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Proinsulin Physiology-  Goal is to understand researchers,  their work: their interests and needs  and why they would be interested in measuring PI.

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Presentation on theme: "Proinsulin Physiology-  Goal is to understand researchers,  their work: their interests and needs  and why they would be interested in measuring PI."— Presentation transcript:

1 Proinsulin Physiology-  Goal is to understand researchers,  their work: their interests and needs  and why they would be interested in measuring PI  SUMMARY,  Review of Insulin, to understand proinsulin significance  Where is Proinsulin and what it makes  What it is and what it makes..  How and Where it is secreted..  When is it elevated and when decreased…  Who does and Why they would want to measure?  Relevant data.

2 R&D insight and relevance of ALPCO’s new Biomarker assays SUMMARY Mouse & Rat Proinsulin and C-peptide  Significance as a biomarker. Physiology, Biochemistry and relevant data of Proinsulin (PI) and C-peptide (CP). In the healthy state, each PI molecule is processed to equally to CP and insulin. Because of this PI concentrations are low, except in diabetic patients, where inefficiencies in processing are evident. As a result, in humans, PI has been shown to be a strong predictor of type II diabetes. Proinsulin is elevated in TIIDM and is symptomatic of beta cell stress and as glucose intolerance progresses, the increase in PI concentration is greater than insulin concentration. Furthermore, as insulin resistance progresses, from normal glucose tolerance to impaired glucose tolerance and eventually to overt type II diabetes, where PI levels are increase. These increases in proinsulin levels are independent of, and relative to, insulin and/or c-peptide concentration. In addition to PI contribution to the etiology of the diabetes it has been suggested that elevated PI levels may be a predictor of cardiovascular disease and atherosclerosis, more so than Insulin concentrations. For example, PI has been shown to increase plasminogen activator inhibitor-1. More importantly, increased PI levels are associated with increased arterial wall thickness, acute myocardial infarction and heart disease.  Mouse and Rat Proinsulin ELISAs- With limited techniques to quantify circulating rodent proinsulin, relatively limited data has been published regarding rodent proinsulin concentrations. There are multiple barriers in the ability to easily and accurately measure rodent C-peptide and Proinsulin. Most notably, these barriers stem from the hyper-variation in the primary sequence of the two C-chain sequences produced from the two rodent insulin genes, as compared to human with only one insulin gene. A few Relevant publications and preliminary data from evaluations will be described.  Mouse and Rat C-peptide ELISAs- Because of the difference in circulating half life and hepatic insulin clearance, measuring C-peptide is often more useful to quantify beta cell function and/or functional mass without having to account for changes in insulin clearance. Quantifying c-peptide is also a useful biomarker when exogenous insulin is administered allowing the measurement of endogenous or de-novo secretion (beta cell function) in response to hyperglycemia, therapy and/or a secretagogue.  Mouse and Rat CP & PI R&D hurdles and resulting data- Measuring both Rat and Mouse I & II is imperative. Real Stimulation Indexes or Changes in [PI] or [CP] are questionable, unless the variability in –I vs. –II secretion AND the variability in assay cross reactivates to each of these forms can be controlled for. Linearity concerns resulting from variations in rat versus mouse matrix. Preliminary data to data Adiponectin, Mouse HMW and Total Adiponectin ELISA.  Significance as a biomarker. Physiology, Biochemistry and relevant data specificity is important in development and in application. Development and preliminary data from relevant models GLP-1, 7-36 (‘active’) and 7-36+9-36 (‘Total’) ELISA  Significance as a biomarker. Physiology, Biochemistry and relevant data Assay needs, sensitivity and specificity needs. Development and preliminary data from relevant models Collin Shaw, MS ALPCO Diagnostics cshaw@alpco.com www.alpco.com

3  Insulin is primarily responsible for blood glucose homeostasis, by sensing circulating [Glucose]  Upon elevation of [Glucose] in β-Cells, Insulin and C-peptide are released, in a 1:1 fashion… normally  Insulin binds to and is internalized by fat & muscle  Inhibits Glucagon production (from αCell) and acts on the liver directly.  Insulin cleared by liver Normally ~70-80% of pulse mass (↓↓ with pathology)  Regulating [Glucose], Carbohydrate metabolism and indirectly, lipid & protein metabolism - Proinsulin - Physiology Review- Insulin Regulates [Glucose] ↓ Glucose ↑ α- cell Glucagon β- cell Insulin Muscle Glu Transport GLUT4-Glycogneogenesis + Liver Glu Production: Glyconeogenesis Gluconeogenesis ­ + + Fat ↔ Glucose GLUT4 -Lipogenesis BetaOxidation Adipose ↓ [GLUCOSE] ↑ In circulation Food Intake Satiety HGO Regulate Islets function Many others ­

4  After processing, Insulin (INS) & C-peptide (CP) are then stored in vesicles, some docked to the ß-cell membrane  In response to ↑ [Glucose] = ↑ [G6P] → ↑ATP/ADP ― K + ATP channel → +∆Ψ → Ca ++channel ↑Ca ++ →  Vesicle Release of INS & CP to be secreted from the ß-cell  Normally equimolar release of C-peptide & Insulin concentrations (1:1)-  C-peptide has a longer half-life in circulation (T1/2= 30 min vs. 3min for Insulin) Secretion of- Insulin, C-peptide & Proinsulin ↑ ATP/ADP C-Peptide Insulin Proinsulin K+K+ K + ATP Passive GLUT transport Glucose Glucokinase 8mM Glu6-P Ca ++ + ∆ Ψ Ca ++

