1. ADIPONECTIN  Organic product?  Goal/understand customers  What it is and What it does..  How and Where it acts..  When is it elevated and when decreased…  Who does and Why they would want to measure Adiponectin?  Relevant data. Collin Shaw ALPCO Diagnostics Sales Manager (Diabetes & Obesity Product focus) cshaw@alpco.com www.alpco.com Publications with ‘Adiponectin’ in Abstract- 2008 to October, 2009= 2,344

2. Adiponectin- (also referred to as ADP, Ad, GBP-28, apM1, AdipoQ and Acrp30)…  acts in an endocrine manner and is exclusively secreted from adipocytes (fat cells) into the bloodstream- where it accounts for approximately 0.01% of all plasma protein at around 5-10 μg/mL  exists as multiple isoforms in circulation, self-associates into larger structures - What it is- Adiponectin, overview ADP  The sum of which is referred to as TOTAL ADIPONECTIN TOTAL ADIPONECTIN (ADP or Ad) = S-S + Alb ( ) + + Globular ? ADIPONECTIN (gADP or gAd) * H. Ebinuma, O. Sato, et al. Sekisui

3. TRIMERIC ADP (LMW)- The basic building block of circulating ADP, Trimeric ADP (aka-LMW) is formed through hydrophobic interactions of individual full length ADP monomers. Hexameric ADP (MMW)- Trimeric ADP is further assembled into Hexameric ADP. Trimer += )( Hexamer Hexameric ADP=6 Monomer ++ = )( Trimeric ADP=3 Alb Albumin Alb += )( Trimer Albumin Bound Trimer=3+Albumin HMW ADP- Hexameric ADP is further assembled into multiple HMW species (12, 18, 24, 30, 36..?) HMW ADP (12=DoDecamer) ( Hexamer += S-S ) + HMW-ADP (12) S-S = ) HMW ADP (18=HexaDoDecamer ( Hexamer TOTAL ADIPONECTIN (ADP or Ad) = S- S + Al b ( ) + + What it is - Adiponectin, isoforms & Nomenclature

4. o ADP has extensive beneficial actions on multiple obesity, diabetic and cardiovascular dysfunctions, through its anti-inflammatory effects and actions on metabolic processing of glucose regulation and fatty acid catabolism. o Multiple ways ADP controls & regulate energy metabolism o Regulate glucose flux - o ↑ insulin sensitivity (Si)- AMPkinase o ↑ glucose uptake, ↓ gluconeogenesis o ↑ GSIS, ↑βC mass/function o adjust lipid catabolism- o ↑ β-oxidation (FA), triglyceride clearance o anti-inflammatory activity- o ↓ TNFα & other ProInflammatory cytokines o ↓PAI-1 production & Activity o ─ ┤ βC apoptosis (in-vitro) o protection from endothelial dysfunction o ↑ NO production, o ─ ┤ cell adhesion molecules, ↓ scavenger receptor expression ↓Cholesterol uptake, o ↓ Endothelial cell necrosis- important facet of atherosclerotic formation o Control Satiety o ↑ Satiety, weight loss- weight reduction effects (some via the brain) o Most biological effects of adiponectin are mediated by the activation of the ‘energy sensor’ AMPK (AMP activated protein kinase) as strong therapeutic target. o Thiazolidinediones (TZD’s) are a class of peroxisome proliferator-activated receptor agonists (PPPARγ) act primarily to increase the secretion of HMW Adiponectin o Rosiglitazone → ↑ ↑ [HMW ADP] + ↓ [Glucose] What it does- Adiponectin is a Good Adipokine. What it does- Adiponectin is a Good Adipokine.

5. o ADP has protective effects on multiple target tissues. o Sk. Muscle, Adipose Tissue, Brain, Heart/Vasculature, Beta Cell, …) Low adiponectin level yields insulin resistance and directly promotes atherosclerosis. Low Adiponectin level yields and deteriorates metabolic disease. ↓ [ADP] &/or ↓ [HMW ADP] Genetic factors ADP Gene polymorphisms Environmental Factors Environmental factors leading to obesity (High fat diet/ lack of exercise ） Inflammation, Insulin Resistance, type 2 diabetes atherosclerosis What it does- Adiponectin is a Good Adipokine. Islet/βC- ↓βC functional Mass ↓GSIS, ↑Apoptosis CNS ↑Food Intake Liver- Regul ate HGP NAFLD, Hep.Stotosis Sk. Muscle- Ins. Resistance ↑Lipid accumulation Vasculature- HypTx (BV constriction) EndoDysf, Thrombosis, Arthrosclerosis, CHF/CHD, Fat- ProInflammatory Cytokines (IL-6, IL-1β, TNFα, MCP-1….) Resistin, Leptin, Visfatin, Hypertorphy, ↑FFA production

6.  Adiponectin binds to multiple receptors, although their relative abundance varies in different target tissues  Most biological effects of adiponectin are mediated by the activation of the ‘energy sensor’ AMPK (AMP-activated protein kinase is a s strong therapeutic target) How & Where it acts- Adiponectin, Major receptors & function 1)AdipoR1 (adiponectin receptor 1) → AMPkinase >sk. muscle, coronary arterioles, aorta.. 2)AdipoR2 (adiponectin receptor 2) → AMPkinase >mostly Liver, some in sk. muscle, arterioles, ca. muscle 3) T-cadherin (T-Cad) Adiponectin enhances the binding of APPL1 (key signaling molecule) which couples ADP receptor signaling to AMPK activation Different adiponectin forms have different affinities to T-Cad, ADPR1 and ADPR2 *T. Kadowaki, et al, JCI. 2006.

