Beneficial impact of Gpnmb and its significance as a biomarker in nonalcoholic steatohepatitis
Akihiro Katayama1, Atsuko Nakatsuka1, Jun Eguchi1, Kazutoshi Murakami1, 2, Sanae Teshigawara1, Motoko Kanzaki1, Tomokazu Nunoue1, Kazuyuki Hida3, Nozomu Wada4,
Tetsuya Yasunaka4, Fusao Ikeda4, Akinobu Takaki4, Kazuhide Yamamoto4, Hiroshi Kiyonari5, 6, Hirofumi Makino1, and Jun Wada1
1Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Kita-ku, Okayama 700-8558, Japan
2Department of General Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Kita-ku, Okayama 700-8558, Japan
3Department of Diabetes and Metabolism, National Hospital Organization Okayama Medical center, Kita-ku, Okayama 701-1154, Japan
4Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Kita-ku, Okayama 700-8558, Japan
5Animal Resource Development Unit and 6Genetic Engineering Team, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima Minami, Chuou-ku, Kobe 650-0047, Japan
Supplementary Figure legends
Supplementary Figure 1 mRNA expression levels of Gpnmb in OLETF rats and
generation of Gpnmb transgenic (Tg) mice. a. Northern blot analyses in various tissues in obese OLETF rats (O) and lean LETO rats (L). b. Western blot analyses of mature
adipocytes and stromal vascular fraction (SVF) in wild type (WT), Gpnmb Tg and Gpnmb-/- mice under high fat high sucrose (HFHS) chow. c. Schematic diagram showing structure of transgene consisting of aP2 promotor, β-globin intron, coding region of Gpnmb cDNA and hGH polyA tail. Primers, AP2, Gpnmb-Tg-AS, Gpnmb-sequence-S1 and GnnmbAS1 were used for the screening for the Gpnmb Tg mice. d. Gpnmb Tg mice #1, #12 and #13 demonstrated 824 and 600 bp PCR products using primer sets indicated in panel c.
Supplementary Figure 2 Body weight (BW), fat pad weight and glucose metabolism in Gpnmb-/- mice. a. Western blot analyses in epididymal fat of Gpnmb-/- and Gpnmb+/+ mice under high fat-high sucrose (HFHS) chow. b. Body weight of Gpnmb-/- and Gpnmb+/+ male C57BL/6JJcl mice fed with HFHS and standard (STD) chow. c. Fat pad weight of Gpnmb-/- and Gpnmb+/+ mice at 25 weeks of age. d. The size distribution and the average size of adipocytes in epididymal adipose tissues of Gpnmb-/- and Gpnmb+/+ mice under HFHS chow.
e. Periodic acid-Schiff (PAS) staining of epididymal adipose tissues of Gpnmb-/- and Gpnmb+/+ mice under HFHS chow. f and g. Glucose and insulin tolerance test of Gpnmb-/- and Gpnmb+/+ mice under HFHS chow at 15 weeks of age. All data are presented as mean ± standard deviation (SD). n=8.
Supplementary Figure 3 Serum lipid profile and evaluation of liver fibrosis in Gpnmb-/- and Gpnmb+/+ mice. a. Total cholesterol, triglyceride, and LDL cholesterol in sera of Gpnmb-/- and Gpnmb+/+ mice fed HFHS chow. b. Sirius red staining of liver tissues of Gpnmb-/- and Gpnmb+/+ mice fed HFHS chow. c. Liver fibrosis areas of Gpnmb-/- and
Gpnmb+/+ mice fed HFHS chow. d. Liver hydroxyproline contents of Gpnmb-/- and Gpnmb+/+
mice fed HFHS chow. All data are presented as mean ± standard deviation (SD). n=5.
**p<0.01 vs. Gpnmb+/+ mice.
Supplementary Figure 4 Body weight (BW), fat pad weight and glucose metabolism in Gpnmb transgenic (Tg) mice. a. Western blot analyses in epididymal fat of Gpnmb Tg and wild type (WT) mice under HFHS chow. b. Body weight of Gpnmb Tg and WT male
C57BL/6JJcl mice fed with HFHS and STD chow. c. Fat pad weight of Gpnmb Tg and WT mice at 25 weeks of age. d. The size distribution and the average size of adipocytes in epididymal adipose tissues of Gpnmb Tg and WT mice under HFHS chow. e. Periodic acid-Schiff (PAS) staining of epididymal adipose tissues of Gpnmb Tg and WT mice under HFHS chow. f and g. Glucose and insulin tolerance test of Gpnmb Tg and WT mice under HFHS chow at 15 weeks of age. All data are presented as mean ± standard deviation (SD).
n=8.
