1. Insulin secretion and resistance (MORE than you ever wanted to know) Tisha Joy August 14, 2013

2. Objectives Briefly describe insulin secretion and resistance in the context of: –Pathophysiology –Clinical relevance –Assays

4. Pre-talk Question 1 1.What is this structure?

5. Pre-talk question 2 2. Which of the following is correct? A. Insulin consists of 2 chains connected by disulfide bonds B. Proinsulin levels are low in insulinoma C. Insulin has minimal first-pass hepatic clearance D. C-peptide is derived from cleavage of preproinsulin

6. Pre-talk Question 3 3. All of the following are correct EXCEPT: A. Insulin secretion is normally characterized by 2 phases B. There is loss of the 1 st phase of insulin secretion with diabetes C. Pulsatility of insulin secretion is maintained with diabetes D. There is a hyperbolic relationship between insulin secretion and insulin resistance

7. Pre-question 4 4. Which of the following is CORRECT? A. Development of type 2 diabetes is due to insulin resistance B. Loss of 30% of beta-cell mass leads to diabetes C.Non-diabetic individuals never exhibit insulin resistance D.Insulin resistance occasionally is an adaptive response

8. Pre-talk Question 5 5. What is the gold standard for determining insulin resistance? A. Insulin tolerance test B. Hyperinsulinemic euglycemic clamp C. Minimal model of FSIVGTT D. HOMA-IR

9. Background Beta cells are found in islets of Langerhans, which are variable in size Normal ~10 6 islets; total weight 1-2 g; constitute 1-2% of the pancreatic mass www.uptodate.com Image from http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/pancreas/anatomy.html

10. Background Beta-cells (70%) – insulin Alpha cells – glucagon Delta cells – somatostatin PP cells – pancreatic polypeptide www.uptodate.com Image from http://www.abpischools.org.uk β cells α cells

11. Insulin 51 aa molecule In portal circulation, insulin concentration is ~200-500pM in fasting state and ~1000- 5000 pM in fed state. Approximately 50-80% of insulin is cleared on 1 st pass through the liver. Estimated basal secretion is 24 units/24 h T 1/2 =10 min Schofield CJ and Sutherland C Diabetic Medicine 2012;29:972-979 Caumo A, Lizio L AJP Endo Metab 2004;287:E371-E385

12. Insulin synthesis www.uptodate.com Ribosome Golgi Secretory granules

13. Insulin synthesis and secretion Adapted from http://www.igis.com Insulin Schofield CJ and Sutherland C Diabetic Medicine 2012;29:972-979 Glucokinase

14. Insulin secretion Adapted from Rorsman P et al Physiology 2000;15:72-77 Caumo A and Luzi L Am J Physiol End Metab 2004;287:E371-E385 1 st phase important in shutting down hepatic glucose production The magnitude of the 1 st phase is dependent on the pancreas’ past history of glucose exposure Why important? Preformed De novo

15. Multiple players in insulin secretion Gut Incretins Glucose Neuropeptide Y Parasympathetic nervous system + Activation of K ATP channels Autocrine + Glucagon - + Insulin secretion

16. Adapted from Rorsman P et al Physiology 2000;15:72-77

17. Insulin secretion Individually, β-cells all have differing pulse times (2-5 min), but when formed into aggregates, the pulse rhythm develops into ~4 min cycles So, the integration of all islets is required to ensure the pancreas releases insulin in a regulated fashion – the integration is mediated by the autonomic nervous system Schofield CJ and Sutherland C Diabetic Medicine 2012;29:972-979

18. Insulin secretion Why is pulsatility important? –Maintains the responsiveness of beta-cells to glucose and insulin by preventing the desensitization of the signalling cascade Schofield CJ and Sutherland C Diabetic Medicine 2012;29:972-979

19. Insulin resistance Decreased tissue effect of insulin per unit delivered Hepatic vs. peripheral In people WITHOUT diabetes or obesity, are there times that they can transiently develop insulin resistance?

20. Insulin resistance Tsatsoulis A et al Metabolism 2013;62(5):622-633

21. “Normal” conditions of IR Tsatsoulis A et al Metabolism 2013;62(5):622-633

22. Sample conditions associated with insulin resistance Obesity (apple vs. pear) DM2 PCOS Lipodystrophies [familial, acquired (HIV)] Morelet et al JCEM 2006;91:2689-2695

23. Control BMI 22.8 Control BMI 34.8 HIV BMI 28.3 Hegele R, Joy T, et al J Lip Res 2007;48:1433-1444 Differences in adipose distribution