5 The pre-cursor molecule of insulin, proinsulin (PI), is synthesized in the Pancreas, by the ß-cells withing the Islets of Langerhans in Proinsulin= A-chain (insulin) + C-chain (c-peptide)+ B-chain (insulin) Upon leaving the Golgi, the 84 AA proinsulin molecule undergoes further intra-cellular processing, to form both C-peptide (33 AA length) and Insulin (51 AA), through the enzymatic activity of: 1)Prohormone Convertase 1/3 (PC1/3) preferentially cleaves at B/C Junction of PI (AA 32-33) PC1/3 Initiates process & is preferential 1 st cleavage PC1/3 is up-regulated by ↑[GLU]…through CPE? 2) Prohormone Convertase 2 (PC2) preferentially cleaves at A/C Junction of PI, (AA 65-66) PC2 is rate limiting. 3) Carboxypeptidase E or H (CPE/H) Removes 2x basic AA’s from Split Partially process Proinsulins. CPE expression is upstream & reqd. for PC1/3 & PC2 expression. T 1/2 Proinsulin Processing → INS+CP (in-vitro) Hu PI (~50’), Rat PI-I (23’) PI-II (32’) Y. Zambre, D. Pipeleers, et al. JCEM 1998 Intact PI vs. Partially processed PI (PP PI) vs. Total PI = PP + intact WHAT IT IS- Proinsulin precursor of C-peptide & Insulin β- cell Split-32-33- Des-64,65- Proinsulin CPE/H Split-65,66- Des- 31,32-Proinsulin Des-64,65-Proinsulin PC1/3 PC2 Proinsulin C A B Split-65,66-Proinsulin CPE/H PC2 Des-31,32-Proinsulin Split-32,33-Proinsulin CPE/H InsulinC-peptide Intact PI PP PI

6  Why devote resources to rodent Proinsulin ELISA?  As Insulin resistance progresses, β-Cells try to compensate to elevated insulin demand in the Fat, Muscle and liver by increasing Insulin secretion resulting in elevated circulating [Insulin]  ↑[FBG] = ↑ Insulin demand= β-Cell Hypertrophy/ Hyperplasia(?)  Eventually β-Cells failure & β-Cells apoptosis= ↓[Insulin]  Hyperproinsulinemia is a feature of human TIIDM and is among the early defects observed in its pathogenesis.  Dogma:  Healthy = PI/Insulin ~5%  Late TIIDM = PI/Insulin ~50-70% TIIDM Progression model- Insulin Resistance, Secretion & Proinsulin TIIDM [Insulin] Insulin Resistance Hyper-Proinsulinemia Model adapted from R. Kulkarni, Joslin. Harvard, MA.

7 Why Measure- Proinsulin Model of Progression from NGT→IGT →TIIDM C-Peptide Insulin Proinsulin NGT IGT TIIDM βC failure =↓ function &/or ↓ in Mass EElevated demand for Insulin results in increased β-Cell stress, resulting in β-Cell dysfunction, in-efficient processing of Proinsulin to Insulin and C-peptide, which is associated with the onset and progression of TIIDM.

8 ß-Cell- secretion Diabetes progression active Insulin Insulin- resistance Insulin Proinsulin Combined Secretion Disorder IIIIII Model Comliments of Andreas Pfützner, IKFE, Mainz Germany

9 Proinsulin → INS+ CP. Clinical evidence (TIIDM)  Clinical Relevance of measuring PI in Diabetes In the healthy state, each proinsulin (PI) molecule is processed to c-peptide and insulin. Because of this proinsulin concentrations are low, except in diabetic patients(5), where inefficiencies in processing are evident. As a result, in humans, PI has been shown to be a strong predictor of type II diabetes (3 Multiple studies to report elevated Proinsulin- ‘Consistent PI/INS ratios in TIIDM PT’s of 25%-50%’ 3 M. Røder, D. Porte, R. Schwartz, S.Kahn, et al. JCEM -1998  TIIDM pts have elevated fasting [Proinsulin]  33pM (TIIDM) vs. 9.4pM (CØ)  PI/Insulin Ratio more in TIIDM, more so than.  PI/I % = 35.9% (TIIDM) vs.12.8% (CØ)  AIRmax negatively correlated with PI/Insulin (r= - 0.74), [Glucose] (r= -0.058), [PI] (r= - 0.048)  Degree of elevation in PI/I% and resulting hyperglycemia are related to the severity of β-dysfunction (capability to secrete Insulin)

10 100 0 515 10 3020 25 90 80 70 60 Zeit [Jahre] Survival [%] [Intact Proinsulin] @ t=0 > 3,1 pmol/l (p<0.001) <1,7 pmol/l 1,7-3,1 pmol/l Zethelius et al., Circulation. 105:2153-8, 2002  Non-diabetic men, n = 874, observation period: 26.7 years  HPLC method measuring [Proinsulin] @ t=0  n=1000, 126 develop DM  Mortality rate of patients 27 years later in three categories of [proinsulin] at t=0  10% mortality at Low [iPI] vs. >30% mortality at elevated [iPI] Insulin Resistance, Secretory isorder & CVD Risk Slide addapted from Andreas Pfützner, IKFE, Mainz Germany

11 Proinsulin → INS+ CP. Clinical evidence (TIIDM)  Clinical Relevance of measuring PI in Diabetes GP 5 SM. Haffner, RR. Bowsher, et al. JCEM 1994  As glucose tolerance deteriorates from NGT → IGT → TIIDM ↑ [PI] (greater than ∆ in [insulin] in subjects  Fasting PI/I% increases with ↑ Metabolic Disorders  zero, 0.060; one, 0.086; two, 0.098; three, 0.177; four, 0.182; P < 0.001  ….

