1. Insulin and the integrity of nerve fibre Dr MV Srishyla Regional medical director Novo Nordisk India 3 rd DFSI conference, Jaipur, India 12 Sep 2004 3 rd annual conference, Diabetic Foot Society of India, Jaipur, India. 12 Sep 2004

2. Nerve electrophysiology & morphology Animal studies Clinical studies

3. Nerve electrophysiology(1) Nerve conduction studies NCV most sensitive index of severity of DSP NAPA index of degree of fiber loss Relationship between DSP and risk factors Pittsburgh epidemiology of diab complications study, 1989 DCCT, 1993 EURODIAB type 1 complications study, 1996 Seattle prospective diabetic foot study, 1997 Focus on identifying risk factors for presence of DSP

4. Nerve electrophysiology(2) Tkac I and Bril V. Diabetes Care Oct 1998 Studied the value of GHb, duration, age, sex and height in predicting the electrophysiologic severity of DSP Used different models of severity GHb significantly related to severity GHb cut-off for highest predictive value GHb 9% (poor control) Mean SNCV & SAMP 6.3% & 18% lower resp in poorly controlled Corresponds with that achieved in DCCT

5. Nerve morphology Perkins B, Greene D and Bril V. Diabetes Care Apr 2001 Studied the value of GHb, duration, age, sex and height in predicting the morphologic severity of DSP Used different models of severity GHb significantly related to severity GHb cut-off for highest predictive value GHb 9% (poor control) Mean FD 33% (3461 Vs 2334) lower in poorly controlled

6. Nerve electrophysiology & morphology Animal studies Clinical studies

7. Study 1 Brussee V, Cunningham FA and Zochodne DW, Diabetes July 2004 “Direct insulin signaling of neurons reverses DN” Benefits of insulin independent of glycemia Provides direct support for neurons and peripheral axons Low doses – reverse conduction slowing (abnormal function) and axonal atrophy (abnormal structure)

8. Study 1: Neurons express insulin receptors Insulin had the capability of signaling sensory neurons FITC-labelled insulin (intrathecal) accessed and labeled individual lumbar dorsal root ganglion neurons. L4-6 DRG Neurons Saline inj Insulin inj ImmunofluoroscenceLight Non-diabetic rats

9. Study 1: Insulin improves conduction abnormalities Sciatic-tibial motor Caudal sensory Higher dose of insulin completely reversed slowing of sensory nerve conduction

10. Study 1: Insulin prevents axonal atrophy Myelinated sural sensory nerve sections 22.7 18.4 Insulin reversed distal sensory axonal atrophy Control rat Diabetic rat

11. Study 1: Effect of anti-insulin antibody Non-diabetic rats Sequestering endogenous insulin generates axonal abnormalities

12. Study 2 Zochodne DW, Sun H and Eyer J, Brain Aug 2004 “Accelerated DN in axons without neurofilaments” Beneficial effects of insulin on axons when neurofilaments (critical latticework of axons) are damaged Superimposed STZ-diabetes on transgenic mouse model with deficient Nf-H protein Nf-replete and Nf-deficient diabetic and non-diabetic mice

13. Study 2: Neurofilament deficiency accelerates diabetic neuropathy - NCV Diabetic mice lacking neurofilaments, Nf-dia+, show a decline in CV betn 4 and 8 weeks of diabetes

14. Study 2: Neurofilament deficiency accelerates diabetic neuropathy - Amplitude

15. Study 2: Effect of insulin – electrophysiology and morphology Nf-Dia+ mice Non-Neurofilament related actions – e.g new protein synthesis, mitochondrial target etc

16. Study 3 Huang et al, Diabetes, Aug 2003 “Insulin prevents depolarization of mitochondrial membrane in the presence of sustained hyperglycemia” Effect of insulin on mitochondrial membrane Mitochondrial dysfunction depolarization / membrane potential measured as whole-cell fluorescent video imaging using rhodamine 123 (R123) CCCP ~ Carbonyl cyanide chlorophenylhydrazone

17. Study 3: Insulin prevents diabetes-induced mitochondrial dysfunction

18. Study 3: Insulin action independent of blood glucose levels Sensory neuron cultures were treated for 6 or 24 h with and without 1.0 nM insulin and mitochondrial polarization status calculated Sensory neuron cultures were treated for 24 h with 10 or 50 mM glucose and with / without 1.0 nM insulin and mitochondrial polarization status calculated

19. Study 3: Insulin action independent of blood glucose levels

20. Summary animal studies Insulin signals neurons directly and has distal protective effects Insulin prevents axonal loss and atrophy through non-neurofilament related actions Insulin prevents depolarization of mitochondrial membrane in sensory neurons

21. Nerve electrophysiology & morphology Animal studies Clinical studies

22. Studies of intensive insulin therapy DCCT, follow-up 6.5 years Oslo study, follow-up18 years

23. Intensive therapy reduced Confirmed clinical neuropathy by 64% Abnormal nerve conduction by 44% Abnormal autonomic function by 53% Nerve conduction velocities Remained stable with intensive therapy Decreased significantly with conventional DCCT study 5% 13% Ann Intern Med 1995;122:561-8

24. Oslo study HbA1c TertilesNone or 1 nerve below ref for NCV None or 1 nerve below ref for NAPA <7.8%92%77% 7.9-8.4%91%73% >8.4%53% HbA1c strong predictor of nerve function HbA1c<8.4% over 18 years assoc with near normal nerve function Diabetes Care 2003;26:2400-4

25. Conclusion

26. Diabetic neuropathy - Mechanisms Insulin restores balance between Nerve degeneration and regeneration

27. Diabetic neuropathy - Mechanisms Insulin ↓ Survival response mechanisms ↓ PI3-kinase ↓ PKB/Akt ↓ Expression/translocation of proapoptotic factors e.g Bcl-2 proteins

28. Mixtard  50 study NovoPen  3 study ICEONiSTART Year 2000 20022003 N 500926531307013236 Age 53.3±1350.3±13.153.67±15.653.17±9.93 Duration 10±6.99.3±6.17.87±5.517.15±5.18 BMI 24.6±4.924.97±4.1524.83±4.0124.83±4.35 FPG 192±63173.9±54.4203.16±48.8190.08±41 PPPG 279±83249.8±77.2293.05±68.3276.95±61 HbA1c 9.8±2.29.6±3.49.6±1.699.08±4.11 NN India Experience Int J Diab Dev Countries 2001;21:133-7;Data on file, 2000; Novo Nordisk Diabetes Update, March 21-23, Colombo, Srilanka 2003: 80-92; iSTART meeting, Rome, 7 May 2004