24. Hegele R, Joy T, et al J Lip Res 2007;48:1433-1444 Control BMI 34.8 Control BMI 22.8 HIV BMI 28.3 Muscle

25. Insulin resistance Tsatsoulis A et al Metabolism 2013;62(5):622-633

26. Insulin secretion and resistance are intertwined

27. Kahn SE et al Diabetes 1993;42(11):1663-1672 http://www.medscape.org/viewarticle/561655

29. Butler AE et a Diabetes 2003:52:102-110 http://www.medscape.org/viewarticle/561655

30. Brunzell JD et al JCEM 1976;42:222-229 http://www.medscape.org/viewarticle/561655 6.4 – 8.3 8.3 – 19.4 5.6 – 6.4

31. Alterations in insulin secretion in T2DM –Absent 1 st -phase insulin response –Reduced 2 nd -phase response –Larger proportion of secreted insulin is basal insulin –Post-meal secretory pulses are smaller in amplitude and cycles are shorter and more irregular (less coupling to glucose) Williams Textbook of Endocrinology, Edition 11. DM2

32. Clinical relevance of insulin synthesis Diagram representing the human preproinsulin molecule showing location of mutations causing ND. The amino acid residues in the signal peptide are indicated in green, the B chain in red, the C-peptide in orange, and the A chain in blue. The dashed circles indicate the basic residues that are the cleavage site for conversion from proinsulin to insulin. The mutations are noted in black circles together with location in the B or A chain. Stoy J et al PNAS 2007;104(38):15040-15044

33. Clinical relevance of insulin secretion Bell GI et al Nature 2001;44:788-791 Glucokinase

34. Assays of insulin secretion and insulin resistance How do you know you have enough insulin or ineffective insulin action?

35. Assays/Measurements Clinical Methods –Fasting blood glucose –Oral Glucose Tolerance Test Research Methods –Plasma insulin concentration –C-peptide concentration –HOMA/QUICKI –Hyperinsulinemic euglycemic insulin clamp –Minimal model analysis of FSIVGTT –Insulin suppression test –Insulin tolerance test –Intravenous Glucose Tolerance Test –Arginine stimulation

36. Measures of insulin secretion and beta-cell mass Fasting blood glucose Fasting insulin OGTT C-peptide HOMA %β Intravenous GTT Arginine stimulation ?PET scan

37. Fasting plasma glucose Simple test Assumes that glucose, insulin, and hepatic glucose production are in steady state Very insensitive Need to lose more than 40-60% of beta-cell mass before blood sugars increase in humans; in rats, need more than 70% of beta-cell mass loss. Clinical use – one of the diagnostic criteria for diabetes

38. Fasting insulin concentration Simple – just a blood test But, peripheral insulin levels are based on secretion, degradation, and distribution…. Influenced by BMI Does not distinguish between endogenous and exogenous insulin So, measuring plasma insulin alone is not useful as a measure of insulin secretion (see later)

39. Oral glucose tolerance test 75 grams of oral glucose given; measurements of fasting glucose usually only at 0 and 120 min False negative – take a walk/exercise during the 2 hours (unusual); not take all of the drink (makes some nauseated) False positive – low CHO load prior to test Can be affected by gastric emptying, gastric bypass Clinical relevance: used as diagnostic test for diabetes – 0 min 7.0 or higher mmol/L 120 min 11.1 or higher mmol/L

40. Stumvoll et al. show derived equations using data from OGTT has reasonable accuracy in predicting 1 st phase & 2 nd phase insulin release Diabetes Care, 2000; 23: 295

41. C-peptide Secreted in equimolar concentration to insulin Can use a technique of C-peptide deconvolution to estimate C-peptide secretion (Eaton-Polonsky approach) – removes the substance kinetics aspect T 1/2 = 35 minutes

42. HOMA %β Calculation based on fasting insulin and glucose levels = 20 x insulin (mU/L) % glucose (mmol/L) – 3.5 Simple calculation Caveat – uses steady state conditions to derive a dynamic situation (r=0.64 with IVGTT) Matthews DR et al Diabetologia 1985;28(7):412-419

43. Measures of insulin resistance Direct –Hyperinsulinemic euglycemic clamp –Insulin suppression test Indirect –Minimal model of FSIVGTT Simple surrogates –HOMA –QUICKI

44. Hyperinsulinemic euglycemic clamp http://diabetesmanager.pbworks.com/w/page/17680151/Assessing%20Insulin%20Sensitivity%20and%20R esistance%20in%20Humans

45. Hyperinsulinemic euglycemic clamp Insulin IV run at constant rate of 5-120 mU/m 2 /min BG monitored q5-10 min until steady state achieved D20 infusion – rate adjusted to keep BGs in normal range K infusion to avoid hypokalemia When steady state = HGP is suppressed. Thus glucose infusion rate = glucose disposal rate More insulin resistant means less glucose infused http://diabetesmanager.pbworks.com/w/page/17680151/Assessing%20Insulin%20Sensitivity%20and%20R esistance%20in%20Humans

