1. Journal Club 亀田メディカルセンター 糖尿病内分泌内科 Diabetes and Endocrine Department, Kameda Medical Center 松田 昌文 Matsuda, Masafumi 2007 年３月 22 日 8:20-8:50 B 棟８階 カンファレンス室

2. Activation of AMP-activated protein kinase (AMPK) by exercise induces several cellular processes in muscle. Exercise activation of AMPK is unaffected in lean (BMI 30 kg/m 2 ), and exercise stimulation of AMPK is blunted in obese rodents. We examined whether obese type 2 diabetic subjects have impaired exercise stimulation of AMPK, at different signaling levels, spanning from the upstream kinase, LKB1, to the putative AMPK targets, AS160 and peroxisome proliferator– activated receptor coactivator (PGC)-1, involved in glucose transport regulation and mitochondrial biogenesis, respectively. Twelve type 2 diabetic, eight obese, and eight lean subjects exercised on a cycle ergometer for 40 min. Muscle biopsies were done before, during, and after exercise. Subjects underwent this protocol on two occasions, at low (50% VO2max) and moderate (70% VO2max) intensities, with a 4–6 week interval. Exercise had no effect on LKB1 activity. Exercise had a time- and intensity-dependent effect to increase AMPK activity and AS160 phosphorylation. Obese and type 2 diabetic subjects had attenuated exercise-stimulated AMPK activity and AS160 phosphorylation. Type 2 diabetic subjects had reduced basal PGC-1 gene expression but normal exercise-induced increases in PGC-1 expression. Our findings suggest that obese type 2 diabetic subjects may need to exercise at higher intensity to stimulate the AMPK-AS160 axis to the same level as lean subjects. Diabetes 56:836–848, 2007

3. Background

4. Copyright ©2005 American Physiological Society Jessen, N. et al. J Appl Physiol 99: 330-337 2005; doi:10.1152/japplphysiol.00175.2005 Muscle contractions cause translocation of the glucose transporter proteins (GLUT4) to the cell membrane and T tubules

5. Copyright ©2005 The Endocrine Society Ishiki, M. et al. Endocrinology 2005;146:5071-5078 Insulin signaling pathways regulating GLUT4 traffic glucose transport regulation AMPK

6. Mitochondrial biogenesis and gene expression mitochondrial biogenesisAMPK Endocrine Reviews 24: 78–90, 2003

7. Abbreviation ACC, acetyl CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide-1-D- ribofuranoside; AMPK, AMP-activated protein kinase; FFA, free fatty acid; IL, interleukin; NRF, nuclear respiratory factor; OGTT, oral glucose tolerance test; PAS, phospho-Akt substrate; PGC, peroxisome proliferator–activated receptor coactivator.

8. Hypothesis and Aim We examined whether obese type 2 diabetic subjects have impaired exercise stimulation of AMPK, at different signaling levels, spanning from the upstream kinase, LKB1, to the putative AMPK targets, AS160 and peroxisome proliferator– activated receptor coactivator (PGC)-1, involved in glucose transport regulation and mitochondrial biogenesis, respectively. Type 2 diabetic subjects with moderate-to-severe obesity (BMI ＞ 30 kg/m 2 ) have impaired AMPK signaling. LKB1 (up) AMPK AS160 glucose transport regulation PGC-1 mitochondrial biogenesis

10. Methods Twelve type 2 diabetic, eight obese, and eight lean subjects exercised on a cycle ergometer for 40 min. Muscle biopsies were done before, during, and after exercise. Subjects underwent this protocol on two occasions, at low (50% VO2max) and moderate (70% VO2max) intensities, with a 4–6 week interval.

11. Basal AMPK, ACC, AS160, and Akt. AMPK subunit, ACC, AS160, and Akt protein content (A) were measured in 8 lean (□), 8 obese (□), and 12 type 2 diabetic (T2DM) (■) subjects. Data are means ± SE. Blots are shown for three subjects/group. Basal protein content

12. Basal AMPK, ACC, AS160, and Akt. AMPK subunit, ACC, AS160, and Akt phosphorylation (B) were measured in 8 lean (□), 8 obese (□), and 12 type 2 diabetic (T2DM) (■) subjects. Data are means ± SE. Blots are shown for three subjects/group. Basal phosphorylation

