptide-1 synthetic analogs: new therapeutic agents for use in the treatment of diabetes mellitus. Curr Med Chem 10:2471–2483 • 5. RodriquezdeFonsecaF,NavarroM,AlvarezE,RonceroI,ChowenJA, Maestre O, Go ´mez R, Mun ˜oz RM, Eng J, Bla ´zquez E 2000 • Peripheral versus central effects of glucagon-like peptide-1 receptor agonists on satiety and body weight loss in Zucker obese rats. Metabolism 49:709–717 • 6. SzaynaM,DoyleME,BetkeyJA,HollowayHW,SpencerRG,GreigNH,EganJM 2000 Exendin-4 decelerates food intake,weight gain, • and fat deposition in Zucker rats. Endocrinology 141:1936–1941 • 17. Ross R 1999 Atherosclerosis: an inflammatory disease. N EnglJ Med 340:115–126