1. Resistance to Leptin Action is the Major Determinant of Hepatic Triglycerides Accumulation in vivo. By Sigal Fishman, MD

2. Insulin resistance/Syndrome X Obesity/abdominal obesity Diabetes (Type 2) Hyperlipidemia (low HDL) Hypertension (increased AGT) Thrombosis (increased PAI-1) Inflammation (Cytokines) NAFLD Leptin resistance (High leptin; develops with insulin resistance)

3. Resistance to both, insulin and leptin action may be implicated in accumulation of hepatic TGResistance to both, insulin and leptin action may be implicated in accumulation of hepatic TG Which one is the major determinant??Which one is the major determinant??

5. Leptin role:

6. Browning JD JCI 2004

7. Insulin action on glucose metabolism PeripheralHepatic glycogenolysis Gluco- neogenesis Glycogen synthesis Glycolysis

8. Clamp studies Hyperinsulinemic clamp assesses peripheral insulin action and hepatic Insulin infused at a fixed rate (3mU/kg/min) In insulin sensitive states, glucose is driven into the cells, higher rate of glucose infusion required to maintain euglycemia In insulin resistant states, the amount of glucose required to maintain euglycemia is much lower. Glucose fluxes in to the peripheral tissues assessed by the use of tracers.

9. Clamp studies Blood glucose is a balance between Tissue glucose uptake (RD) and Hepatic glucose production (HGP)+ Glucose infusion rate (GIR). When hepatic glucose production goes down, glucose infusion rate should be increased to maintain euglycemia.

10. Liver Muscle 25% glucose Insulin action + _ Diet a. Glycolysis b. Glycogen syntase a. Glycogenolysis b. Gluconeogenesis

11. Insulin glucose isotopes infusion Blood Sample

12. VF- IV catheter Day of the clampRecoveryDay -3Day 0 Tritiated glucose infusion Insulin 3mu/kg/min+glu Somatostatin H2OH2O 3

13. Old Young r=-0.57 p=0.01 Relationship between hepatic TG and hepatic insulin action

14. Leptin reduces hepatic TG and improves hepatic insulin sensitivity in young lean rats 1

15. Effect of chronic leptin delivery on the hepatic TG P<0.01 vs. all *

16. Effect of chronic leptin delivery on the insulin-mediated suppression of HGP P<0.001 vs. all

17. Role for SCD-1 in mediating leptin action: Monounsaturated Fatty acid Saturated Fatty Acyl CoA Oxidation TG VLDL Storage Acetyl-CoAMalonyl-CoA CPT-1 SCD-1 ACC Cohen P. J Nutr 2004

18. Effect of chronic leptin delivery on hepatic SCD-1 expression p<0.001 vs. young SCD-1 young leptin pair-fed 0 0.5 1 1.5

19. Effect of chronic leptin delivery on hepatic ACC-1 expression p<0.001 vs. young ACC-1 0 0.2 0.4 0.6 0.8 1 1.2 1.4 young leptin pair-fed

20. Effect of chronic leptin delivery on hepatic ACC2 expression P<0.05 vs. young ACC2 0 1 2 3 4 5 6 7 young leptin Pair-fed

21. Barzilai et al. JCI. 100:3105, 1997 Effect of chronic leptin delivery on hepatic Malonyl Co-A levels P<0.01 vs. all *

22. Jiang G. JCI 2005

23. Leptin: Improves hepatic insulin sensitivity. Decreases hepatic TG stores What happens in leptin resistant states?

24. Leptin’s effect on insulin suppression of glucose production in aging -100 -80 -60 -40 -20 0 YoungOld % Suppression Leptin vs. pair-fed *

25. TG levels after leptin administration in aging rats

26. Young– pair-fed Young– leptin Old– pair-fed Old – leptin 0 1 2 3 4 5 6 7 8 9 0246810 12 HGP (mg/kg/min) Hepatic TG Content ( mg/gr ) * # ** ## 1A 1B

27. Improvement in hepatic insulin action by visceral fat removal is associated with reduction in hepatic TG content in old obese rats…but also with improvement in leptin sensitivity 2

28. Epidydimal fat removal

29. Visceral fat Associated with insulin resistance, abnormal glucose tolerance and diabetes across all ages. Labile fat depot. VF is associated with increased risk of hypertension, thrombosis and dyslipidemias. VF, in adolescents, correlates with insulin resistance. Increased visceral fat seen in aging.

30. Are the fat depots biologically distinct? Sprague-Dawley rats were sacrificed after 12 hours fast. RNA was isolated from perinephric (visceral fat) and subcutaneous adipose tissues. Experiments were performed using rat genomic microarrays (RGU34A), a platform containing 9000 genes (Affymetrix, Santa Clara, CA). Results of gene array expression involving genes implicated in insulin resistance (PPAR- , leptin) or it’s syndrome (angiotensinogen and plasminogen activating inhibitor-1 {PAI-1}), were confirmed and quantified by real time PCR. Some of the genes that are involved in glucose metabolism but were not part of the gene array platform, such as Resistin and Acrp 30, were studied by real time PCR.