Supplementary Figure 5 Serum lipid profile and evaluation of liver fibrosis in Gpnmb Tg and WT mice. a. Total cholesterol, triglyceride, and LDL cholesterol of Gpnmb Tg and WT mice fed HFHS chow. b. Sirius red staining of liver tissues of Gpnmb Tg and WT mice fed HFHS chow. c. Liver fibrosis areas of Gpnmb Tg and WT mice fed HFHS chow. d. Liver hydroxyproline contents of Gpnmb Tg and WT mice fedHFHS chow. All data are presented as mean ± standard deviation (SD). n=8. **p<0.01 vs. WT mice.
Supplementary Figure 6 Quantitative RT-PCR in liver tissues. a. mRNA expression of Sod1, Sod2, Cat, Gpx1, Cybb, and Ncf1 were not altered in Gpnmb-/- and Gpnmb+/+ mice fed with high fat high sucrose (HFHS) chow. b. mRNA expression of Sod1 and Cat, anti-oxidative stress genes, were significantly increased in Gpnmb Tg mice. c and d. The oxidative stress in the liver demonstrated by malondialdehyde (MDA) was ameliorated in Gpnmb Tg mice compared with wild type (WT) mice fed with HFHS chow. All data are
presented as mean ± standard deviation (SD). n=8. *p<0.05, **p<0.01 vs. WT mice.
Supplementary Figure 7 mRNA and protein expression of Gpnmb. a. Relative mRNA expression of Gpnmb in epididymal fat and liver in Gpnmb transgenic (Tg) and wild type (WT) mice. All data are presented as mean ± standard deviation (SD). n=5 b. Western blot of Gpnmb and calnexin in plasma membrane and cytosolic fractions in liver tissues of Gpnmb-/- and Gpnmb Tg mice.
Supplementary Figure 8 mRNA expression of COL1A1, MMP3, ACTA1, cell morphology and oxidative stress in LI90 cells transfected with p3xFLAG-mGpnmb. a.
Relative mRNA expression of COL1A1, MMP3, ACTA1 and mGpnmb in LI90 cells transfected with p3xFLAG-mGpnmb. b. Immunofluorescence staining using anti-FLAG antibody in LI90 cells transfected with p3xFLAG-mGpnmb. c. Dihydroethidium staining and fluorescence intensity in LI90 cells transfected with p3xFLAG-mGpnmb. All data are presented as mean ± standard deviation (SD). n=3. **p<0.01 vs. LI90 cells transfected with p3xFLAG-Vector.
Supplementary Figure 9 Serum GPNMB levels in the patients with nonalcoholic fatty liver disease (NAFLD). a. Serum GPNMB levels and NAS score in the patients with NAFLD.
They were classified into three groups according to the NAS score. b. Serum GPNMB levels and necroinflammatory grading in the patients with NAFLD. They were classified into three groups (grade 1-3) according to the degree of liver steatosis and inflammation.
Supplementary Figure 10 Schematic drawing for the function of Gpnmb. Gpnmb ameliorates fat accumulation and fibrosis in the liver of diet-induced obesity mice by reducing the oxidative stress. Gpnmb in hepatic macrophages and stellate cells interacts with calnexin and the serum soluble GPNMB is elevated in the patients with non-alcoholic
steatohepatitis.
Supplementary Figure 11 Uncropped images for the blots shown in Figure 1b, Supplementary Figure 2a, Supplementary Figure 3a, and Figure 4a.
Supplementary Figure12 Uncropped images for the blots shown in Figure 5.
Supplementary Figure 1
aP2 promoter β-globin Gpnmb polyA
AP2
Gpnmb-sequence-S1 Xho I
KpnI EcoRI/SalI SphI/EcoRINotI
5.5kb 0.6kb 1.7kb 0.5kb
Gpnmb-Tg-AS
GpnmbAS1
c