13. Exercise had a time- and intensity-dependent effect to increase AMPK activity.

14. Effect of exercise on AMPK and ACC phosphorylation. Biopsies were done at basal (□), after 10 (□) and 40 min (■) of exercise, and 150 min postexercise (□). Data are means ± SE in 8 lean, 8 obese, and 12 type 2 diabetic (T2DM) subjects. Data are expressed as arbitrary units (A and C) and as fold change (B and D). *P < 0.05 vs. basal of respective group; †P < 0.05 vs. lean group at 40 min. Blots are shown for one subject/group. B, basal; R, rest postexercise. Exercise had a time- and intensity-dependent effect to increase AMPK activity. Reduced activity

17. Exercise had a time- and intensity-dependent effect to increase AMPK activity.

18. AMPK activity. AMPK1, AMPK2, and total AMPK activities were measured before (□), after 40 min of exercise (■), and 150 min postexercise (■). Data are means SE. Data are expressed as kinase activity (A, C, and E) and as fold change (B, D, and F). n 6–12 in each time point (samples were not available for all the assays). *P < 0.05 vs. basal of respective group; †P < 0.05 vs. lean group in respective time point. T2DM, type 2 diabetes.

19. LKB1 expression and activity. Equal amounts of protein (40 g) were used for blotting of LKB1 and MO25 (A). Blots are shown for two subjects per group. LKB1 activity was measured as described in RESEARCH DESIGN AND METHODS (B). B, basal; T2DM, type 2 diabetes. Exercise had no effect on LKB1 activity.

20. AS160 phosphorylation. Immunoblots are shown for two subjects in the basal state and after 40 min of exercise, after immunoprecipitation with AS160 and probing with PAS antibody (A). Biopsies were done at basal (□), after 10 (□) and 40 min (■) of exercise, and 150 min postexercise (□). Data are means SE in 8 lean, 8 obese, and 12 type 2 diabetic (T2DM) subjects. Data are expressed as arbitrary units (B) and as fold change (C). *P < 0.05 vs. basal of respective group; †P < 0.05 vs. lean at 150 min postexercise. Blots are shown for one subject/group. B, basal; R, rest postexercise. Exercise had a time- and intensity-dependent effect to increase AS160 phosphorylation. Reduced activity

21. Akt phosphorylation. Biopsies were done at basal (□), after 10 (□) and 40 min (■) of exercise, and 150 min postexercise (■). Akt-Ser473 (A) and Thr308 (B) were measured as described in RESEARCH DESIGN AND METHODS. Data are means ±SE in 8 lean, 8 obese, and 12 type 2 diabetic (T2DM) subjects. *P < 0.05 vs. basal of respective group. Blots are shown for one subject/group. B, basal; R, rest postexercise.

23. PGC-1 and NRF-1 gene expression. Basal PGC-1 (A) and NRF-1 (C) gene expression. Effect of exercise on PGC-1 (B) and NRF-1 (D) gene expression. Gene expression was measured at baseline, after 40 min of exercise, and 150 min postexercise. Data are means ± SE in 8 lean, 8 obese, and 12 type 2 diabetic (T2DM) subjects. *P < 0.05 vs. type 2 diabetes group; †P < 0.05 vs. basal of respective group; ‡P 0.05 vs. basal in obese group. Type 2 diabetic subjects had reduced basal PGC-1 gene expression. but normal exercise-induced increases in PGC-1 expression

24. Exercise had no effect on LKB1 activity. Exercise had a time- and intensity-dependent effect to increase AMPK activity and AS160 phosphorylation. Obese and type 2 diabetic subjects had attenuated exercise-stimulated AMPK activity and AS160 phosphorylation. Type 2 diabetic subjects had reduced basal PGC-1 gene expression but normal exercise- induced increases in PGC-1 expression. SUMMARY

25. Our findings suggest that obese type 2 diabetic subjects may need to exercise at higher intensity to stimulate the AMPK-AS160 axis to the same level as lean subjects. CONCLUSIONS

27. J Clin Endocrin Metab. First published ahead of print January 23, 2007 as doi:10.1210/jc.2006-1882 The Dipeptidyl Peptidase IV Inhibitor Vildagliptin Suppresses Endogenous Glucose Production and Enhances Islet Function after Single Dose Administration in Type 2 Diabetic Patients Short title: Vildagliptin, EGP, and Islet Function Bogdan Balas 1, Muhammad R Baig 1, Catherine Watson 2, Beth E Dunning 3, Monica Ligueros-Saylan 4, Yibin Wang 4, Yan-Ling He 2, Celia Darland 1, Jens J Holst 5, Carolyn F Deacon 5, Kenneth Cusi 1, Andrea Mari 6, James E Foley 4, Ralph A DeFronzo 1. 1 Division of Diabetes, Department of Medicine, University of Texas Health Science Center at San Antonio; 2 Novartis Institutes for Biomedical Research, Cambridge, MA; 3 PharmaWrite, LLC, Princeton, NJ; 4 Novartis Pharmaceuticals Corp. East Hanover, NJ; 5 Panum Institute, University of Copenhagen, Copenhagen, Denmark; 6 Institute of Biomedical Engineering, National Research Council, Padova, Italy

