Humanin protects β-cells from apoptosis
induced by Cytokines in vitro
Phuong Thao Hoang, Vladislava Paharkova-Vatchkova,
Laura Cobb, KuK-Wha Lee
Division of Pediatric Endocrinology
Type 1 Diabetes Mellitus
• Type 1 DM is an autoimmune T-cell mediated
disorder resulting from destruction of pancreatic
islet β-cells
• Onset of spontaneous T1DM in human and
animal models is preceded by progressive
leukocyte infiltration into islets (insulitis).
• Cytokines have been shown to induce β-cell
death via mechanisms that involve apoptosis or
programmed cell death.
• Evidence suggests that apoptosis is the
principle mode of β-cell death during
development of T1DM
The Pancreatic Islet
Model for T1DM Pathogenesis
β−cell mass
(Normoglycemia)
REDUCTION IN β-CELL MASS
Autoimmunity
Apoptosis of β-cells
PRESERVATION OF β-CELL MASS
Immune modulation
Prevention of β-cell apoptosis
β-cell regeneration/replication
β-cell mass
Hyperglycemia
Central Question:
How do we improve cell survival amidst
autoimmunity and replication / regeneration?
PNAS 2003 100:13042
• 24 AA polypeptide
• Found in normal, non-affected brain of Alzheimer’s disease
patients
• Antagonizes neurotoxicity caused by Alzheimer’s disease
relevant insults and is a survival peptide.
• Humanin is transcribed from the region of the 16S
mitochondrial ribosomal RNA (rRNA)
• Binds and inactivates the pro-apoptotic factor Bax
• We cloned Humanin as a binding partner for IGFBP-3
Humanin is a potent neuronal survival factor
Ikonen et al 2003 PNAS
Hypothesis:
Humanin is a survival factor for β cells
Research Design and Methods
• NIT-1 mouse insulinoma cells were treated
with cytokines with and without Humanin.
Apoptosis was evaluated by Caspase
activation assay.
• Stat3 and ERK activation were assessed
by Western blotting.
Dose-dependent Protection of β cells
from Serum-starvation induced Apoptosis
1.4
Apoptosis ELISA (AU)
1.2
1.0
0.8
**
0.6
**
**
0.4
0.2
0
SF
1
10
100
HN (nM)
500
1000
10000
Cytokines induce apoptosis in NIT-1 cells
Caspase 3/7 Activity (AU)
* p< 0.05
** p< 0.005
2.5
**
2
**
*
TNF-α
IFN-γ
1.5
1
0.5
0
SF
IL-1β
Humanin reduces IFNγ-induced apoptosis in β cells
CASPASE 3/7 ACTIVITY (AU)
*
1 .4
* p<0.05
1 .2
1
*
*
0 .8
0 .6
0 .4
0 .2
0
HN
-
+
-
+
IFN-γ
-
-
+
+
Dose-dependent protection of β cells from
cytokine-induced apoptosis by Humanin
Caspase 3/7 Activity (RLU)
1.2
* p<0.05
** p<0.005
**
1
0.8
*
0.6
*
0.4
**
**
100
1000
*
**
100
1000
0.2
0
HN (pmol)
TNFα
0
10
-
0
10
+
Humanin reduces apoptosis-induced by
TNF-α and IFN-γ combination treatment in β cells
Caspase 3/7 Activity (AU)
**
4
3.5
*
3
*
2.5
2
1.5
1
0.5
0
HN
TNF-α + IFN-γ
-
-
-
-
+
+
+
HN induces STAT3 activation
(min)
•STAT3 is a transcription factor.
•STAT3 is activated when its Tyr705 is phosphorylated by Jak, and
modulates transcription.
•Overexpression of dominant negative STAT3 inhibits anti-neuronal death
activity of HN. (Hashimoto et al. 2005)
Humanin activates ERK and Stat3 in β cells
0
15’
30’
4h
8h
24h
(Time)
pERK
Total ERK
pStat3
Total Stat3
Summary
• Humanin protects against serum-starvation
induced apoptosis in vitro
• Humanin inhibits cytokine-induced β cell
apoptosis in vitro
• ERK and Stat3 activation may be a
mechanism by which Humanin induces
survival.
• Humanin is a unique survival peptide with
interesting links to the IGF-system, and is a
potential therapeutic agent in Type 1
Diabetes.
Thank you
•
•
•
•
Kuk-Wha Lee
Pinchas Cohen
Laura Cobb
Vladislava PaharkovaVatchkova
• David Hwang
• Jyotsna Keni
•
•
•
•
Hidekazu Hosono
Hiromi Nakamura
Paulette Yamada
Michael Hakimi
Funding:
• McCabe T32
• ICPTP T32
Pancreas/Liver
Transdifferentiation
ES
DE
Ins+
Directed Differentiation
of ES cells
A Potential Role For Humanin?
Human Islet
Transplantation
Beta cell Replication
and Regeneration
β