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Piero MarchettiUniversity of Pisa
Effects of gliclazide on oxidative stress
in the pancreatic beta-cell
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Defining T2DM
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Defining T2DM
…varying degrees of insulin resistance and insulin secretory deficiency
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Defining T2DM
…varying degrees of insulin resistance and insulin secretory deficiency
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The natural history of T2DM
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T2DM: the fundamental factor is…
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T2DM: the fundamental factor is…
…the beta-cell!
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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
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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
![Page 10: Piero Marchetti University of Pisa Effects of gliclazide on oxidative stress in the pancreatic beta-cell](https://reader036.vdocuments.us/reader036/viewer/2022062408/56649ec75503460f94bd36a9/html5/thumbnails/10.jpg)
~ 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
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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
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The normal beta-cell
• Half-life of ~30 days
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The normal beta-cell
• Half-life of ~30 days
• Apoptosis is the major mechanism of death
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The normal beta-cell
• Half-life of ~30 days
• Apoptosis is the major mechanism of death
normal
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The normal beta-cell
• Half-life of ~30 days
• Apoptosis is the major mechanism of death
normalapoptotic
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The normal beta-cell
• Half-life of ~30 days
• Apoptosis is the major mechanism of death
• New beta-cells by– replication– neogenesis
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Butler P et al, Diabetes 2003
Del Guerra S et al, Diabetes 2005
The beta-cell in T2DM
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Summary1
• Compared to the normal situation, in T2DM there is:– decreased beta-cell mass – reduced insulin secretion function (in particular
in response to glucose)
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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
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Beta-cell oxidative stress in human T2DM beta-cells
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Beta-cell oxidative stress in human T2DM beta-cells
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Beta-cell oxidative stress in human T2DM beta-cells
8-OHdG immunostaining
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Beta-cell oxidative stress in human T2DM beta-cells
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Beta-cell oxidative stress in human T2DM beta-cells
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Beta-cell oxidative stress in human T2DM beta-cells
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Beta-cell oxidative stress in human T2DM beta-cells
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Beta-cell oxidative stress in human T2DM beta-cells
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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
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Glucose Toxicity
Robertson RP, JBC 2004
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Summary2
• In T2DM, there is increased oxidative stress at the beta-cell level, which contributes to:– mass reduction – insulin secretion defects
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The beta-cell in T2DM
normal
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The beta-cell in T2DM
Genetic factors
Acquired factors(glucotoxicity, lipotoxicity,IAAP, others)
normal
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The beta-cell in T2DM
Genetic factors
Acquired factors(glucotoxicity, lipotoxicity,IAAP, others)
normal
Type 2 diabetes
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The beta-cell in T2DM
Genetic factors
Acquired factors(glucotoxicity, lipotoxicity,IAAP, others)
normal
Type 2 diabetes
WHAT’S THE ROLE OFSULPHONYLUREAS?
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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
![Page 36: Piero Marchetti University of Pisa Effects of gliclazide on oxidative stress in the pancreatic beta-cell](https://reader036.vdocuments.us/reader036/viewer/2022062408/56649ec75503460f94bd36a9/html5/thumbnails/36.jpg)
The role of sulphonylureas
• Some studies have reported increased sulphonylurea-induced beta-cell apoptosis in-vitro
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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
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The role of gliclazide
Diabetes Metab Res Rev 2007
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• 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
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Collagenase digestionCollagenase digestion
Density gradient Density gradient purificationpurification
Preparation of isolated human islets
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• 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
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• 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
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0
20
40
60
80
100
120
S.I.
NG
NG+GLIB
NG+GLICL
Stimulation index (% of controls)
*
The role of gliclazide
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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
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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
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The role of gliclazide(antioxidant properties)
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• 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)
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The role of gliclazide
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0
5
10
15
20
25
nitrotyrosine
NG
HG/NG
HG/NG+GLIB
HG/NG+GLICL
Nitrotyrosine (ng/ml)
*
**
The role of gliclazide
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The role of gliclazide
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The role of gliclazideCtrl H2O2
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The role of gliclazideCtrl H2O2
H2O2+GLIBH2O2+GLICL
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The role of gliclazide
0102030405060708090
100
100 150 200
ctrl
gliclazide 1
gliclazide 5
glibenclamide 1
glibenclamide 5
% viability
H2O2 (µmol/l)
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Summary3
• Gliclazide has several beneficial effects on beta-cells, including:– better survival– improved function
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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
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Piero MarchettiUniversity of Pisa
Effects of gliclazide on oxidative stress
in the pancreatic beta-cell