12  Clinical Relevance of measuring PI in Diabetes Proinsulin is elevated in TIIDM and is symptomatic of beta cell stress (3,4) and as glucose intolerance progresses the increase in Proinsulin concentration is greater than insulin concentration. Furthermore, as insulin resistance progresses, from normal glucose tolerance to impaired glucose tolerance and eventually to overt type II diabetes, where proinsulin levels are increase(5). These increases in proinsulin levels are independent of, and relative to, insulin and/or c-peptide concentration. C.McDonald, J.Shapiro. Transplantation 2004 Edmonton Protocol- 1 year post transplant (2004 )  ↓ [Total PI] in transplant pt’s vs. Controls  ↑ [Total PI] insulin dependant pts  ↑ [Total PI] in Trx pt’s if insulin independence lost  ? Assays no longer on market (Dako) A. Kippen, K. Rose, et al, JBC - 1997 isotope dilution assay (IDA) to determine precise insulin, C-peptide, TIIDM (vs CØ) had elevated Insulin (22 vs. 92pM), CP (335 vs. 821) & PI (6- 37pM) values PI/ Insulin Ratio 36% (TIIDM) vs. 24% (CØ) CP/ Insulin Ratio 17.6% (TIIDM) vs. 10.7% (CØ) have elevated fasting [Proinsulin] (33pM vs. 9.4 pM) Proinsulin → INS+ CP. Clinical evidence (TIIDM)

13  A. Klimek, et al, 2009 Am J of Transplantation 9:2119-25 Fig. B) ↑ PI/CP ratio in islet autotransplant (TP/CP) Fig. A) ↑ total PI / total PI+Insulin (TP/TP+I) Clinical Data as a markers- Proinsulin/ C-peptide  Pre-pancreatectomy vs 1 yr post-autotransplant:  PI/CP levels were ~6-fold higher after 1 yr, wide response range was very wide.  T2DM (n=?): PI/CP was ~2.6, which was 1.8 greater than nondiabetic controls (1.6 %) and similar to allotransplant recipients (2.4%).  Fig. 4) Autotransplant recipients receiving 10,000 IE/kg (2.2%, n=8) Discussion point:  PI/CP should be measured immediately following max stimulation of b-cell secretion to minimize effects of renal clearance.  Proinsulin kit: Total PI (Linco), 100% CR w/ des 31,32 islet autotransplantallotranspantnon-DM control n=12n=13n=17 PI/CP 8.9%2.4%1.4% TPI / Insulin+TPI 35.9%24.1%13.9% HbA1c (%) 7.1 5.4 f[Glucose] 5.57.75.1 [Insulin] pM 22.5 (1.6-134) 51.4 (11.5-106.6) 58.5 (24.1-124.2) [C-peptide] pM 126 (7.2-217.9) 695 (161-1157) 693 (373-1040) [Proinsulin] pM (total) 7.3 (0.3-82.3) 16.8 (5.5-28) 8.4 (4.0-21.8)

14 In-Vitro relevance and conflicting results- R.Carr, B. Ahren, et al. AJP Endo Met. 2002  M&M- Morbidly obese patients treated with bariatric surgery, 15% (3-5mo) and 25% (~1yr) weight reduction  Results @15% mo- 20% ↑ADPtotal / 15% WR and 35% ↑ADP total/25% weight loss  ↑ Insulin sensitivity (Euglycemic HyperInsulinemic Clamp) ↑ ß-cell function by the acute insulin (AIRarg)  PI & PI/Insulin ratios (Insulin (linco SW RIA), Proinsulin (Total Proinsulin RIA), Adiponectin (Linco RIA) Proinsulin → INS+ CP. Clinical evidence (TIIDM) M. Swarbrick, PJ Havel, et al. Diabetologia. 2008  M&M- Obese patients treated with Roux-en-Y Gastric Bypass Surgery  Results @12 mo  ↑ Insulin sensitivity (HOMA-IR), ↑Total [ADP], ↑ ↑ [HMW ADP].  No change in Proinsulin/Insulin Ratio (Linco RIA)

15  Clinical Relevance of measuring PI in CVD In addition to proinsulin contribution to the etiology of the diabetes it has been suggested that elevated Proinsulin levels may be a predictor of cardiovascular disease and atherosclerosis (6), more so than Insulin concentrations. For example, proinsulin has been shown to increase plasminogen activator inhibitor-1(PAI-1). More importantly, increased proinsulin levels are associated with increased arterial wall thickness, acute myocardial infarction and heart disease (7-11). Proinsulin → INS+ CP. Clinical evidence (CVD) 7 S.Haffner, M.Saad, et al. STROKE- 1998  OGTT, IMT, CCA, ICA  PI may represent a marker of atherosclerosis (Athrx) rather than a causal factor for Athrx….[PI] strong correlation to IMT.  Suggests to confirm CR of insulin assay for PI. 8* DJ Schneider, TK Nordt & BE Sobel. DIABETES 1992.  PI increases PAI-1 activity, attenuating endogenous fibrinolysis and accelerating atherosclerosis  Proinsulin Augments PAI-1 expression & activity.  PI= ↑PAI-1 mRNA expression &↑ endothelial cell PAI-I activity (In-vivo)  Not dependant on PI→ Insulin conversion or Insulin Receptor 6 Steven M. Haffner; Anthony J.G. Hanley, CIRCULATION - 2002  isotope