31. Out of approximately 8,000 full-length sequences and approximately 1,000 EST clusters 1660 were expressed 297 were up/down- regulated in each chip Atzmon et. Al Horm Metab Res. 2002; 34:622

32. Atzmon et. Al Horm Metab Res. 2002 34:622

33. Visceral and SC fat are biologically distinct  Visceral fat and subcutaneous fat are biologically distinct.  Some of the significant changes are in the expression of fat-derived peptides that may have a role in insulin resistance (PPAR, leptin, resistin, and adiponectin) or its syndrome (PAI-1 and AT) and in factors affecting body fat distribution (leptin,  adrenergic receptors, PPAR, IGF-1, GH).

34. Factors affecting expression of FDP Is there a role for nutrients in the expression of FDP? How do nutrients affect the two fat depots? Is the effect of nutrients on the two depots different? How do adipocytes “sense” excess nutrients?

35. FFA Hexosamine Biosynthetic Pathway Glucose Glucose-6-P Glc-1-P UDP-Glc Glycogen F -6-P Triose -P Glycolysis GlcN-6-P UDPGlcNAc GFAT 1-3% FFA Glucosamine Glycosylation sp1

36. SC 0 2 4 6 8 10 12 14 16 Saline Glucose Insulin GlcN+In PAI-1 25 30 35 40 45 50 Gene expression (adjusted by GAPDH) $@ $$ VF Nutrients, nutrient sensing, and induction of fat-derived peptides $$ $ $$$ Saline Glucose Insulin GlcN+In Resistin 0 10 20 30 40 50 60 Gene expression (adjusted by GAPDH) SCVF *@*@ Saline Glucose Insulin GlcN+In $$ @ $ Gene expression (adjusted by GAPDH) Leptin 0 2 4 6 8 10 12 SCVF $$ *@*@ Gene expression (adjusted by GAPDH) $$@ Saline Glucose Insulin GlcN+In Angiotensinogen 0 2 4 6 8 10 12 14 16 18 20 SCVF Gene expression (adjusted by GAPDH)  * $$ $@@ Saline Glucose Insulin GlcN+In TNF- 0 1 2 3 4 5 6 SCVF Gene expression (adjusted by GAPDH) $@ $ @ Saline Glucose Insulin GlcN+In Acrp30 0 2 4 6 8 10 12 14 16 SCVF Gene expression (adjusted by GAPDH) $$ *@*@ ADA, 2003

37. What are the biological reasons for the risks determined by increased VF? 1)VF expresses higher harmful fat-derived peptides. 2)Nutrients induce the expression of fat-derived peptides more in VF than other fat depots.

38. The metabolic syndrome Apo B Apo B Insulin Insulin Glucose Glucose TG TG Skeletal Muscle IR LPL LPL NEFAs NEFAs Liver HL HL NEFAs NEFAs Increased Visceral Fat Glucose FFA Nutrient sensing (HBP?) TNF-  Leptin PAI-1IL-6adiponectin TNF-  TNF-  AdiponectinResistinleptin

39. Insulin’s Suppression of Hepatic Glucose production After VF removal 0- 2- 4- 6- 8- 10- 12- HGP (mg/kg/min) * P<0.01 Old AL Old VF-

40. Reduction in Hepatic TG After VF removal 0 TG (mg/g liver) * P<0.01 vs. VF- Old AL Old VF- Old SC- 2- 4- 6- 8-

41. Plasma FFA levels in this model (old VF-) do not relate to changes in hepatic TG FFA (mmol/l) old VF- old AL Basal: 1.06 ± 0.13 0.83 ± 0.6 clamp: 0.77 ± 0.11 0.65 ± 0.1

42. Removal of visceral fat in rats results in coordinated changes in leptin level and leptin gene expression of SC fat. VF- Leptin ß-actin VF+ VF- Leptin (expression)-VF- 0 20 40 80 100 60 MSC * E P M SC 0 2 3 4 5 6 Leptin (ng/ml) * Old ALVF- 1 Decrease levels of hormone may be an index for improvement in its action!

43. VF removal might improved leptin sensitivity: Hormone level decreased,combined with reduction in SC gene expression No change in food intake! In this model we can not dissociate again, insulin action from leptin role

44. Improvement in hepatic insulin Does not improve hepatic TG content in leptin resistant ZDF rats 3

45. Removal of visceral fat improves glucose tolerance in Zucker diabetic rats. ZDVF+ZDVF- 4 6 8 10 12 Glucose (mM) * 4 6 8 10 12 14 16 EGP (mg/kg/min) * 0 25 50 75 100 125 Insulin (µU/ml) GIR 0 2.1  (mg/kg/min) ZDVF+ZDVF-

46. TG levels after VF extraction in ‘leptin resistance’ Zucker rats. Hepatic TG stores improves by leptin independent of insulin sensitivity

47. Old – sham operation Old – VF- 17 22 2 3 4 5 6 7 8 9 10 02468 12 HGP (mg/kg/min ) Hepatic TG Content (mg/gr) ZDF– sham operation ZDF – VF- * # $ ** ## 2A 2B

48. Summery Leptin and insulin resistance occur together with obesity and overfeeding. Leptin decreases hepatic TG stores by decreasing lipogenesis and increasing ß-oxidation.(leptin studies in young) With leptin resistance hepatic TG stores are not decreased (in old obese animals). Reversal of insulin resistance is associated with decreasing hepatic TG stores. (Visceral fat removal) It is the leptin action and not the insulin action that modulates hepatic TG stores (in Zucker NASHI rats).

49. Nir Barzilai Radhika Humuzumdar Gil Atzmon Xiao-Hui Ma Xiao-man Yang Hong qiang Liang Thank you!!!