1. ALYSSA HASTY, PHD ASSISTANT PROFESSOR MOLECULAR PHYSIOLOGY & BIOPHYSICS Metabolic Impact of Adipose Tissue

2. The Simplified Model

3. Obesity Trends Among U.S. Adults between 1985 and 2002 Definitions: Obesity: having a very high amount of body fat in relation to lean body mass, or Body Mass Index (BMI) of 30 or higher Body Mass Index (BMI): a measure of an adult’s weight in relation to his or her height, specifically the adult’s weight in kilograms divided by the square of his or her height in meters Definitions: Obesity: having a very high amount of body fat in relation to lean body mass, or Body Mass Index (BMI) of 30 or higher Body Mass Index (BMI): a measure of an adult’s weight in relation to his or her height, specifically the adult’s weight in kilograms divided by the square of his or her height in meters

4. Diseases Associated with Obesity Diabetes: 80% related to obesity Hypertension: prevalence is >40% in obesity Heart disease: 70% related to obesity Cancer: Obesity accounts for 15-20% of cancer- related deaths Death: Obese individuals have a 50-100% increased risk of death from all causes compared to lean individuals (most of this risk is due to cardiovascular disease)

5. Central obesity Glucose intolerance Hypertension Insulin resistance High TG Low HDL-C Small, dense LDL particles The Metabolic Syndrome © 1998 PPS

6. 0 5 10 15 20 25 30 CHDPrevious MIPrevious Stroke WITH Metabolic Syndrome All Subjects NGT IFG/IGT Type 2 Diabetes (n=3,928) (n=1,808) (n= 685) (n=1,430) Without Metabolic Syndrome 0 5 10 15 20 25 30 CHDPrevious MIPrevious Stroke Prevalence of CHD, MI, and Stroke in Relation to the Presence of Metabolic Syndrome Isomaa et al. 2001. Diabetes Care. 24:683-689. Prevalence

7. Traditional CHD Risk Factors Elevated Plasma Cholesterol Reduced Plasma HDL Levels Diabetes Hypertension Smoking Age Lipid Lowering only results in a relative risk reduction of 30% New Markers Inflammatory Adipokines

8. New Inflammatory Markers to assess Risk CRP IL-6 TNF  Serum Amyloid A  Non-esterified fatty acids Adipose tissue is an important source of inflammatory cytokine production

9. White Adipose Tissue (WAT) Many different adipose tissue beds throughout the whole organism Many distinct cell types: adipocytes, pre- adipocytes, fibroblasts, macrophages, vascular cells Heterogeneous metabolic capabilities, depending on visceral or subcutaneous location of fat depot Secrete adipokines with systemic effects

10. J. Magn. Reson. Imaging Vol.21, 4 Pages: 455-462 Gender Differences in Adipose Tissue Distribution Female Male Original Image SCAT Subcutaneous Adipose Tissue HIGHLIGHTED WHITE VAT Visceral Adipose Tissue HIGHLIGHTED WHITE

11. White Adipose Tissue WAT is comprised of 2 Fractions: 1) adipocytes 2) SVF which consists of:  preadipocytes  endothelial cells  macrophages WAT is central to energy storage in the body and the mobilization of this energy store is highly regulated.

12. Preadipocyte Mature adipocyte http://www.hsph.harvard.edu/GSHLAB/adipos.html Basic Adipose Tissue Expansion

13. Adipocyte Growth: Hypertrophy vs. Hyperplasia  Hypertrophy (increase in size) “Lipogenesis”  Result of excess triglyceride accumulation in existing adipocytes  Hyperplasia (increase in number)  ‘adipogenesis’ results from the recruitment of new adipocytes from precursor cells in adipose tissue and involves proliferation and differentiation Hausman et al. Obesity Reviews (2001) 2, 239-254