16  In-Vitro Relevance of measuring PI in Diabetes In-Vitro observations of elevated Proinsulin-  When culturing human Islets and Rat 1⁰ βC in low glucose (2.5mM) vs. high glucose (20mM)  >2x elevation in secretion of PI (ppPI /des 31-,32), from 2-3% (0.5%) to 5-6% (2-3%)  (Pipeleers, JCEM, 1996) suggest ↑ Pi release is not Processing defect, but just supply demand of new granules not processed yet  INS-1 Cell line has ↓ rates of PI → INS+CP conversion than Rat 1⁰ βC  (Halban, Biochem J, 1996) can increase INS PI conversion by ↑ PC (AdPC2 and Ad-PC1/3)  Proinsulin → INS+ CP. Clinical evidence (TIIDM)  ↑PI/Insulin in pts w/ TCF7L2 mutation Diabetologia 51:597-6001 2008, Diabetes 2008.

17  Minimal Rodent data for Proinsulin in Diabetes et al.  With limited techniques to quantify circulating rodent proinsulin, relatively limited data has been published regarding rodent proinsulin concentrations. A. Assmann, R. Kulkarni, American Diabetes Association 2008 Oral Abstract (215-OR)  Using 1⁰ Human Islets and beta-cells derived from mouse models lacking either insulin receptor (betaIRKO) or insulin receptor substrate-1 or -2 (IRSKO-1 or IRSKO-2)  Theory: Expression of Proinsulin processing enzymes (PC1/3 & PC2) are regulated by βcell Insulin Receptor.  Results from betaIRKO and IRSKO mice  ↑↑ Accumulation of intracellular Proinsulin (confocal microscopy) in both models  ↓ 50% in PC2 expression.  CPH expression ↓ ~70% (betaIRKO) and no residual in IRSKO-2.  No change in PC2 expression in IRS-1KO mice  ↑ βcell Stress markers in response to glucose, markers of ER stress are up-regulated. Proinsulin → INS+ CP. Clinical evidence (TIIDM)  Since there was no way to measure Mouse Insulin in circulation, the researchers at Harvard were not able to measure the changes in circulating proinsulin in these models.  Project is being recovered now that our Mouse Proinsulin assays are available.

18  Mouse and Rat Proinsulin assays fulfill a demand in Basic Science Research and hopefully reinvigorate the demand to measure human Proinsulin as a necessary biomarker/tool in studying and predicting the TIIDM, Obesity, CVD Milieu called Metabolic Syndrome. Proinsulin → INS+ CP. Clinical evidence (TIIDM)

19 C-peptide Physiology-  Goal is to understand researchers,  their work: their interests and needs  and why they would be interested in measuring C-peptide (CP)  SUMMARY,  to understand CP significance, Circulation of C-peptide relative to Insulin  Who does and Why they would want to measure …  Multiple forms of CP, PI & insulin in rodents vs. humans  Relevant data.  Publications with ‘C-peptide’ in Abstract- 2008 to October, 2009= 757

20 Why Measure- C-peptide relative to Insuiln C-Peptide Insulin Proinsulin  Insulin relative to C-peptide, or CP alone, used to asses Insulin resistance, Beta Cell Function &/or Hepatic Insulin clearance (reduced in TIIDM).  Half life: T 1/2 of Insulin is about 3 minutes, where as C-peptide has a much longer t 1/2 …30 minutes Liver clearance ~70% 1 st phase insulin secretion. Peripheral tissues (muscle/Fat) internalize quickly for activity. Because of the difference in circulating half life and hepatic insulin clearance, measuring C-peptide is often more useful to quantify beta cell function and/or functional mass without having to account for changes in insulin clearance. (liver clearance of Insulin, diminished in TIIDM/ IR) Liver Hepatic Portal Vein VC→ Heart→Arteries Sample collected, Veins mostly

21  Other reasons WHY MEASURE C-peptide:  Insulin Dependant DM (TIDM), Auto Immune destruction of β-Cells/Islets. Measure of residual β- cell function in TIDM, as C-peptide is an indicator of low amounts of beta cell release of insulin…any release of insulin would be cleared from circulation quickly,  Rodents, to confirm Stz treatment.  In TIDM, there are instances where interfering anti-insulin antibodies develop. Measuring C- peptide allows for the measurement of beta cell function with out the interference of auto- insulin Abs.  Quantifying c-peptide is also a useful biomarker when exogenous insulin is administered allowing the measurement of endogenous or de-novo secretion (beta cell function) in response to hyperglycemia, therapy and/or a secretagogue;  Eg. Differentiating therapeutic insulin from endogenous beta cell function.  Eg. Xeno-transplantation, allowing differentiation of de-novo beta cell function (human Islets transplanted in mice) vs. the residual endogenous mouse insulin secretion rate from mouse islets. Why Measure- C-peptide

22  Why Measure C-peptide Some Basic Principles on measuring in Rodents vs. Humans  Humans have 1x Insulin gene (INS) = 1x PI, 1x Ins. & 1x CP  Rodents have 2x INS Genes (INS-1 & INS-2) Rat = 2x PI, 2x Ins & 2x CP Mice = 2x PI, 2x Ins & 2x CP - Proinsulin precursor of C-peptide & Insulin The pre-cursor molecule of insulin, proinsulin (PI), is synthesized by the ß-cells of the Islets of Langerhans in the pancreas. The 84 amino acid proinsulin molecule undergoes further intra-cellular processing to form both C-peptide (33 amino acids in length) and insulin (51 amino acids). While these two hormones are secreted from the ß-cell in equimolar concentrations, c-peptide has a longer half-life in circulation (T1/2= 30 min vs. 3min).

