Cell Metabolism, Volume 4

Supplemental data
Granuphilin is activated by SREBP-1c and involved in impaired
insulin secretion in diabetic mice
Toyonori Kato, Hitoshi Shimano, Takashi Yamamoto, Tomotaka Yokoo, Yuko Endo,
Mayumi Ishikawa, Takashi Matsuzaka, Yoshimi Nakagawa, Shin Kumadaki, Naoya
Yahagi, Akimitsu Takahashi, Hirohito Sone, Hiroaki Suzuki, Hideo Toyoshima, Alyssa
H. Hasty, Satoru Takahashi, Hiroshi Gomi, Tetsuro Izumi, and Nobuhiro Yamada

Supplemental figures
Figure S1Copy Number of hSREBP-1c

2500

1200hSREBP-1c
mSREBP-1c

Copy Number of mSREBP-1c

A

1000

2000

800

1500

600
1000

400

500

200
0Cer
eb
rum
Cerebellum
H
ip
pocam
H
pu
yps
ot
halam
us
Is
let

0200

N.S

100

Is
let

yp
ot
hal

am
us

0m
pus

eb
ru
m
C
er
eb
ellu
m
H
ip
poca
m
H
pu
yps
ot
halam
us
Is
let

C
er

er

Cer

eb
rum
eb
ellu
H
m
ip
pocam
H
pu
ypsot
halam
us
Is
let

0

*300

H50

*TgRIP-SREBP-1cippoca

100

WT-Control

H

150

400ebrumebellum0

TgRIP-SREBP-1c

N.SCer50

WT-Control

FAS expression (% of WT-Control)

100200Cer

TgRIP-SREBP-1c

IRS-2 expression (% of WT-Control)

WT-Control

150CNPY Expression (% of WT-Control)

BFood Intake (g/24 hr)C

58 weeks
24 weeks

43210

WTTgRIP-SREBP-1c

Figure S1 Transgene (human nuclear SREBP-1c) expression in brain and pancreatic
islets of transgenic mice under rat insulin promoter I(A)mRNA levels of the human and mouse SREBP-1c on brain fractions and pancreatic

islets from male age-matched (15-18 weeks-old) WT-Control or TgRIP-SREBP-1c mice as
estimated by real-time PCR.

hSREBP-1c levels were calculated as copy numbers.

(B)

mRNA levels of NPY, IRS-2, and FAS on the indicated tissues from the indicated mice as
estimated by real-time PCR.
(C)

* p < 0.01 (vs WT-Control mice). N.D; not detectable.

Analysis of food intake from male 8 weeks-old or 24 weeks-old mice over 24 hrs

using metabolic cage system.

Results are expressed by mean ± SEM.

performed in triplicate from three to four mice per replicate.

Studies were

Figure S2

Figure S2

DNA sequences of mouse granuphilin promoter

Mouse granuphilin promoter sequences.

The consensus sequences of transcriptional

phE

102000

500

LG
HG

20RN
Ai
-7
84

20600

LacZ
i

200
1000

ilina

400
1200

ph600

Insulin secretion (pg/ng DNA/hr)

800
1400

ranu

15ATP/ADP Ratio

RN
Ai-7
84
RN
Ai-1
95
5CFP

RN
Ai
-784

ilina

La
cZiphFP

B 1000G5ilinaranu

G0LacZi

ranu

G

Relative mRNA Levels(Normalized to cyclophilin)

ranu

FPphilLa
cZ in-aiRN
Ai-784RN
Ai-1
95
5GGAGGFP

DG

Insulin Content(ng/ng DNA/hr)factors are underlined (dotted: potential consensus).

Figure S3

Granuphilin

α-tubulin

GFP
Granuphilin-a-Ad
LacZi-Ad
RNAi-784-Ad#800*400*KCl

LG
HG

1510

Figure S3

Effects of adenovirus-mediated over-expression and knockdown ofgranuphilin on insulin secretion in murine pancreatic islets
Islets were isolated from male C57BL/6 mice (10 weeks-old) and were infected withadenoviral-GFP, -granuphilin-a or -granuphilin-RNAi (RNAi-784, -1955) (500 MOI) for 48
hrs.

(A) Immunoblot analysis on total proteins of the islets with antibody against mouse

granuphilin, and α-tubulin as a loading control.

(B)

the islets as estimated by real-time PCR. (C)LG-, HG-, and KCl-stimulated insulin

mRNA level of granuphilin fromsecretion in the islets treated with adenoviral-GFP (white bars), -granuphilin-a (black bars),-LacZ-RNAi (light grey bars), and –granuphilin-RNAi-784 (dark grey bars).

* p <0.05(vs GFP) and # p < 0.05 (vs LacZi). (D) Cellular insulin contents from the islets.
Cellular ATP/ADP ratio from the islets.

(E)

Results were normalized to cellular DNA content(C and D) and are expressed by mean ± SEM. Studies were performed in triplicate withsets of islets pooled from three to four mice per replicate.

