1362397068 insulin and integrity of the nerve fiber
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insulin and integrity of the nerve fiber
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1362397068 insulin and integrity of the nerve fiber
- 1. Insulin and the integrity of nerve fibre Dr MV Srishyla Regional medical director Novo Nordisk India 3rd DFSI conference, Jaipur, India 12 Sep 2004 3rd 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% vs > 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% vs > 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 Immunofluoroscence Light Non-diabetic rats
- 9. Study 1: Insulin improves conduction abnormalities Sciatic-tibialmotorCaudalsens 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 Tertiles None 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% 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 ICEON iSTART Year 2000 2000 2002 2003 N 5009 2653 13070 13236 Age 53.3±13 50.3±13.1 53.67±15.6 53.17±9.93 Duration 10±6.9 9.3±6.1 7.87±5.51 7.15±5.18 BMI 24.6±4.9 24.97±4.15 24.83±4.01 24.83±4.35 FPG 192±63 173.9±54.4 203.16±48.8 190.08±41 PPPG 279±83 249.8±77.2 293.05±68.3 276.95±61 HbA1c 9.8±2.2 9.6±3.4 9.6±1.69 9.08±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
Notas do Editor
- Ladies and gentlemen, Good morning. I’m extremely honoured to be speaking today in front of you and the chairman the very eminent Prof Boulton. Just a prelude to the very unique and very special session, perhaps first time in India any conference organisers have thought up and included this topic. I owe a special thanks to Dr Kelkar, the secretary of DFSI for having chosen me to baptise this topic into DFSI meetings. I’m pretty sure DFSI will bring in more experts in neurosciences, both basic biological and clinical, to deliver such a lecture in the future. Since we’re attempting such a topic the first time and also keeping in view the interest and background of the audience, I’ve tried to strike a balance between some basic research and its applied aspects in clinic. The graphic in this slide also stands for the storyline I’ve adopted.
- In order to tell you about how insulin benefits the integrity of nerve fibre, I’ve selected parameters which you can relate to easily from your practice experience and also comprehend the clinical implications of the studies which I’ll be detailing here. I’ll begin with the nerve physiology and morphology as the parameters of nerve integrity I’ve chosen belong there.
- Incidentally, all of us are familiar with nerve conduction studies that yield information on two parameters – the nerve conduction velocity or NCV and the nerve action potential amplitude or NAPA. NAPA is the more variable, but certainly a better index as it tells us more about the Fibre density in diabetic neuropathy or DSP, diabetic poly sensorimotor neuropathy. Now look at the clinical significance of these 2 parameters. Cross-sectional and prospective studies in diabetic neuropathy over the past decade and a half have focussed on ability of risk factors to predict the presence of DSP But the importance of these parameters was realized when 2 other studies focussed on the ability of the very same risk factors to predict the severity of DSP through these parameters of nerve conduction and nerve morphology. What were these studies?
- Incidentally, 2 studies from the same group led by Vera Bril of Univ of Toronto. This is the first one. Published in Oct 1998 issue of Diabetes Care, this paper proved that clinicians could also predict the severity of diabetic neuropathy based on NCS (Nerve conduction study) parameters based on the degree of glycemic control. In the 1st paper that appeared they reported on the value of HbA1c in predicting the electrophysiologic severity. The sample size was small, but the analysis was robust. They used different models of severity (5 models in all) and found that HbA1c could predict severity of DN in both univariate and multivariate regression analysis. The significance held true when HbA1c was handles as both a continuous and discrete variable. How? As shown in the 4th bullet point, their population could be divided into poorly- and well-controlled at a HbA1c cut-off of 9%. Patients with a HbA1c of &gt;9% had lower NCS values as indicated here. The numbers that came out of the paper corresponded with DCCT and by adopting these criteria, you can confer what the authors called “a clinically meaningful degree of prevention (of DN)’
- This is the subsequent paper taken out by the same group, this time on nerve morphology (Fiber density) with very similar conclusions. That is a brief background to why I’ve chosen studies that have used NCS parameters in this presentation. These are very elegant studies that demonstarte glucose-independent effects of insulin. Thus this presentation also goes to show the benefits of insulin beyond glycemic control, this time in the context of DN.
- Let us first look at 3 animal studies, all recent ones
- This Canadian group demonstarted that Insulin signals neurons at the level of nerve cell bodies (Perikarya, here the dorsal root ganglia /DRG neurons) and exert distal axon-protective effects. They proved the trophic nature of insulin Insulin was used in sub-hypoglycemic doses proving its benefits independent of glycemic control.
- Their study revealed 4 findings. This was the first. That insulin receptors are present on the DRG neurons. Insulin was given intra-thecal as a Fluorescein isothiocyanate insulin. There was one control group which was given FITC-labeled saline. The picture here is a light microscopy picture. saline Inj controls did not take up the stain. See the lower panel, Insulin-inj Neurons have taken up the stain, mostly confined to the cell membranes (cytoplasm) confirming the availability of insulin receptors here and they inferred that Insulin could signal neurons directly.