23 - Proinsulin precursor of C-peptide & Insulin Split-32-33- Des- 64,65-Proinsulin CPE/H Split-65,66- Des- 31,32-Proinsulin Des-64,65-Proinsulin PC1/3 PC2 Proinsulin-II C A B Split-65,66- Proinsulin CPE/H PC2 Des-31,32- Proinsulin Split-32,33-Proinsulin CPE/H Insulin-IIC-peptide- II Split-32-33- Des- 64,65-Proinsulin CPE/H Split-65,66- Des- 31,32-Proinsulin Des-64,65-Proinsulin PC1/3 PC2 Proinsulin-I C A B Split-65,66- Proinsulin CPE/H PC2 Des-31,32-Proinsulin Split-32,33-Proinsulin CPE/H Insulin-IC-peptide-I

24  Why do we need a separate assay for Mouse CP and PI vs. Rat CP & PI? 1.HYPERVARIATION OF C-peptide 1⁰AA sequence- Most notably, these barriers stem from the hyper-variation in the primary sequence of the two C-peptide proteins produced from the two rodent insulin genes, as compared to human with only one insulin gene. 2.Rat and Mouse Insulin I & II are relatively similar in primary sequences as compared to the Mouse and Rat C-peptide I & II and the precursor Proinsulin I & II. Conserved and not so conserved- 1⁰AA Sequence of Insulin and C-peptide Conserved and not so conserved- 1⁰AA Sequence of Insulin and C-peptide.

25  Conserved and not so conserved- 1⁰AA Sequence of Insulin and C-peptide.  Insulin 1⁰AA sequence is a highly conserved sequence over mammals, reptiles, birds & fish, testament to function. C-Peptide demonstrates relative hyper variability 1⁰AA Sequence of Insulin and C-peptide 1⁰AA Sequence of Insulin and C-peptide.

26  Why do we need a separate assay for Mouse CP and PI vs. Rat CP & PI? 1.HYPERVARIATION OF mouse and rat C-peptide 1⁰AA sequence…. 2.Regulation & secretion of Rodent INS-1 & INS-2 products (PI, CP & Ins) are not equimolar Transcription- Since INS-1 & INS-2 are different genes, they have different promoters to regulate gene transcription. Mostly Translational regulation- Secretion products from rodent INS-1 & INS-2 (PI, CP & INS) are regulated at the translational level. Fetal life Dogma of which is more dominant (I or II) opposite from mouse to rat. Mouse II ~70% vs. mouse I ~30% Rat I ~ 70% vs. Rat II ~30% Embryonic Life Relative transcription and secretion of MS and Rt INS-I vs. II.(Steiner PNAS 1993) MS PI-II/TP ranged between 27-61% (e8.5-e14.5) MS PI-1/TP ranged between 39-73% (e8.5-e14.5) Conserved and not so conserved- 1⁰AA Sequence of Insulin and C-peptide Conserved and not so conserved- 1⁰AA Sequence of Insulin and C-peptide.

27  Measuring both Rat and Mouse I & II is imperative.  If not measuring Total relatively equal, the baseline vales as well as changes (upon Secretion) can interfere with experimental results Conserved and not so conserved- 1⁰AA Sequence of Insulin and C-peptide Conserved and not so conserved- 1⁰AA Sequence of Insulin and C-peptide. 100% CR w/ -I & -II Rodent- I Rodent -II Total [PI & CP] Glu. Stim. Index (βC Function) Fast [PI] pM 20+ =40 [CP] pM 200+ =400 Equal Change in -I and -II GSIS [PI] pM 80+ =1603.0 [CP] pM 800+ =16003.0 Non equimolar Change in -I and -II secretion, not equal to total GSIS [PI] pM 20+200=2204.5 [CP] pM 200+2000=22004.5 Non equimolar Change in -I vs -II, but total is same GSIS [PI] pM 140+20=1603.0 [CP] pM 1400+200=16003.0 50% CR w/ -I & 100% CR w/ -II Total [PI & CP] Glu. Stim. Index (βC Function) % Diff vs. total [PI & CP] 30 -25% 300 -25% 1203.0-25% 12003.0-25% 100% CR w/ -I & 15% CR w/ -II Total [PI & CP] Glu. Stim. Index (βC Function) % Diff vs. total 23 -43% 230 -43% 923.0-43% 9203.0-43% 902.0-44%1435.2-11% 9002.0-44%14305.2-11% 2106.0-5%501.2-77% 21006.0-5%5001.2-77%