46. Hyperinsulinemic euglycemic clamp Gold standard It directly measures whole body glucose disposal at a given level of insulinemia under steady-state conditions Disadvantages: –Cumbersome, labor-intensive, expensive –Feasibility limited (n should be small) –Time consuming http://diabetesmanager.pbworks.com/w/page/17680151/Assessing%20Insulin%20Sensitivity%20and%20R esistance%20in%20Humans

47. Insulin suppression test After overnight fast, infusion of somatostatin (250 μg/h) or octreotide (25 μg bolus followed by 0.5 μg/min) given to suppress endogenous secretion of insulin and glucagon. Simultaneously, insulin (25 mU/m 2 /min) and glucose (240 mg/m 2 /min) are infused into same antecubital vein In contralateral arm, blood samples for glucose and insulin taken q30 min for 2.5 h and then q10 min from 150-180 min (latter is steady-state) SSPG (steady state plasma glucose) will be higher in IR patients http://diabetesmanager.pbworks.com/w/page/17680151/Assessing%20Insulin%20Sensitivity%20and%20R esistance%20in%20Humans

48. Insulin suppression test Highly reproducible Less labor intensive and technically demanding compared to clamp but still requires more than one person and is cumbersome Feasibility limited http://diabetesmanager.pbworks.com/w/page/17680151/Assessing%20Insulin%20Sensitivity%20and%20R esistance%20in%20Humans

49. Indirect – Minimal model of FSIVGTT After overnight fast, IV bolus glucose (0.3 g/kg body weight) is infused over 2 min. IV insulin (4 mU/kg/min) is infused over 5 min beginning 20 min AFTER the IV glucose bolus. Frequent blood samples for plasma glucose and insulin over the next 180 min Values entered into a computer model (MINMOD) http://diabetesmanager.pbworks.com/w/page/17680151/Assessing%20Insulin%20Sensitivity%20and%20R esistance%20in%20Humans

50. Slightly less labor-intensive since there are no infusions requiring constant adjustment Equations also can be used to calculate beta cell function (unlike the prior tests) Not as accurate as clamp or ISTT Can be done in larger n, potentially Indirect – Minimal model of FSIVGTT http://diabetesmanager.pbworks.com/w/page/17680151/Assessing%20Insulin%20Sensitivity%20and%20R esistance%20in%20Humans

51. HOMA HOMA - IR = homeostasis model of assessment for insulin resistance = Glucose (mmol/L) x Insulin (mU/L) 22.5 Normal HOMA IR is 1 R=0.88 with hyperinsulinemic euglycemic clamp Simple, quick, can be done in large populations, relatively inexpensive http://diabetesmanager.pbworks.com/w/page/17680151/Assessing%20Insulin%20Sensitivity%20and%20R esistance%20in%20Humans

52. QUICKI Quantitative insulin sensitivity check index (QUICKI) 1 = [log(fasting insulin) + log (fasting glucose)] Correlates better with clamp data over a wide range of IR better than minimal model or HOMA IR Simple, validated, easy, quick, inexpensive http://diabetesmanager.pbworks.com/w/page/17680151/Assessing%20Insulin%20Sensitivity%20and%20R esistance%20in%20Humans

53. Post-talk Question 1 1.What is this structure?

54. Post-talk question 2 2. Which of the following is correct? A. Insulin consists of 2 chains connected by disulfide bonds B. Proinsulin levels are low in insulinoma C. Insulin has minimal first-pass hepatic clearance D. C-peptide is derived from cleavage of preproinsulin

55. Post-talk Question 3 3. All of the following are correct EXCEPT: A. Insulin secretion is normally characterized by 2 phases B. There is loss of the 1 st phase of insulin secretion with diabetes C. Pulsatility of insulin secretion is maintained with diabetes D. There is a hyperbolic relationship between insulin secretion and insulin resistance

56. Post-question 4 4. Which of the following is CORRECT? A. Development of type 2 diabetes is due to insulin resistance B. Loss of 30% of beta-cell mass leads to diabetes C.Non-diabetic individuals never exhibit insulin resistance D.Insulin resistance occasionally is an adaptive response

57. Post-talk Question 5 5. What is the gold standard for determining insulin resistance? A. Insulin tolerance test B. Hyperinsulinemic euglycemic clamp C. Minimal model of FSIVGTT D. HOMA-IR

58. Thank you

59. Dodson and Steiner Curr Op Struc Biol 1998;8:189-194