- This was their next finding. I’ve shown here 2 graphs – the motor NCV (sciatic-tibial) on top and the sensory NCV (caudal). Look at the first 2 bars. At baseline which is 3 months after after STZ-induced diabetes, compared to the control rats, diabetic rats had a significantly lower (upto 50%) NCV. Subsequent 1 month of saline inj had no effect on the NCV. But subsequent 1 month of insulin in 2 doses – 0.1 U or 0.2 U per day improved the NCV. So much so that at higher doses, insulin completely reversed the slowing of nerve conduction.
- If the previous slide was on NCV, this is on nerve morphology. You can clearly see the axon loss and atrophy in the diabetic rat versus control on the right picture – a cross-section of sural sensory nerve. The bars clearly indicate how diabetes reduces nerve fiber density (expressed on the y-axis as mean caliber) and insulin reverses the defect.
- The researchers proved that the DRG neurons contained insulin receptors by exposing them to anti-insulin antibody which reduced the NCV. Rat anti-albumin did not induce the same effect.
- Now let us look at a second study from the same group. This time they demonstrated that insulin could preserve the integrity of the structural lattice of axons. Once again a very elegant study. They were fortuitous to procure transgenic mice that were deficient in a high mol wt neurofilament protein (a serendipitous creation of the French group of Joel Eyer) so that we had a neurofilament-deficient mouse phenotype here. This group superimposed STZ-diabetes on this mouse to test the hypothesis whether insulin could reverse this phenotype.
- Their first finding shown in the title here. There are 4 lines in this graph. The bottom 2 belong to the Nf-deficient mice – NCV is slowed by inducing Nf-deficiency and diabetes further worsens the NCV. You can see that the NCV has taken a sharp downward dip at 4 weeks after onset of diabetes
- The same hold true for nerve amplitude too.
- Look here at what insulin does to such a phenotype. On the left is the graph which shows the effects of insulin on 4 mice. The administration of insulin has led to an immediate reversal of the defect. On the right is the same effect of insulin on the nerve fiber density.
- This is the last animal study data I wanted to present. Now we all know the crucial role of mitochondria in cells affected by diabetes. This group from Manchester UK showed that Insulin could preserve the integrity of mitochondria in neurons affected by diabetes. The mitochondrial depolarization was measured by special techniques and the increase or decrease of fluorescence triggered by exposure to a substance known as CCCP was monitored. I’ll be using these fluorescence tracings to explain the effects of diabetes and insulin on the mitochondrial membrane in the subsequent slides.
- This was their first finding. I’ve shown 3 line graphs in the upper panel. These are obtained from cultured DRG neurons from the exparimental animals. Look at the left-most graph. CCCP has been able to trigger a fluorescence-trace indicating that mitochondrial membrane is preserved. The second picture shows that diabetes stunted the mitochondrial membrane function. Look at the 3rd graph. Insulin treatment reveresed the defect you see in diabetes. The bar graph below on the left is showing the same effect as in the upper panel. But the bottom-left picture is different. Mitochondria play an important role in calcium intracellular homeostasis thus contributing to the preservation of physiological ionic fluxes. This graph shows that while diabetes increass intracellular calcium, insulin reverses the effect.
- This experiment also proved that the effects of insulin were glucose-independent. The response of mitochondria in sensory neuron cultures of diabetic rats to CCCP was increased in the presence of insulin. The graph on the right shows that this was due to indeed insulin and not glucose.
- When the group studied the blood glucose parameters – Glucose, HbA1c, Sorbitol, myoinositol – none were affected by the dose of insulin employed. This implies that insulin acts independent of glucose levels and directly on neurons.
- To summarize, what does insulin do at a cellular level in diabetic neuropathy?...
- I’ll just touch upon the clinical studies. two of them and we all know them well.
- Self-explanatory
- Self-explanatory
- Oslo study report published last august, is the longest follow-up we have on diabetic neuropathy as an outcome in a long-term study. When the Oslo study group divided the study population in tertiles, they found that a HbA1c below 8.4% cut-off meant a near-normal nerve function. Look at the first two rows. &gt;90% with no defects in NCV, &gt;70% with no defects in nerve amplitude action potential. So they concluded that HbA1c is a strong predictor of nerve function and HbA1c&lt;8.4% over 18 years assoc with near normal nerve. Both DCCT and Oslo study used intensive insulin therapy which provides clinical proof of the benefits of insulin on the integrity of nerve fibre.
- I’ll conclude by highlighting the mechanisms of the beneficial effects of insulin on the nerve fibre integrity.
- Self-explanatory
- Self-explanatory
- &lt;number&gt; This is my last slide. We at NN have been conducting such large population data-capture programs almost every year since 2000. These are point-of-time observational characteristics , baseline characteristics at the time we initiate the programs. It shows that the avrage patient of type 2 diabetes in a doctor’s clinic in India has a FPG of 170-200 mg/dl, HbA1c &gt; 9% and 7-10 years into diabetes. Nearly 50% of these report with complications. At least two of tehse studies were on insulin-naïve patients (ICEON and iSTART). We believe that most of these patients who participated in these studies are today on our insulins and hopefully their nerves are protected!