200100

C

400
C57BL/6 LacZi-Ad
KK-Ay-LacZi-Ad
KK-Ay-Granu-RNAi-Ad

SREBP-1(+/+)-Control
SREBP-1(+/+)-PA
SREBP-1(-/-)-ControlSREBP-1(-/-)-PA*300*200100*2001000M
af
A
Rab3
Syntaxin-1
M
un ac1
8
SN -1
AP
25FA
SUCP2

*SREG BPra -1nu cphi
M lin
af
A
RSy abnt 3axM inu n 1ac1
SN 8-1AP25FA
SUC
P2500

*

B

00F

M
afA
Rab3
Sy
ntax
in-1
M
un ac1
8
SN -1
AP
25FA
SUC
P2300
TgRIP-SREBP-1c-Granu-RNAi-Ad

Relative mRNA Levels(Normalized to cyclophilin)

TgRIP-SREBP-1c-LacZi-Ad

Relative mRNA Levels(Normalized to cyclophilin)

M
af
A
Ra
b3
Sy
ntax
in-1
M
un ac18SN -1
AP25FA
SUC
P2

Relative mRNA Levels(Normalized to cyclophilin)

400 WT-Granu-RNAi-Ad

Relative mRNA Levels(Normalized to cyclophilin)

M
af
A
Ra
b3
Sy
nt
ax
in-1
M
un ac18SN -1
AP25FA
SUC
P2

Relative mRNA Levels(Normalized to cyclophilin)

300Rab3
Sy
ntax
in-1
M
un ac18SN -1
AP25FA
SUCP2EM
afA

Relative mRNA Levels(Normalized to cyclophilin)

Figure S4
WT-LacZi-Ad
A
500400300 **500

400300C57BL/6 LacZi-Ad
KK-Ay-LacZi-Ad
KK-Ay-Granu-RNAi-Ad

500*400**

Control-LacZi-Ad
Control-Granu-RNAi-Ad
PA+LacZi-Ad
PA+Granu-RNAi-Ad*300*

2001000D

MF-LacZi-Ad
MF-Granu-RNAi-Ad
HF-LacZi-Ad
HF-Granu-RNAi-Ad
HF/HS-LacZi-Ad
HF/HS-Granu-RNAi-Ad*200***1000**

2001000Figure S4(A)Gene expression profiles

mRNA levels of the fusion machinery for exocytosis of insulin granules andSREBP-1c target genes from WT-Control or TgRIP-SREBP-1c mice islets as estimated byreal-time PCR.

* p < 0.01 (vs WT-LacZi). (B)mRNA levels of indicated genes fromC57BL/6 or KK-Ay mice islets as estimated by real-time PCR.
C57BL/6-LacZi).

(C)* p < 0.01 (vsmRNA levels of indicated genes from C57BL/6 or ob/ob miceislets as estimated by real-time PCR. * p < 0.01 (vs C57BL/6-LacZi).

(D)

mRNA

levels of indicated genes from diet-inducibed obese (DIO) mice islets as estimated byreal-time PCR.

* p < 0.01 (vs MF-LacZi). (E)mRNA levels of indicated genes fromSREBP-1-null (SREBP-1(-/-)) and wild-type littermate (SREBP-1 (+/+)) mice islets asestimated by real-time PCR.

* p < 0.01 (vs wild-type littermate (SREBP-1(+/+))-Control). (F) mRNA levels of indicated genes from PA-treated or control islets asestimated by real-time PCR.

* p < 0.01 (vs Control-LacZi).

Each islets were infectedwith adenoviral-LacZ-RNAi (LacZi) or -granuphilin-RNAi (Granu-RNAi) (500 MOI) for48 hrs.

Results are expressed by mean ± SEM. Studies were performed in triplicate withsets of islets pooled from three to four mice per replicate.

Figure S5A
Insulin Secretion (pg/ng DNA/hr)

1400

B
C57BL/6 KK-Ay

1200
RNAi

1000
p <0.05600

p <0.08

Granuphilin*α-tubulin

*

400
200

C1200

LacZ LacZ BP-1c

SREBP-1(Nuclear)

8000Insulin Secretion (pg/ng DNA/hr)

C57BL/6+LacZi-Ad
KK-Ay+LacZi-Ad
KK-Ay+SREBP-1-RNAi-Ad

LG

HG

KCl

C57BL/6+LacZi-Ad
ob/ob+LacZi-Ad
ob/ob+SREBP-1-RNAi-Ad

D
C57BL/6 ob/ob
RNAi

1000

SREBP-1(Nuclear)

p <0.05800
Granuphilin

600*p <0.05*4002000LGHGKClα-tubulin

LacZ LacZ BP-1c

Figure S5Effects of knockdown of SREBP-1 on insulin secretion in pancreatic islets

from different diabetic model mice
Islets were isolated from male C57BL/6 mice, KK-Ay or ob/ob mice (10 week-old) andwere infected with adenoviral-LacZ RNAi (LacZi-Ad) or SREBP-1 RNAi (500 MOI) for
48 hrs.

(A)

LG-, HG-, and KCl-stimulated insulin secretion in the indicated islets;

C57BL/6-LacZi (white bars), KK-Ay-LacZi (black bars), KK-Ay-SREBP-1-RNAi (greybars).

* p <0.05 (vs C57BL/6-LacZi).

(B)

Immunoblot analysis of SREBP-1 andmouse granuphilin from the indicated islets with α-tubulin as a loading control. (C) LG-,HG-, and KCl-stimulated insulin secretion in the indicated islets; C57BL/6-LacZi (whitebars), ob/ob-LacZi (black bars), ob/ob-SREBP-1-RNAi (grey bars).

* p <0.05 (vsC57BL/6-LacZi). (D) Immunoblot analysis of SREBP-1 and mouse granuphilin fromthe indicated islets with α-tubulin as a loading control.

Results were normalized tocellular DNA content (A and C) and are expressed by mean ± SEM.

Studies wereperformed in triplicate with sets of islets pooled from three to four mice per replicate.