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Piero Marchetti University of Pisa Effects of gliclazide on oxidative stress in the pancreatic beta-cell
Piero MarchettiUniversity of Pisa
Effects of gliclazide on oxidative stress
in the pancreatic beta-cell
Defining T2DM
Defining T2DM
…varying degrees of insulin resistance and insulin secretory deficiency
Defining T2DM
…varying degrees of insulin resistance and insulin secretory deficiency
The natural history of T2DM
T2DM: the fundamental factor is…
T2DM: the fundamental factor is…
…the beta-cell!
To be discussed
• The beta-cell in normal conditions and in type 2 diabetes
• The importance of oxidative stress induced beta-cell damage
• The role of gliclazide
To be discussed
• The beta-cell in normal conditions and in type 2 diabetes
• The importance of oxidative stress induced beta-cell damage
• The role of gliclazide
~ 3,000 cells75% Beta cells25% non-Beta cells
200 µm
Micrograph: Lelio Orci, Geneva
Islet of Langerhans
Presented by Pr Philippe Halbanat the 1st Amsterdam Diabetes Meeting, March 30-April 1, 2006
10 µm~ 10,000granules
Micrograph: Lelio Orci, Geneva
The normal beta-cell
Presented by Pr Philippe Halbanat the 1st Amsterdam Diabetes Meeting, March 30-April 1, 2006
The normal beta-cell
• Half-life of ~30 days
The normal beta-cell
• Half-life of ~30 days
• Apoptosis is the major mechanism of death
The normal beta-cell
• Half-life of ~30 days
• Apoptosis is the major mechanism of death
normal
The normal beta-cell
• Half-life of ~30 days
• Apoptosis is the major mechanism of death
normalapoptotic
The normal beta-cell
• Half-life of ~30 days
• Apoptosis is the major mechanism of death
• New beta-cells by– replication– neogenesis
Butler P et al, Diabetes 2003
Del Guerra S et al, Diabetes 2005
The beta-cell in T2DM
Summary1
• Compared to the normal situation, in T2DM there is:– decreased beta-cell mass – reduced insulin secretion function (in particular
in response to glucose)
To be discussed
• The beta-cell in normal conditions and in type 2 diabetes
• The importance of oxidative stress induced beta-cell damage
• The role of gliclazide
Beta-cell oxidative stress in human T2DM beta-cells
Beta-cell oxidative stress in human T2DM beta-cells
Beta-cell oxidative stress in human T2DM beta-cells
8-OHdG immunostaining
Beta-cell oxidative stress in human T2DM beta-cells
Beta-cell oxidative stress in human T2DM beta-cells
Beta-cell oxidative stress in human T2DM beta-cells
Beta-cell oxidative stress in human T2DM beta-cells
Beta-cell oxidative stress in human T2DM beta-cells
Beta-cell oxidative stress in human T2DM beta-cells
0
0,2
0,4
0,6
0,8
1
1,2
Mn-SOD Cu/Zn-SOD
Controls
T2DM
gene expression
Marchetti P et al, JCEM 2004
Glucose Toxicity
Robertson RP, JBC 2004
Summary2
• In T2DM, there is increased oxidative stress at the beta-cell level, which contributes to:– mass reduction – insulin secretion defects
The beta-cell in T2DM
normal
The beta-cell in T2DM
Genetic factors
Acquired factors(glucotoxicity, lipotoxicity,IAAP, others)
normal
The beta-cell in T2DM
Genetic factors
Acquired factors(glucotoxicity, lipotoxicity,IAAP, others)
normal
Type 2 diabetes
The beta-cell in T2DM
Genetic factors
Acquired factors(glucotoxicity, lipotoxicity,IAAP, others)
normal
Type 2 diabetes
WHAT’S THE ROLE OFSULPHONYLUREAS?
To be discussed
• The beta-cell in normal conditions and in type 2 diabetes
• The importance of oxidative stress induced beta-cell damage
• The role of gliclazide
The role of sulphonylureas
• Some studies have reported increased sulphonylurea-induced beta-cell apoptosis in-vitro
The role of sulphonylureas
• Some studies have reported increased sulphonylurea-induced beta-cell apoptosis in-vitro
Maedler K et al, JCEM 2005
ctrl nategl glibencl
The role of gliclazide
Diabetes Metab Res Rev 2007
• Human islets were prepared by collagenase digestion and density gradient purification
number age (yrs) gender BMI (Kg/m2)
16 57.9 ± 5.7
10M
6F
24.6 ±
3.2
The role of gliclazide
Collagenase digestionCollagenase digestion
Density gradient Density gradient purificationpurification
Preparation of isolated human islets
• Culture for 5 days in: – continuous normal glucose medium (NG, 5.5
mM) or high (HG, 16.7 mM) and normal glucose media, alternating every 24h
– with or without 10 µmol/l glibenclamide (GLIB) or 1/10 µmol/l gliclazide (GLICL)
The role of gliclazide
• Assessment of:– acute glucose-stimulated insulin secretion– beta-cell apoptosis and mitochondrial
morphology (electron microscopy)– gene expression– protein expression– oxidative stress (nitrotyrosine concentration)
The role of gliclazide
0
20
40
60
80
100
120
S.I.
NG
NG+GLIB
NG+GLICL
Stimulation index (% of controls)
*
The role of gliclazide
0
20
40
60
80
100
120
S.I.
NG
NG+GLIB
NG+GLICL
HG/NG
HG/NG+GLIB
HG/NG+GLICL
Stimulation index (% of controls)
* * **
The role of gliclazide
0
5
10
15
20
25
30
35
apoptosis
NG
HG/NG
HG/NG+GLIB
HG/NG+GLICL
Beta-cell apoptosis (%)
*
*
The role of gliclazide
The role of gliclazide(antioxidant properties)
• Scott NA et al, Eur J Pharmacol 1991• Noda Y et al, Res Commun Mol Pathol Pharmacol
1997• Jennings PE and Belch JJ, Metabolism 2000• Renier G et al, J Diabetes Complications 2000• Fava D et al, Diabet Med 2002• Onozato ML et al, Kidney Int 2004• Alper G et al, Endocr Res 2005• Li L and Renier G, Metabolism 2006
The role of gliclazide(antioxidant properties)
The role of gliclazide
0
5
10
15
20
25
nitrotyrosine
NG
HG/NG
HG/NG+GLIB
HG/NG+GLICL
Nitrotyrosine (ng/ml)
*
**
The role of gliclazide
The role of gliclazide
The role of gliclazideCtrl H2O2
The role of gliclazideCtrl H2O2
H2O2+GLIBH2O2+GLICL
The role of gliclazide
0102030405060708090
100
100 150 200
ctrl
gliclazide 1
gliclazide 5
glibenclamide 1
glibenclamide 5
% viability
H2O2 (µmol/l)
Summary3
• Gliclazide has several beneficial effects on beta-cells, including:– better survival– improved function
Conclusions
• The pancreatic beta-cell is central to the development and progression of diabetes
• Several factors and mechanisms contribute to beta-cell damage in T2DM, including increased oxidative stress
• Gliclazide has protective effects on the beta-cell, due, at least in part, to its antioxidant properties
• Additional long term, prospective studies in patients are needed to confirm the in-vitro data
Piero MarchettiUniversity of Pisa
Effects of gliclazide on oxidative stress
in the pancreatic beta-cell