7. Who and Why measure ADP - Adiponectin Physiology- Who does and Why they would want to measure ADP?  Biotech/Pharma- How does our drug candidate work? Drug Discovery, Target/Lead Validation, Safety/Toxicity Departments, Clinical Studies…. Does a drug increase ADP (&/or HMW)? If so, good. If not, what else does it do. Therapeutically Measure ADP to evaluate efficacy & Safety of treatments (drug candidates) for treating DM, CVD &/or Metabolic Syndrome  Academic/B.S.R.- understand etiology of Metabolic Syndrome Use Multiple models (transgenic animal/cell line), KI/KO models…to study (patho)biology Since ADP is beneficial, how does their model change ADP levels. Study various known therapies to study and identify (if) a pathway involves ∆’s in ADP.

8. Who and Why measure ADP - Adiponectin Physiology-  Many studies have reported HMW ADP, ratio of multimers (e.g. HMW/Total) vs. total ADP, to be more relevant to the pathogenesis of type 2 diabetes, obesity, metabolic syndrome, and vascular disease.  HMW ADP, has a longer t1/2, is the most Active & may have most effects  The HMW form of adiponectin exhibits the highest AMP kinase phosphorylation  Evidence that HMW ADP has the major insulin-sensitizing effects of the multiple bioactive forms-  HMW levels, or its ratio index (HMWR), are more useful than the total adiponectin level for predicting insulin resistance and the development of metabolic syndrome  Elevated total [Adiponectin] is good. Higher [HMW Adiponectin] is even better.  Predictive of TIIDM. Levels of [Adiponectin] are Inversely correlated to the increase in pathology (a.k.a. ↓[ADP]=↑metabolic disease)  ↓ [Adiponectin] = ↑ TIIDM, Metabolic Syndrome and Obesity  ↓ [Adiponectin] = ↑ Coronary Artery Disease, Coronary Heart Disease

9.  Thiazolidinediones (TZD’s) (eg. Rosiglitizone) act primarily to increase the secretion of HMW Adiponectin. TZD’s are a class of peroxisome proliferator-activated receptor agonists (PPPARγ). JA Wagner, et al. Clinical Pharmacology and Therapeutics. 2009  M&M: TIIDM patients treated 26 wks of +/- PPAR γ.  PPAR γ Rx → ↑ ↑ [ADP] (total ADP)  ↓ [Glucose],↓ HbA1c, ↓ hematocrit, and  ↓ triglycerides, ↓ TC & LDL, ↑HDL-C,  ↑ in levels of blood urea nitrogen, creatinine Who and Why measure ADP- Adiponectin Physiology- Who and Why measure ADP- Adiponectin Physiology- Results: Changes in [ADP] at 6-8 wks of therapy, predict changes in HbA1c at 24-52 wks. “Early (6–8 weeks) increases in levels of adiponectin after treatment with PPAR agonists showed a negative correlation (r = −0.21, P < 0.0001) with subsequent changes in levels of HbA1”… “These analyses confirm previously demonstrated relationships between adiponectin levels and metabolic parameters and support the robust predictive utility of adiponectin across the spectrum of glucose tolerance.” Main Point: clinical trials utilize changes in HbA1c as the therapeutic endpoint, ADP predicts this outcome in 6 wks.

10. HMW ADP, ratio of multimers (e.g. HMW/Total) vs. total ADP, more relevant to the pathogenesis of insulin resistance, type 2 diabetes, obesity, metabolic syndrome, and vascular disease. *von Eynatten, M., et al. Atherosclerosis 2007. *Ebinuma, H., Kadowaki, T., et al. Diabetes Care 2006. Figure 1—ROC curves of plasma total adiponectin levels and HMWR values for the prediction of insulin resistance, defined as a HOMA-IR index 2.5 (n 171). The AUC for the HMWR values was significantly larger than that for plasma total adiponectin levels (0.713 [95% CI 0.620–0.805] vs. 0.615 [0.522– 0.708], P 0.0160). Fig. 2. ROC curves of ADP (total, HMW & HMWR)& predictive values for the extent of coronary atherosclerosis. Prediction of CAD extent was based on discrimination between subjects with moderate (Extent Score 75 percentile) coronary wall irregularities. HMW adiponectin: 0.673 [95%CI 0.600–0.746] vs. total adiponectin: 0.568 [95%CI 0.491–0.646], P < 0.001). HMW/total-adiponectin ratio: 0.718 [95%CI 0.644–0.792] vs. total ADP 0.568 [95%CI 0.491–0.646], P = 0.010). No statistically significant difference between the AUC for HMW adiponectin and the HMW/total-adiponectin ratio (P = 0.265). Relevant clinical Data HMW or HMW/Total > than Total ADP-

