1. Mechanisms for Improved Insulin Sensitivity by Branched-chain Amino Acids Deprivation Reporter : Fei Xiao, PhD Shanghai Institute for Nutritional Sciences, Chinese Academia of Sciences

2. Prevalence of diabetes Background

3. Insulin signaling Nature. 2001, 414:799-806 Insulin Receptor, IR Insulin Receptor Substrate, IRS AKT

4. Insulin resistance: a physiological condition where insulin becomes less effective at lowering blood glucose a common feature of many metabolic diseases Background

5. The DPP Research Group, NEJM,2002 Background Various strategies to treat insulin resistance

6. Background Branched-chain amino acids (BCAAs) L-leucine L-isoleucine L-valine

7. The role of leucine in insulin sensitivity Increased serum leucine level: * Improves the whole body glucose metabolism * Does not alter susceptibility to diet-Induced obesity J. Nutr. 139: 715–719, 2009. * Increases insulin resistance in models of obesity Cell Metab 9:311-326, 2009 Diabetes 54:2674-2684, 2005 Diabetes 56:1647-1654, 2007 ？

8. Metabolic diseases (insulin sensitivity/glucose metabolism) Nutrient (BCAAs) Genetic factors sensing Metabolism regulation X Research interest of our work

9. Outline Part Ⅰ The role of leucine deprivation in insulin sensitivity Part Ⅱ Effects of individual BCAAs on insulin sensitivity and glucose metabolism in mice Part Ⅲ Looking for new genes regulating insulin sensitivity by leucine deprivation model

10. The role of leucine deprivation in insulin sensitivity Part Ⅰ

11. Leucine deprivation Fat mass Lipolysis in WAT UCP1 in BAT CNS food intake Serum Insulin Blood Glucose Normal In Our Lab Cell Metab 5:103-114, 2007 Diabetes 59:17-25 ？ Part Ⅰ

12. Hypothesis Leucine deprivation may improve insulin sensitivity Part Ⅰ

13. Experimental Design control 85% control (-)leu control 0d Collect tissues Change different diets Mice were acclimated to control diet for 7 days Control Control (-)leu (-)leu Pair-fed Pair-fed 7d 14d Part Ⅰ

14. Blood Glucose (mg/dl) Result 1: (-) leu improves insulin sensitivity in vivo 0 50 100 150 200 # * Serum Insulin (ng/ml) 0 0.2 0.4 0.6 # * ctrl (-) leu pf HOMA-IR 0.5 1 1.5 0 # * Fasting * : p<0.05 vs. control #: p<0.05 vs. pf Part Ⅰ

15. Result 2: (-) leu improves insulin sensitivity in vivo 020406080100120 Time (min) ITT Blood Glucose (mg/dl) Time (min) 0 100 200 020406080100120 400 500 300 0 50 100 150 200 Blood Glucose (mg/dl) GTT * * # # * * ctrl(-) leupf * # # # * * * # * : p<0.05 vs. control #: p<0.05 vs. pf Part Ⅰ

16. Result 3: (-) leu improves insulin sensitivity in vivo - + p-IR t-IR p-AKT t-AKT ctrl Ins Liver WATMuscle Arbitrary Units * * 100 200 0 0 p-IR p-AKT 100 200 p-IR p-AKT p-IR p-AKT 300 0 100 300 ctrl + Ins (-) leu + Ins * * 200 * * (-) leu - + ctrl(-) leu - + ctrl(-) leu *: p<0.05 vs. control Part Ⅰ

17. Results 4 :(-) leu improves insulin sensitivity under insulin-resistance conditions C HFD (-) leu HFD Blood Glucose (mg/dl) 0 50 100 200 250 150 Time (min) Blood Glucose (mg/dl) 0 200 400 600 0 Time (min) ctrl db/db (-) leu db/db ctrl WT 40801202060 100 ITT * * * * # # # 040801202060100 * * * * ctrl * * * * * * *: p<0.05 vs. HFD #: p<0.05 vs. ctrl *: p<0.05 vs. ctrl db/db #: p<0.05 vs. ctrl WT * * Part Ⅰ

