Insulin and the integrity of nerve fibre

Dr MV SrishylaRegional medical director

Novo Nordisk India

3rd DFSI conference,Jaipur, India12 Sep 2004

3rd annual conference, Diabetic Foot Society of India, Jaipur, India. 12 Sep 2004

•Nerve electrophysiology & morphology•Animal studies•Clinical studies

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

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

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

•Nerve electrophysiology & morphology•Animal studies•Clinical studies

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)

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

Study 1: Insulin improves conduction abnormalitiesSc

iatic

-tibi

al

mot

orCa

udal

sens

ory

Higher dose of insulin completely reversed slowing of sensory nerve conduction

Study 1: Insulin prevents axonal atrophy

Myelinated sural sensory nerve sections

22.718.4

Insulin reversed distal sensory axonal atrophy

Control rat

Diabetic rat

Study 1: Effect of anti-insulin antibody

Non-diabetic rats

Sequestering endogenous insulin generates axonal abnormalities

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

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

Study 2: Neurofilament deficiency accelerates diabetic neuropathy - Amplitude

Study 2: Effect of insulin – electrophysiology and morphology

Nf-Dia+ mice

Non-Neurofilament related actions – e.g new protein synthesis,

mitochondrial target etc

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

Study 3: Insulin prevents diabetes-induced mitochondrial dysfunction

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

Study 3: Insulin action independent of blood glucose levels

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

•Nerve electrophysiology & morphology•Animal studies•Clinical studies

Studies of intensive insulin therapy

• DCCT, follow-up 6.5 years• Oslo study, follow-up18 years

•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

Oslo studyHbA1c Tertiles None or 1 nerve

below ref for NCVNone 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 functionHbA1c<8.4% over 18 years assoc with near normal nerve function

Diabetes Care 2003;26:2400-4

Conclusion

Diabetic neuropathy - MechanismsInsulin restores balance

between Nerve degeneration

and regeneration

Diabetic neuropathy - Mechanisms

Insulin↓

Survival response mechanisms↓

PI3-kinase ↓

PKB/Akt↓

Expression/translocation of proapoptotic

factors e.g Bcl-2 proteins

Mixtard 50 study

NovoPen3 study

ICEON iSTART

Year 2000 2000 2002 2003N 5009 2653 13070 13236Age 53.3±13 50.3±13.1 53.67±15.6 53.17±9.93Duration 10±6.9 9.3±6.1 7.87±5.51 7.15±5.18BMI 24.6±4.9 24.97±4.15 24.83±4.01 24.83±4.35FPG 192±63 173.9±54.4 203.16±48.8 190.08±41PPPG 279±83 249.8±77.2 293.05±68.3 276.95±61HbA1c 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