28. GLP-1 analogue

30. Dipeptidyl Peptidase IV Inhibitors

31. Aims/hypothesis: Vildagliptin is a selective DPP-4 inhibitor that augments meal stimulated levels of biologically active GLP-1. Chronic vildagliptin treatment decreases postprandial glucose levels and reduces HbA1c in type 2 diabetes (T2DM). However, little is known about the mechanism(s) by which vildagliptin promotes reduction in plasma glucose concentration. Methods: 16 T2DM (age=48±3y; BMI=34.4±1.7 kg/m 2 ; HbA1c=9.0±0.3%) participated in a randomized, double-blind, placebo-controlled trial. On separate days patients received 100 mg vildagliptin or placebo at 5:30PM followed 30 min later by a meal tolerance test (MTT) performed with double tracer technique (3- 3 H-glucose intravenously and 1- 14 C- glucose orally). Results: Following vildagliptin, suppression of endogenous glucose production (EGP) during 6 hours MTT was greater than with placebo (1.02±0.06 vs 0.74±0.06 mg/kgmin, p=0.004), and insulin secretion rate (ISR) increased by 21% (p=0.003 vs PBO) despite significant reduction in mean plasma glucose (213±4 vs 230±4 mg/dl, p=0.006). Consequently, ISR[AUC] ÷ plasma glucose[AUC] increased by 29% (p=0.01). Suppression of plasma glucagon during MTT was 5-fold greater with vildagliptin (p<0.02). The decline in EGP was positively correlated (r=0.55, p<0.03) with the decrease in fasting plasma glucose (Δ= -14 mg/dl). Conclusions: During MTT, vildagliptin augments insulin secretion and inhibits glucagon release, leading to enhanced suppression of EGP. During the postprandial period, a single dose of vildagliptin reduced plasma glucose levels by enhancing suppression of EGP. Abstract

32. Aims/hypothesis Vildagliptin is a selective DPP-4 inhibitor that augments meal stimulated levels of biologically active GLP-1. Chronic vildagliptin treatment decreases postprandial glucose levels and reduces HbA1c in type 2 diabetes (T2DM). However, little is known about the mechanism(s) by which vildagliptin promotes reduction in plasma glucose concentration.

33. 16 T2DM (age=48±3y; BMI=34.4±1.7 kg/m 2 ; HbA1c=9.0±0.3%) participated in a randomized, double-blind, placebo-controlled trial. On separate days patients received 100 mg vildagliptin or placebo at 5:30PM followed 30 min later by a meal tolerance test (MTT) performed with double tracer technique (3- 3 H- glucose intravenously and 1- 14 C-glucose orally). Methods

35. Plasma DPP-4 activity and plasma GLP-1, GIP, glucose, insulin, and C- peptide concentrations during the meal tolerance test after ingestion of vildagliptin and placebo. Data are the mean ± SE.

36. Change from baseline in plasma glucagon concentration during the meal tolerance test and during the postabsorptive period after ingestion of vildagliptin and placebo. Data are the mean ± SE.

37. Insulin secretion rate (ISR) and ISR(AUC)/Plasma Glucose(AUC) during the meal tolerance test after ingestion of vildagliptin and placebo. Data are the mean ± SE.

38. Effect of vildagliptin and placebo on glucose sensitivity of insulin secretion from the Mari model. Data are the mean ± SE.

39. Change from baseline in the rate of endogenous glucose production during the meal tolerance test and during the postabsorptive period after ingestion of vildagliptin and placebo. Data are the mean ± SE.

40. Total body rate of glucose disappearance and glucose clearance during the meal tolerance test and during the postabsorptive period after ingestion of vildagliptin and placebo. Data are the mean ± SE.

41. During MTT, vildagliptin augments insulin secretion and inhibits glucagon release, leading to enhanced suppression of EGP. During the postprandial period, a single dose of vildagliptin reduced plasma glucose levels by enhancing suppression of EGP. Conclusions