18. Summary 1 leucine deprivation improves insulin sensitivity under normal and insulin-resistant conditon Mechanisms ? Part Ⅰ

19. Nature 431:200-205, 2004 Role of mTOR/S6K1 signaling in insulin sensitivity mTOR ： mammalian target of rapamycin S6K1 ： ribosomal protein S6 kinase 1 Part Ⅰ

20. Results 5 :(-) leu increases insulin sensitivity by decreasing mTOR/S6K1 signaling in vivo Liver p-S6K1 t-S6K1 p-S6 t-S6 p-mTOR t-mTOR ctrl (-) leu 0 50 100 150 200 Blood Glucose (mg/dl) 04080120 Time (min) ITT 2060100 + Ad-CA-S6K1 * * * - Ad-CA-S6K1 *: p<0.05 vs. control On a leucine-deficient diet A B Part Ⅰ

21. GCN2 General control nonderepressible (GCN)2 A serine protein kinase Function as a sensor for amino acid deprivation ● ● ● Leucine deprivation p-IR/p-IRS/p-AKT p-mTOR /p-S6K1 GCN2 ? Part Ⅰ

22. Results 6:(-) leu increases insulin sensitivity by activation of GCN2 p-GCN2 t-GCN2 Liver Arbitrary Units 0 100 200 * ctrl (-) leu 04080120 Time (min) 2060100 0 50 100 150 Blood Glucose (mg/dl) Gcn2 -/- ITT Gcn2 +/+ *: p<0.05 vs. control Part Ⅰ

23. AMPK AMP-activated protein kinase Energy sensor ● ● Target of many drugs ● Leucine deprivation p-IR/p-IRS/p-AKT p-mTOR /p-S6K1 GCN2 AMPK ? Part Ⅰ

24. Results 7 :(-)leu improves insulin sensitivity via activation of AMPK DN-AMPK Ins p-ACC - - - + ++ p-IR t-AMPK t-IR t-ACC t-IRS1 p-IRS1 (Tyr612) HepG2 p-AKT t-AKT 0 + DN-AMPK + Ins 100 Arbitrary Units 50 150 * * * p-IR p-IRS1 p-Akt HepG2 +leu -leu * p-AMPK t-AMPK Liver Arbitrary Units 0 100 200 * ctrl(-) leu - DN-AMPK + Ins *: p<0.05 vs. control Part Ⅰ

25. Summary2 Leucine deprivation increases hepatic insulin sensitivity via GCN2/mTOR/S6K1 and AMPK pathways model Leucine deprivation p-IR/p-IRS/p-AKT p-mTOR /p-S6K1 p-GCN2 p-AMPK Part Ⅰ

26. Conclusion One Elucidate the mechanisms underlying increased insulin sensitivity by leucine deprivation GCN2/mTOR/S6K1 and AMPK pathways Demonstrate a novel function for GCN2 in the regulation of insulin sensitivity ● ● Part Ⅰ The paper titled “leucine deprivation increases hepatic insulin sensitivity via GCN2/mTOR/S6K1 And AMPK pathways” was published in Diabetes, 60:746-756,2011.

27. L-isoleucine (ile) L-valine (val) How about the other two branched-chain amino acids ?

28. Effects of individual branched-chain amino acids on insulin sensitivity and glucose metabolism in mice Part Ⅱ

29. Results 8: (-)val and (-)ile improves insulin sensitivity in vivo 200 150 100 50 Time (min) 0 Blood Glucose (mg/dl) 04080120 ITT * * * * A B ctrl (-) val ctrl (-) ile Blood Glucose (mg/dl) Time (min) 0 100 200 04080120 400 500 300 GTT * * * * 0 100 200 400 500 300 Blood Glucose (mg/dl) Time (min) 04080120 GTT * * * * 0 50 100 150 200 Blood Glucose (mg/dl) Time (min) 04080120 * * * * ITT * * Part Ⅱ