28  A. Klimek, et al, 2009 Am J of Transplantation 9:2119-25  Fig. B) ↑ PI/CP ratio in islet autotransplant (TP/CP)  Fig. A) ↑ total PI / total PI+Insulin (TP/TP+I) Clinical Data as a markers- Proinsulin/ C-peptide  Fig. 4) Autotransplant recipients receiving 10,000 IE/kg (2.2%, n=8) Discussion point:  PI/CP should be measured immediately following max stimulation of b-cell secretion to minimize effects of renal clearance.  Proinsulin kit: Total PI (Linco), 100% CR w/ des 31,32 islet autotransplantallotranspantnon-DM control n=12n=13n=17 PI/CP 8.9%2.4%1.4% TPI / Insulin+TPI 35.9%24.1%13.9% HbA1c (%) 7.1 5.4 f[Glucose] 5.57.75.1 [Insulin] pM 22.5 (1.6-134) 51.4 (11.5-106.6) 58.5 (24.1-124.2) [C-peptide] pM 126 (7.2-217.9) 695 (161-1157) 693 (373-1040) [Proinsulin] pM (total) 7.3 (0.3-82.3) 16.8 (5.5-28) 8.4 (4.0-21.8)

29  Fiorina et al 2008 Diabetes Care 31;436-8  Allotransplant recipients monitored at baseline and monthly follow-up  Peak islet function = highest c-peptide level  HOMA-IR ~ 2-fold higher in short-term vs long-term at peak islet function (p=0.02), i.e. before failure is evident  Higher PI/CP in failed (“short-term,” n=20) vs success (“long-term,” n=17) allotransplant at peak function (nonsignificant) and at last measurement (failure vs success) (~4.5-fold higher, p=0.04)  Proinsulin kit: Total PI (Dako), 65% CR with des 31,32  Truyen et al 2005 Diabetologia 48(11):2322-9. (Belgian Diabetes Registry) Clinical Data as a markers- Proinsulin/ C-peptide  De Pauw, et al, 2008 Clin Chem 54(12):1990-8  Normoglycemic/ prediabetic 1st degree relative of T1DM (n=46) had higher PI/CP than nondiabetic relative (n=292):  3.87% (2.32 – 5.82%) vs 2.10% (1.68 – 3.81%), p<0.001  Time-Resolved Fluorescence Immunoassay (TRFIA) for PI and CP  Proinsulin kit: Total PI (TRFIA): 65% CR with des 31,32  Vandemeulebroucke et al 2009 Diabetes Metab 35(4):319-27. (Belgian Diabetes Registry)

30  Thank you for your time. Mouse and Rat C-peptide Mouse and Rat C-peptide.

31  Human Insulin-  ELISA/RIAs –ALPCO ELISA, Millipore-ELISA, Millipore-RIA, Mercodia ELISA  Automated Analyzer –Abbott, Beckman-Coulter, Invitronc, Roche, Siemens Centaur, Siemens Immulite, Tosoh  Mouse & Rat Insulin-  There are three 5 µl double mAb rodent Insulin ELISAs Available  Mercodia, Crystal Chem & ALPCO mouse and Rat Insulin assays  Linco, mAb/pAb format, 10 µl sample size.  Mouse & Rat Proinsulin-  Until Fall 2009, non available.  Mercodia offered a Rat proinsulin for a few years, but it was recalled. Competitive Advantages of ALPCO-Insulin

32 In comparison of 10 commercial methods (RIA, ELISA, chemistry analyzers) to the established reference ‘gold standard’ method (IDMS) for insulin measurement, ALPCO ELISA is the only method to report ≤ 5% individual sample biases exceeding the 15.5% desirable allowable bias for insulin measurement based on biologic activity. ‘When the mean of replicate measurements of each sample was used to minimize the influence of imprecision, we found that only 1 of 10 methods had 95% of individual sample biases within 15.5% of the IDMS values.” RIA had amongst the highest error ‘Millipore RIA had an approximately constant bias over most of the measuring range, with a progressively increasing bias at lower concentrations’. RIA standards were unstable as reported by the manufacturer. -HUMAN Insulin Market Place

33 -Why Measure- Mouse & Rat Insulin Market Place  Crystal Chem. Very Popular in North America Regular High Background as compared to first and second standards. Resulting in samples recover below zero OD (non Detect) ↑CV’s (↓performance & ↓reproducibility) Many customer have modified protocol to incubate over night and still don’t get separation. Short (3mo) shelf life More $, also must order Mouse Standard separate ($65)  Linco/Milipore, has been on the market the longest, since 70’s. Kits Developed by Ron Gingrich. pAb/ mAb format (change frequently), High Volume (RIA 100µl, EIA, >10µl.) Long Incubations, not easy to use. Recently, ‘standards have problems regularly’… Degrading Standards, tech support not there…millipore support aint what it was with Linco  Mercodia, We grew this market from 1991 till 2007. Flat Curve, ↑background= ↑CV’s + samples recover below zero OD (non Detect) ↓performance/ ↓reproducibility More $, buy controls separately. Ease of use (multiple) More Pipetting Steps. Prices Change frequently

34 -Why Measure- Insulin Market Place 3) ALPCO-Mouse and Rat Insuiln  better separation at the low end of the curve.  All assays include a bi-level control set, so controls don’t need to order separately (save $).  The conjugate vial was large, could not be spun down and it was very difficult to capture all 600µl. The conjugate solution is now in a cryo-vial, making capturing all 600µl much easier.  All standards come reconstituted standards; we believe this improves long term stability of the standards.  Standard Values are now static, do not change lot to lot, as they did before.  Sealable pouch for 96 well plates, with sturdier strips and a locking mechanism.  There is one less pipetting step (pipette sample into blank well, easier to track loading 5µl), some sample sizes are smaller and the overall correlations (to Mercodia) range from 0.8-0.96, Depending on the sample type.