14. Hormones, Adipokines, enzymes, molecules and other factors reportedly associated with Adipose Tissue  -1 acid glycoprotein (AGP) 3  -hydroxysteroid dehydrogenase (3  HSD) 3  -HSD 5  reductase 7  hydroxylase 11  HSD 17  HSD Acylation-stimulatin protein (ASP or C3adesArg) Adenosine Adipocyte differentiation factor (ADF) Adipogenin Adiponectin Adiponutrin Adipohilin (adipose differentiation-related protein [ADRP]) Adipose protein 2 (aP2 or adipocyte-specific fatty acid-binding protein, otherwise known as 422 protein) Adipose triglyceride lipase (ATGL) Adipsin (ADN; complement factor D, C3 convertase activator and properdin factor D) Agouti protein Androgens Angiotensin I and II Angiotensin-converting enzyme (ACE) Angiotensinogen (AGT) Annexin (lipocortin) Apelin Aoplipoproteins E, C1 and D Aquaporin 7 (AQP7) Autotaxin (lyosphospholipase D) Bone morphogenic protein (BMP) C-reactive protein (CRP) Calumenin Calvasculin Cathepsin D and G Ceramide Cholesteryl ester transfer protein (CETP) Chymase Collagen Type VI  3 Complement factor C3 and B Cytochrome p450-dependent aromatase (P450arom) E2F proteins Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 Eicosanoids Endothelin Entactin/nidogen Eotaxin Epidermal growth factor Estrogen Fasting-induced adipose factor Fatty acid translocase Fatty acid transport protein Fibroblast growth factor Free Fatty Acids Galectin-12 Gelsolin Glucose transporter 4 Glutamine Glycerol Haptoglobin Hippocampal cholinergic neurostimulating peptide Hormone-sensitive lipase Insulin-like growth factor Insulin-like growth factor binding protein Interleukin-1, -6, -8, and -10 Interleukin-1 receptor antagonist Lactate Leptin Lipin Lipocalins Lipoprotein lipase Lysophospholipid Macrophage migration inhibitory factor Metalloproteases Metallothionein Monobutyrin Monocyte chemoattractant protein-1 Necdin Nerve growth factor Neuronatin Nitric oxide synthase Nuclear factors Omentin Osteonectin (secreted protein, acidic and rich in cystein/SPARC) Pentraxin-3 p85phosphatidylinositol 3-kinase Perilipins Phosphoenolpyruvate carboxykinase Phospholipid transfer protein Pigment epithelium-derived factor Plasminogen activator inhibitor Pre-adipocyte factor-1 Prolactin Protein kinases Protease inhibitors (e.g., cystatin C and Colligin-1) Prostaglandin E, I2 and F2 prostacyclins (PGI2/PGF2) Regulated on activation, normal T-cell expressed and secreted (RANTES) Renin Resistin (FIZZ3 or serine/cystein-rich adipocyte-specific secretory factor [ADSF]) Resistin-like molecules Retinol Retinol-binding protein Sergin protease inhibitors Serum amyloid A Stearoyl-CoA desaturase Stromal cell-derived factors (e.g., stromal cell-derived factor 1 precursor or pre-B growth stimulating factor) Tissue factor Tonin Transforming growth factor- Tumor necrosis factor- UDP-glucuronosyltransferase A242B15 Uncoupling proteins Vascular endothelial growth Visceral adipose tissue-derived serpin Visfatin (pre- cell colony-enhancing factor [PBEF]) Bays H & Ballantyne C; Future Lipidol 2006; 1(4): 389-420

15. Free Fatty Acids (FFA) Adipose tissue serves as a buffer for FFA levels following feeding (similar to the way liver buffers blood glucose levels) FFA are released from WAT when systemic energy needs are not being met FFA levels are a predictor of future development of type 2 diabetes

16. Effects of elevated plasma FFA levels Insulin resistance in muscle Inhibition of normal function of insulin to suppress hepatic glucose production Impair insulin-stimulated glucose uptake FFA are substrate for hepatic triglyceride synthesis leading to increased plasma VLDL and triglyceride levels Impair endothelial function