11.  Eg. Of measuring Adiponectin for Academic/Clinical/BSR- “HMW levels, or its ratio index (HMWR), are more useful than the total adiponectin level for predicting insulin resistance and the development of metabolic syndrome” MM. Swarbrick, PJ. Havel, et al. Diabetologia 2006.  M&M- Obese patients treated with Roux-en-Y Gastric Bypass Surgery  Measure [ADP] (and many others) before and after (t= 1mo & 12 mo) GBP.  RESULTS-  ↑ Insulin sensitivity (HOMA-IR),  @1 month, if measured total, would see no change in [ADP]  @12 mo ↑Total [ADP], ↑ ↑ [HMW ADP], even though ↓in over all fat mass  The increase of HMW, but not that of total adiponectin, predicted the relative decrease of insulin resistance (HMW: p=0.0044; total: p=0.0775, after adjustment for covariates, 12mo post GBP). Relevant clinical Data -HMW or HMW/Total > than Total ADP-

12. Mice are Good Model for Diabetes Research.  In mouse elevated total [Adiponectin] is good. Higher [HMW Adiponectin] is even better.  Mice exhibit similar sexual dimorphisms in adiponectin complex profiles (♀↑ HMW vs. ♂)  Trimer form does not appear in same ratio in mice.  Mice have similar clearance rates and clearance pathways for adiponectin  In mice, HMW adiponectin is positively correlated to insulin sensitivity and inversely correlated to T2DM  The relative levels of the higher-order structures are sexually dimorphic where females have increased proportions of Total and HMW. What it is- Adiponectin, overview *A. Xu, et al. JBC 2005

13. Mouse HMW & Total ELISA (Method Comparison)  HMW/Total Adiponectin ELISA Methods compared: 1) Gel filtration chromatography  ELISA Xu A, et al. 2005 Testosterone selectively reduces the high-molecular-weight form of adiponectin by inhibiting its secretion from adipocytes. J Biol Chem 280: 18073-80. *Fractionated sera is applied to the ELISA plate  Sample groups: o db/db mice + Rosiglitazone treatment (n=8) o db/db mice + vehicle (n=7) 2) Gel filtration chromatography  Western blot Schraw T, Scherer P, et al. 2008 Plasma adiponectin complexes have distinct biochemical characteristics. Endocrinology 149(5): 12270-82.  Total Adiponectin: o Commercial ELISA (Millipore) o Used to assign concentration to WB data for HMW (i.e. HMW concentration is extrapolated from Millipore ELISA data)  Groups: o db/db mice + Rosiglitazone (Rosi) treatment (n=6) o db/db mice + Vehicle (V) treatment (n=7) o Lean controls (LC) (n=8)

14. Mouse HMW & Total ELISA (Method Comparison) Results Method Comparison #1 M1) Gel filtration chromatography  ELISA Aimin Xu, U. Hong Kong (CONFIDENTIAL) Db/Db Mouse + Vehicle + TZD (Rosi) Results: Similar ↑total, HMW & HMW/Total Strong Correlations Standardization is an issue

15. Mouse HMW & Total ELISA (Method Comparison) Results Method Comparison #2 M2) Gel filtration chromatography  WB ratios, Fractions quantified by ELISA Db/Db Mouse +Rosi + Veh Lean Control Results: Similar Diff in ADP Rosi Tx- ↑total, HMW & HMW/Total Db/Db- ↓ total, HMW & HMW/Total Strong r 2 Correlation, not for HMW/Total Standardization is an issue Total ADP HMW ADP HMW/Total ADP ADP

16. Mouse HMW & Total ELISA (Method Comparison) Results Method Comparison #2

17. Studies 2 & 3: Gel/WB and Millipore ELISA vs ALPCO/Sekisui ELISA  Summary & Conclusions ALPCO/Sekisui ELISA and the Millipore ELISA show a good correlation for total adiponectin ALPCO/Sekisui ELISA and extrapolated WB data also show good correlation for HMW adiponectin Differential over-recovery of total and HMW by the ALPCO/Sekisui results in a less favorable correlation for HMW/Total adiponectin Likely causes include differences in antibody specificity and lack of standardization of calibration material Both the Gel/WB and the ALPCO/Sekisui ELISA show significant inter-group differences in HMW, Total, and HMW/Total Adiponectin, demonstrating the clinical utility of the ALPCO/Sekisui ELISA