35 Multiplexing?? - Theoretically, a very nice idea! -Small sample volume – many analytes. -Potential Issues: -Specificity & Sensitivity - Reproducibility/Variability commonly overlooked in multiple labs…especially with multiple end-users compiling experimental data on one model. YOUR SAMPLES ARE VERY PRECIOUS- after spending lots of Time, $ & Creativity on developing models, techniques & Experiments… measuring [biomarker] CONSISTANTLY accurate is of upmost importance. Running in-house control pools is optimal- QC inter & intra assay CV’s. (<5% good, ≥10% cant trust)

36 Immunoassays Beyond the Ordinary www.alpco.com | 800-592-5726 TA: Diabetes & Obesity- Mouse & Rat Insulin ELISA option Each is tailored for specific applications. A tool for each job!

37 -Why Measure- Insulin Market Place 3) ALPCO- “Thanks for letting me try the rat insulin ELISA. It was great. I tried ones from Mercodia, Crystal Chem and Cayman Chem also, and the one from ALPCO was by far the best.” M. Mosedale, UCSD.

38 Immunoassays Beyond the Ordinary www.alpco.com | 800-592-5726 Technical Tidbits- How to get the best results from you ALPCO (or any) immunoassay Here are a few of the most popular calls:  ‘High Variability’  Common Cause: most likely Washing! Wash the microplate X # of times with Working Strength Wash Buffer (see Reagent Preparation) with a microplate washer (Cleaned Regularly). Alternatively, use a Squeeze Bottle /Wash bottle to fill the wells, and then discard the liquid inverting and firmly tapping the microplate on absorbent paper towels between washes. ‘Blot Dry’- After the final wash with either the microplate washer or wash bottle, remove any residual Wash Buffer and bubbles from the wells by inverting and firmly tapping the microplate on absorbent paper towels (see Microplate Locking Diagram below).  ‘Problems with standard curve’  Common Cause: Plate Rotation, Temperature

39 Immunoassays Beyond the Ordinary www.alpco.com | 800-592-5726 Technical Tidbits- How to get the best results from your ALPCO assay Shaking during incubations http://www.alpco.com/techissue.asp?IssueNo=8

40 Immunoassays Beyond the Ordinary www.alpco.com | 800-592-5726 Technical Tidbits- How to get the best results from ALPCO (or any) assay Here are a few of the most popular calls:  ‘High Variability’  Common Cause: Washing! (See Handout)  ‘Problems with standard curve’  Common Cause: Plate Rotation, Temperature  ‘Controls not in Range’  Common Cause: Improper Regression Analysis

41 Immunoassays Beyond the Ordinary www.alpco.com | 800-592-5726 Technical Tidbits- How to get the best results from your ALPCO assay Using the proper regression analysis is key Cubic Spline Regression Control Value: 3.7 ng/ml Linear Regression Control Value: 6.9 ng/ml Each immunoassay is designed differently, important to use the regression analysis in assay protocol!

42 Immunoassays Beyond the Ordinary www.alpco.com | 800-592-5726 Technical Tidbits- How to get the best results from ALPCO (or any) assay Here are a few of the most popular calls:  ‘High Variability’  Common Cause: Washing! (See Handout)  ‘Problems with standard curve’  Common Cause: Plate Rotation, Temperature  ‘Controls not in spec’  Common Cause: Improper Regression Analysis  ‘High background OD’  Common Cause: Inadequate Washing, Contamination  (See Handout) Plate washer, Squeeze Bottle

43 Immunoassays Beyond the Ordinary www.alpco.com | 800-592-5726 Technical Tidbits- How to get the best results from your ALPCO assay Know your limits! The range of the assay is often defined by the limitations in antibody sensitivity, matrix interference, etc. Extra standards Above or below the assay’s standards will extend the curve, but often in a non-linear way. Non-linear separation will result in very high error and CVs. If your in need of extra sensitivity (or any thing else), please give us a call… …we can talk OFF LABEL. We are full of ideas and always willing to help!

44 Proinsulin → INS+ CP. Clinical evidence (TIIDM)  Clinical Relevance of measuring PI in Diabetes In the healthy state, each proinsulin (PI) molecule is processed to c-peptide and insulin. Because of this proinsulin concentrations are low, except in diabetic patients(5), where inefficiencies in processing are evident. As a result, in humans, PI has been shown to be a strong predictor of type II diabetes (3-6). GP 4 L.Mykkänen, SM Haffner, CN Hales, T Rönnemaa, M Laakso- Diabetes 1994  Normoglyemic patients (n= 138, age 53-61), tested for Insulin resistance (Si).  Decreased Insulin sensitivity correlated with elevated Proinsulin / insulin ratios. Fasting ↑PI/I % Correlated with ↓ Si (r = 0.31) (P < 0.001) PI/I% correlated + with quartiles of Si (low Si to =0.048, 0.078, 0.078 & 0.068 high Si ) Correlation w/ Si- [Insulin] (r= -0.64), [intact PI] (r= -0.43), [split 32,33 ppPI] (r= -0.54)  As patients insulin Secretion in response to Glucose decreases ( ↓ AIRmax)  ↓ PI/I % = ↑Acute Insulin Response (AIRmax) (r= -0.40)  ↓ PI/I% correlated - with quartiles of ↑AIR max (low to high=0.088, 0.068, 0.058 & 0.058)  independent of age, sex, BMI, waist-to-hip ratio & SI  NGT = ↑ PI processing, not induce ↑PI/Ins resistance  “In conclusion, in normoglycemic subjects, insulin resistance (low SI) was associated with a low rather than a high proinsulin-to-insulin ratio. Subjects who maintained normoglycemia with a low AIR had an increased proinsulin-to-insulin ratio compared with those who needed high AIR to maintain normoglycemia. These results suggest that, in subjects with normal glucose tolerance, insulin resistance does not induce increased proinsulin relative to insulin secretion, but rather is associated with enhanced processing of proinsulin.”