17. Control of Release of FFA From White Adipose Tissue WAT Diet Lipid storage Lipolysis Starvation Insulin Free Fatty Acid Insulin Resistance Liver Triglycerides Phospholipids Ketone bodies HypertriglyceridemiaAtherosclerosis

18. Secretory Products of Adipose Tissue Adipose Tissue IGF-1 IGFBP TNF-α Interleukins TGF β FGF EGF Fatty Acids Lactate Adenosine Prostaglandins Glutamine Unknown Factors PAI-1 Angiotensinogen Ang II Estrogen Adiponectin Resistin Bone Morphogenic Protein Adapted from: Roth, J, et al, Obesity Research, Vol 12, supplement Nov 2004:88S-101S Leptin

19. Adipokines Vascular Disease Related  Angiotensinogen  PAI-1 Insulin Resistance Related  ASP (Acylation-stimulating protein)  TNF   IL-6  Resistin  Leptin  Adiponectin

20. Angiotensinogen (AGE) Links obesity with hypertension Positive correlation of blood pressure with AGE levels Primarily produced in liver, but also by WAT Deficiency partially protects against diet-induced obesity

21. Plasminogen Activator Inhibitor 1 (PAI-1) Impairment of fibrinolytic system contributes to cardiovascular complications of obesity WAT is main tissue source of PAI-1 Produced by pre-adipocytes, primarily in visceral WAT Acts to inhibit fibrinolysis (is pro-thrombotic) Also influences cell migration and angiogenesis Could impair pre-adipocyte migration leading to WAT growth

22. TNF  Proinflammatory cytokine Produced by adipocytes and macrophages in WAT Over-expressed in obesity Link between TNF  and insulin resistance Alters insulin signaling and MAPK pathways in vitro and in vivo

23. IL-6 10-30% of circulating IL-6 is from WAT Highly correlated with body mass and inversely related to insulin sensitivity Alters insulin signaling in the liver IL-6 KO mice develop mature-onset obesity and glucose intolerance

24. Resistin Discovered in 2001 Expressed in adipocytes in mice and in macrophages in humans Increased in obesity Recombinant resistin  Promotes insulin resistance in mice  Decreased insulin stimulated glucose uptake in WAT

25. Visfatin Discovered in 2004 Specifically expressed in visceral fat as opposed to subcutaneous fat Binds to insulin receptor on an allosteric site to insulin Can mimic insulin in down-stream insulin signaling pathways Plasma levels are 1/100 th level of insulin and not controlled by nutritional status These original findings were not reproducible, and the paper was later recalled!

26. Leptin Secreted by adipocytes Regulates food intake – satiety factor Leptin receptor is expressed in hypothalamus

27. ob/ob and db/db mouse models Spontaneous mutations first noted in 1950 morbid obesity (3x normal weight) hyperinsulinemia (50-fold increase) hyperglycemia Infertile slightly increased cholesterol

28. Leptin and Leptin Receptor F Both ob/ob and db/db mice originally developed from spontaneous mutations F The genes mutated in these mice were later identified as leptin and its receptor F ob/ob and db/db mice are obese, hyperphagic, hyperglycemic, and hyperinsulinemic and are commonly used models for studies of diabetes ob/oblittermate

29. Parabiosis: ob/ob with wild type Wild Typeob/ob

30. Parabiosis: ob/ob with wild type Wild typeOb/ob Suppressed weight gain in ob/ob mouse

31. Parabiosis between wt and db/db Wild typedb/db

32. Parabiosis between wt and db/db Wild typedb/db

33. Parabiosis between wt and db/db Wild typedb/db

34. Parabiosis between wt and db/db Wild typedb/dbRIP

35. 1994: Cloning of Leptin 167 aa protein belonging to cytokine family Circulates free or bound soluble receptor Expressed in WAT, stomach and placenta 84% homology between mouse and human leptin Leptin administration to ob/ob mice alleviates all aspect associated with the deficiency in mice

