AMPK-mediated KATP channel trafficking is a key
mechanism for glucose-dependent regulation of
-cell excitability
Seoul National University College of Medicine
Department of Physiology
Sun Hyun Park, Ajin Lim, Young Sun Ji, Ju Hong Jeon, Suk Ho Lee, Won-Kyung Ho
The role of pancreatic KATP channel in insulin secretion.
high ATP
low ATP
open
close
KATP channels:
Molecular sensors of cellular metabolism; targets for anti-diabetics
SUR1
Kir6.2
•Kir6.2:
ATP binding site (inhibition)
PIP2 binding site (activation)
•SUR1:
MgADP binding site (activation)
Sulfonylurea binding site (inhibition)
KATP current is determined by ATP-dependent gating (changes in Po)
Po
KATP current
1.0
0.8
0.6
0.4
0.2
0.0
1
10
100
1000
10000
ATP concentration (M)
high PO
Changes in Po
High ATP
Low ATP
low PO
KATP channel activating mechanisms known so far
are known to increase gating by decreasing ATP sensitivity
high PO
Lowering
ATP sensitivity
low PO
Current (I) is determined by
Open probability (PO) and channel number (N)
I= N X PO
high PO
low PO
low N
high N
Question 1
Is KATP channel density regulated by glucose concentrations?
KATP current = N x Po
2.0
Changes in N
1.5
1.0
0.5
0.0
1
10
100
1000
ATP concentration (M)
10000
high glucose
low glucose
Kir6.2
trafficking
to surface memb
Which signaling mechanism are activated during
glucose deprivation to promote KATP channel trafficking?
1. Effect of Compound C, AMPK inhibitor
Kir6.2(S)
Kir6.2(T)
Kir2.1(S)
2. Effect of AICAR, AMPK activator
Kir6.2(S)
Kir6.2(T)
Kir2.1(S)
Kir6.2/DiI/DAPI
Lim et al., (2009) Diabetes
Physiological significance is uncertain:
AMPK was not activated at fasting glucose level in INS-1 cells
INS-1 cells
pAMPK
AMPK
Question 2
•Does AMPK-dependent trafficking
occur in-vivo during fasting?
Fasted
SUR1
Fed
5 m
10 m
10 m
Kir6.2
5 m
Glucose: 244 mg/dl (13.5 mM) 138 mg/dl (7.7 mM)
AMPK is activated at fasting glucose not in vitro, but in vivo
Islets
INS-1 cells
pAMPK
pAMPK
AMPK
AMPK
Hypothesis:
Endogenous ligands promote AMPK activation in normal fasting state
Leptin
a pivotal regulator of energy homeostasis
ob/ob mouse
Leptin-deficient ob/ob mice show
-hyperinsulinemia
-diabetes
insulin
leptin
KATP channel trafficking is impaired in leptin deficiency
ob/ob fasted + Lep
Kir6.2
SUR1
ob/ob fasted
10 m
Islets
INS-1 cells
pAMPK
pAMPK
AMPK
AMPK
ob/ob Islets
AMPK can be activated in normal fasting state in the presence of leptin
Leptin promotes AMPK activation and KATP channel trafficking
Cont
Control
Leptin promotes AMPK activation and KATP channel trafficking
Question 3
•Signaling Mechanisms involved in
leptin-induced AMPK activation?
Leptin-induced AMPK activation is mediated by CaMKK
Leptin increases NSC by activating TRPC4, but not TRPC5
Activation of AMPK and KATP by leptin is blocked by siTRPC4
Low glucose
Leptin
TRPC4/CaMKK
decrease ATP
AMPK
KATP trafficking
KATP opening
increase in KATP surface density
Stabilize pancreatic -cells to inhibit insulin secretion
Park et al., PNAS, 2013 Jul
Gating
mechanism
Trafficking
mechanism
Acknowledegment
Members of Cell Physiology Lab
이석호
지영선
한영은
Dr. Lim
Dr. Park
류신영
Park et al., PNAS, 2013 Jul
Question 4
•Do leptin-induced AMPK activation and
KATP channel trafficking have functional
significance?
-cell RMP and pAMPK responds to physiological range of
glucose in the presence of low concentration of leptin
6 mM Glucose
fasting
-leptin
+leptin
pAMPK
-leptin
fed glucose
fasting
+leptin
fed glucose
-cell RMP and pAMPK responds to physiological range of
glucose in the presence of low concentration of leptin
6 mM Glucose
Physiological range
Glucose (mM)
11 mM Glucose
-cell RMP correlates linearly with pAMPK
pAMPK
Energy status (glucose) and hormonal signal (leptin)
are integrated into AMPK activity
glucose (ATPi) dependence of -cell RMP depends on leptin
-leptin
+leptin
fed glucose
fasting glucose
Energy status (glucose) and hormonal signal (leptin)
are integrated into AMPK activity
Actin depolymerization is induced by glucose deprivation,
and this effect is AMPK-dependent.
A
B
AMPK-dependent KATP channel trafficking is inhibited by Rac inhibition
A
GD
GD + NSC
Kir 6.2
11G
AMPK/Rac
B
11G+Leptin Leptin + NSC
Kir 6.2
11G
K+
C
AMPK-dependent KATP channel trafficking is inhibited by Myosin II inhibition



+
+

+
+
C
Kir6.2-S
Kir6.2-T
Leptin
-20
RMP (mV)
Blebb
Leptin
Blebbstatin
11G
+Leptin
actin
-30
-40
-50
-60
-70
1800
*
3
*
2
1
0
Blebb
Leptin



+
+

1900
2000
2100
2200
blebbistatin
s
2300
-20
RMP (mV)
B
Control
11G
+Leptin
Kir6.2-S
A
+
+
-30
-40
-50
-60
-70
15.1
15.2
15.3
ks
15.4
15.5
MRLC underlies AMPK-dependent KATP channel trafficking
siAMPK
Leptin
+

+

+


+
+
B
scRNA
+


siMRLC
Leptin
pMRLC
Kir6.2-S
MRLC
Kir6.2-T
pMLCP
Kir2.1-S
MLCP
Kir2.1-T
pAMPK
MRLC
AMPK
actin

+

+

+

+
+
C
Leptin ()
Leptin (+)
scRNA
+


siMRLC
scRNA
actin
*
*
*
4
0
+


+

+

+


+
+
2
1
0
scRNA
siMRLC
Leptin
***
6
G (nS/pF)
2
scRNA
siAMPK
Leptin
D
*
3
Kir6.2-S
pMRLC/MRLC
A
+


+

+

+


+
+
***
3
0
scRNA
siMRLC
Leptin





+
+

+

+
+
PTEN


Leptin

+ 

G129E
B
Y138L
A
C124S
AMPK/PTEN pathway mediates KATP channel trafficking
D
Kir6.2

WT
pMRLC
MRLC
pMLCP
C124S
MLCP
Kir6.2-S
Kir6.2-T
C
E
GDP
GTPS
PTENC124S
Leptin
Rac2-GTP
Rac2 (T)

+


+










+


+