45  Release is bi-phasic. Glucose stimulated Insulin Secretion (GSIS) results in a rapid 1 st phase release (Readily Releasable Pool), demonstrated within 15-30minutes, followed by a longer period of second phase release (from reserve pools) of Insulin and C-peptide. Secretion of- Insulin, C-peptide & Proinsulin R.Carr, B. Ahren, et al. AJP Endo Met. 2008 S.Andrikopoulos. Am J Phys, Endo & Met. 2008

46  WHY MEASURE Insulin- In-Vivo  Asses Beta Cell Functional (Functional Mass), how does our drug candidate or experimental model change the capability of the beta cell to respond to ↑[glucose]… more insulin secretion better. 1.OGTT- (Oral Glucose Tolerance Test)- ingest glucose/food and measure ∆ in [insulin] 2.IVGTT- (Intravenous) or IPGTT (Intraperotoneal)-Inject glucose and measure ∆ in [insulin] (No –Incretin effect) 3.Hyperglycemic Clamp/ AIR.glu- ( Acute Insulin Response) Bolus then Infuse ↑ [ Glucose] and measure ∆ in [insulin] 4.AIR.arg- Glucose Potentiation of Arginine (Bolus) induced Insulin Secretion- Best correlations to β-cell max  Most accurate way to measure βCell Functional Mass is Hyperglycemic Clamp, AIRarg, (aka Maximally Induced Insulin Secretion to Arg).  M&M= Sample @ t=-30-0, then again at t= 1, 5, 15, 30, 45, 60, 75 for measurement of Glucose & Insulin.  @t=0, Inject IV Glucose bolus (~375mg/kg) followed by variable infusion of 50% (wt/vol) glucose infusion to CLAMP arterial glucose ~250mg/dl (for 0-70min)  @t=60min, Inject L-Arginine (ARG) (1mM/kg). Why Measure- Insulin, In-Vivo EG of Hyperglycemic Clamp & AIRarg Max to asses βCell functional mass Asses 1 st phase Insulin response to Glucose.  A –Hyperglycemic Clamp  C- AIR.arg-  D-Correlation to βCell Mass *A. Matveyenko, et al. DIABETES 2009

47  WHY MEASURE Insulin- In-Vivo  Insulin Sensitivity (Si)/ Insulin Resistance (IR)  Insulin Resistance is a condition where the pancreas produces insulin in response to elevated glucose, but the muscle, fat and liver cells do not respond to insulin by increasing the Glucose transporter (GLU4) in the cell to transfer glucose into that cell.  Progression= Normal Glucose Tolerance (NGT) → Impaired Glucose Tolerance (IGT) of Impaired Fasting Glucose (IFG) aka pre diabetic → Type 2 Diabetes (TIIDM) aka overt insulin resistance  How MEASURE Si- In-Vivo  Glucose tolerance test (GTT). This test measures blood glucose after people fast (O/N) and ingest Sugar. In humans, a blood glucose level between 140 and 199 mg/dL means glucose tolerance is not normal but is not high enough for a diagnosis of diabetes.  HOMA- [Glucose] (mg/dl) X [Insulin] (µU/ml) / 405 (or Divide by 22.5 for glucose in mmol/L)  Hyperinsulinemic euglycemic clamp (Gold Standard): Measures the amount of glucose necessary (rate of Glucose infusion (GIR)) for increased insulin level without causing hypoglycemia. Through a peripheral vein, insulin is infused at 10-120 mU per m2 per minute. In order to compensate for the insulin infusion, glucose 20% is infused to maintain blood sugar levels between 5 and 5.5 mmol/l. The rate of glucose infusion is determined by checking the blood sugar levels every 5 to 10 minutes. Why Measure- Insulin, In-Vivo

48  WHY MEASURE Insulin- In-Vivo  Asses Insulin secretion (GSIS)-Asses Beta Cell Function- how does our drug candidate or treatment change the capability of the beta cell to respond to ↑[glucose]… more insulin secretion better. 1.Culture Islet Cell Line (INS-1, MIN-6, RIN..) No established human Beta Cell line 2.Harvest Pancreas and measure Total Insulin content (Islet Content) 3.Harvest Pancreas, isolate & culture 1⁰ islets/β-Cells (Measure Supernatant +/- Lysate) A) Static Incubation, Incubate in Low [Glucose] (~2 - 5mM), High [glucose] (~16.7 - 20mM) (and or drug candidate) and measure ∆ [insulin] 2)Perifusion- incubate in chamber with continuous flow of media (Stop Cock to adjust infusing media)- ↓ Glucose, ↑ Glucose and/or X Candidate. Why Measure- Insulin, In-Vitro *N.Fiaschi-Taesch, et al. DIABETES 2009


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