36. Possible Physiologic Roles of Leptin Obesity Anorexia Diabetes Reproduction Bone Mass Immune System Glomerulosclerosis Hematopoeisis Aging sweet-sensing modulator Angiogenic activity Hypertension

37. Adiponectin (Acrp 30, AdipoQ) Discovered in mid-1990’s Protein highly expressed in adipocytes and circulates at high concentrations Collagenous tail and globular head Plasma concentrations are reduced in obesity and insulin resistance TNF  and IL-6 inhibit adiponectin expression Administration of recombinant adiponectin ameliorates IR in obese and lipodystrophic mice Adiponectin is anti-atherogenic

39. Mobilization of Triglycerides Stored in Adipose Tissue Low levels of glucose in the blood trigger the mobilization of triglycerides through the action of epinephrine and glucagon. cAMP pathway activate hormone sensitive lipase to cause hydrolysis of triglycerides into glycerol and FFA

40. Modulators of Triglyceride Synthesis and Storage  Insulin – promotes conversion of carbohydrates into triglycerides  Epinephrine: activates release of fatty acids from adipose tissue  Glucagon: stimulates release of fatty acids from adipose tissue  Pituitary Growth Hormone  Adrenal Cortical Hormones

41. Lipoprotein Lipase LPL: lipolytic activity in the capillary bed; hydrolyzes NEFA from lipoproteins (chylomicrons and VLDL) Secreted by adipocytes, macrophages, muscle but active in capillary endothelial beds Has catalytic activity, but also functions as ligand for anchoring of lipoproteins to cell surface

42. Hormone Sensitive Lipase lipolytic activity inside adipocytes releases NEFA from storage when energy is needed Hydrolyses TG but also cholesteryl esters, retinyl esters and steroid esters

43. Triglyceride  Glycerol backbone with 3 fatty acid chains  Fatty acids can be similar or varied  Stored in cytosol of adipocytes  Carbon atoms are more reduced than sugars – thus provide more energy

44. Together, ATGL and HSL are responsible for >95% of TG hydrolysis from WAT Lipolysis of TG in WAT

46. Perilipins Found within phospholipid monolayer surrounding neutral lipid containing lipid vacuole Phosphorylated by PKA on multiple residues Prevent lipolysis under basal conditions

47. Fatty Acid Binding Proteins (FABP: ap2 and mal-1) Expressed in WAT, liver, intestine, heart, and brain Sequester fatty acids in cytosol to protect cell from harmful effects of intracellular-FFA Interact with HSL to favor translocation of lipid from cytosol to lipid droplet Ap2-/- mice do develop diet-induced obesity, but do not develop IR or diabetes

48. Hormone Stimulated Lipolysis Paradigm Lipolytic stimulation leads to phosphorylation of HSL and perilipin by PKA Phosphorylation of perilipin causes them to lose their lipolysis-blocking capability Phosphorylation of HSL promotes its translocation from the cytosol to the lipid droplet Required for maximal TG storage in WAT

49. Yeaman, Biochem. J 379:11

50. Osuga. 2000. PNAS 97:787 Hormone Sensitive Lipase deficient mice Mice do NOT become obese Mice have increased Diacylglycerol in their WAT and muscle Adipocytes in BAT were enlarged (5-fold) BAT mass was enlarged Males were sterile WAT retained 40% of TG lipase activity

51. Perilipin Knockout mice Decreased adipose tissue mass High levels of basal lipolysis Resistant to diet- induced obesity Javier Martinez-Botas Nature Genetics 26, 474 - 479 (2000)

52. Mechanism for Hormone-mediated lipolysis Hormones include catecholamines, adrenaline, and noradrenaline Receptors are called G-protein coupled receptors called β-adrenergic receptors When activated, they transmit signals to adenylyl cyclase leading to cAMP production cAMP activates PKA PKA phosphorylates HSL and perilipin

53. Gs/cAMP/PKA/HSL Pathway to Lipolysis

54. Kimmel, Biochimie 87: 45-49Biochimie

55. Flex Time: Adipose Tissue Macrophages