30. Result 9: (-)val and (-)ile decrease mTOR/S6K1 and increase AMPK signaling Liver ctrl (-) Val p-S6K1 t-S6K1 p-S6 t-S6 p-mTOR t-mTOR 200 100 0 Liver ctrl (-) Ile p-S6K1 t-S6K1 p-S6 t-S6 p-mTOR t-mTOR Arbitrary Units * * * * * * 200 100 0 p-AMPK t-AMPK p-AMPK t-AMPK * p-s6k1 p-mTOR p-s6 p-AMPK * p-s6k1 p-mTOR p-s6 p-AMPK Part Ⅱ

31. 150 100 50 0 * * Blood Glucose (mg/dl) 0.4 0.2 0 Serum Insulin (ng/ml) * * 0.5 1 HOMA-IR 0 * * ctrl (-) val (-) ile Fasting Results 10: Effects of individual BCAA deficiency on glucose metabolism *: p<0.05 vs. control Part Ⅱ

32. 200 150 100 50 0 * * Blood Glucose (mg/dl) 1.5 1 0.5 Serum Insulin (ng/ml) 0 ** g6pase 0 100 200 Relative mRNA (%) Fed pepck * * * * Results 11: Effects of individual BCAA deficiency on glucose metabolism ctrl(-) val(-) ile （ - ） leu *: p<0.05 vs. control Part Ⅱ

33. Results 12: BCAA deprivation for 1 day improves whole body insulin sensitivity 200 Blood Glucose (mg/dl) 100 0 * * * * 200 Blood Glucose (mg/dl) 100 0 * * * * 200 Blood Glucose (mg/dl) 100 0 * * * * Time (min) 04080120 Time (min) 04080120 Time (min) 04080120 (-) leu(-) val(-) ile ctrl(-) BCAA *: p<0.05 vs. control Part Ⅱ

34. Conclusion Two Leucine deprivation represents a general effect of BCAAs on regulation of insulin sensitivity ● ● The effect of BCAAs deprivation differs in glucose metabolism Part Ⅱ The paper titled “Effects of individual branched-chain amino acids deprivation on insulin sensitivity and glucose metabolism in mice ” was published in Metabolism, 2014.

35. Looking for new genes regulating insulin sensitivity by leucine deprivation model (gene chip) Part Ⅲ

36. Hepatic Gene Chip of lecine-deprived male mice 0 200 400 600 800 PrlR mRNA level * liver con(-) leu （ - ） leu/con Gene number Upregulate more than twice 984 Downregulate more than twice 1163 Prolactin receptor (PRLR) Part Ⅲ

37. Prolactin Receptor (PRLR) Prolactin ： A hormone best known for its role in lactation. PRLR: Present in nearly all organs and tissues. Numerous biological functions of PRLR have been identified. (Regulates glucose levels by modulating the secretion of insulin ） a direct effect on insulin sensitivity ？ Part Ⅲ

38. working model PRLR p-STAT5 p-IR / p-AKT db/db Insulin sensitivity Normal condition Insulin resistance Insulin sensitivity Leucine deprivation GCN2/ mTOR/S6K Part Ⅲ

39. Conclusion Three Identify a novel function for hepatic PRLR in the regulation of insulin sensitivity Provide important insights in the nutritional regulation of PRLR expression ● ● Part Ⅲ The paper titled “PRLR Regulates Hepatic Insulin Sensitivity in Mice via STAT5” was published in Diabetes, 62:3103-3113,2013.

40. Our lab: Feifan Guo, Ph.D Chunxia Wang, Ph.D Junjie Yu, Ph.D Shanghai Chen Kai Li Hao Liu Yajie Guo Jiali Deng Yuzhong Xiao Yalan Deng Acknowledgments Previous members: Ying Cheng, PhD Qian Zhang, Ph.D Ying Du, Ph.D Tingting Xia, Ph.D Qingshu Meng Zhiying Huang Bin Liu Douglas cavener (Penn State Univ) Brad lowell (Harvard Med School) Xiang Gao (Nanjing Univ) Yong Liu (INS, CAS) Hongguang Sheng (CAS clinical center) Houkai Li, PhD Ziquan Li, Ph.D