AP2 x Gpnmb-Tg-AS (824 bp) Gpnmb-sequence-S1 x GpnmbAS1 (600bp) M 1 2 3 4 5 6 7 8 9 10 11 12 13
14 15
M 1 2 3 4 5 6 7 8 9 10 11 12 13
d
28S Gpnmb 18S
Differentially expressed Gpnmb in various tissues of OLETF rats
a
b
Western blot analysesMature adipocytes and stromal vascular fraction
WT (HFHS)
Gpnmb Tg (HFHS)
Adipocytes SVF Adipocytes SVF Adipocytes SVF
Gpnmb-/- (HFHS) 65 kDa
Gpnmb
100 kDa
β-actin
WT (HFHS)
Gpnmb Tg (HFHS)
Gpnmb-/- (HFHS)
Adipocytes SVF Adipocytes SVF Adipocytes SVF 75 kDa
Supplementary Figure 2
a b
Body weight (g)
Gpnmb-/-(HFHS) Gpnmb+/+ (HFHS) Gpnmb-/-(STD) Gpnmb+/+ (STD)
d
e
100μm 100μm Gpnmb-/-(HFHS)
Gpnmb+/+(HFHS)
0 50 100 150 200 250 300 350
0 15 30 60 90 120 Glucose tolerance test
40 80 120 160 200
0 15 30 60 90 120 (1.0 IU/kg weight)
Insulin tolerance test
f
Blood glucose (mg/dl) Blood glucose (mg/dl)
(min) (min)
Gpnmb-/-(HFHS) Gpnmb+/+ (HFHS) Gpnmb-/-(HFHS)
Gpnmb+/+ (HFHS)
g
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Adipocyte size of epididymalfat (%)
Gpnmb-/-(HFHS) Gpnmb+/+ (HFHS)
(μm²)
Western blot analyses (Epididymal fat)
GAPDH
Gpnmb-/- (HFHS) Gpnmb+/+
(HFHS) 100 kDa
65 kDa Gpnmb
0.0 10.0 20.0 30.0 40.0 50.0
5w 9w 13w 17w 21w 25w
c
Fat pad weight (g)
Gpnmb-/-(HFHS) Gpnmb+/+ (HFHS)
Gpnmb-/-(STD) Gpnmb+/+ (STD) 0.0
0.5 1.0 1.5 2.0 2.5
0 3,000 6,000 9,000 12,000
(μm²)
Mean size
Supplementary Figure 3
b
c d
0.0 20.0 40.0 60.0 80.0 100.0
Liver Hydroxyproline contents (μM) Gpnmb-/-(HFHS)
100μm
Gpnmb+/+(HFHS)
100μm
0.0 1.0 2.0 3.0 4.0
5.0 **
Fibrosis areas (%)
Gpnmb+/+
(HFHS) Gpnmb-/-
(HFHS)
Gpnmb+/+
(HFHS) Gpnmb-/-
(HFHS) 0 15 30 45 60 75
LDL-cholesterol (mg/dl)
0 40 80 120 160 200
Gpnmb-/- (HFHS)
Gpnmb+/+
(HFHS)
Total cholesterol (mg/dl)
**
a
0 15 30 45 60 75
Triglyceride (mg/dl)
Gpnmb-/- (HFHS)
Gpnmb+/+
(HFHS)
Gpnmb-/- (HFHS)
Gpnmb+/+
(HFHS)
**
Supplementary Figure 4
a b
Gpnmb Tg (HFHS)WT (HFHS) Gpnmb Tg (STD) WT (STD)
Body weight (g)
Western blot analysse (Epididymal fat) Gpnmb
β-actin
WT (HFHS)
Gpnmb Tg (HFHS)
Fat pad weight (g)
Gpnmb Tg (HFHS) WT (HFHS)
GpnmbTg (STD) WT (STD)
c d
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0
8.0 Gpnmb Tg (HFHS) WT (HFHS)
Adipocyte size of epididymalfat (%)
(μm²)
e
100μm 100μm Gpnmb Tg (HFHS)
WT (HFHS)
f
Glucose tolerance testg
Blood glucose (mg/dl)
(min) Gpnmb Tg (HFHS) WT (HFHS) 0
50 100 150 200 250 300
0 15 30 60 90 120
(1.0 IU/kg weight)
Insulin tolerance test
Blood glucose (mg/dl)
(min) Gpnmb Tg (HFHS) WT (HFHS) 40
80 120 160 200
0 1530 60 90 120
0 3,000 6,000 9,000
12,000 Mean size
(μm²)
0 0.5 1 1.5 2
15.0 20.0 25.0 30.0 35.0 40.0 45.0
5w 9w 13w 17w 21w
100 kDa
Supplementary Figure 5
Gpnmb Tg (HFHS)
100μm WT(HFHS)
100μm
Liver Hydroxyproline contents (μM)
Fibrosis areas (%)
WT (HFHS) Gpnmb Tg
(HFHS)
WT (HFHS) Gpnmb Tg
(HFHS)
b
c d
0.0 0.5 1.0 1.5 2.0 2.5
3.0 **
0.0 20.0 40.0 60.0
80.0 **
0 10 20 30 40
0 40 80 120 160
0 10 20 30 40 50 60
Total cholesterol (mg/dl)
a
p=0.065
Triglyceride (mg/dl) LDL-cholesterol (mg/dl)
Gpnmb Tg (HFHS)
WT (HFHS)
Gpnmb Tg (HFHS)
WT (HFHS)
Gpnmb Tg (HFHS)
WT (HFHS)
0 0.5 1 1.5 2 2.5
0 0.5 1 1.5 2
a
Supplementary Figure 6
Sod1 Sod2 Cat Gpx1 Cybb Ncf1
**
*
Relative ｍRNA expressionRelative ｍRNA expression
Sod1 Sod2 Cat Gpx1 Cybb Ncf1
b
0.0 2.0 4.0 6.0 8.0
Gpnmb Tg (HFHS)
WT (HFHS)
Liver MDA concentration (mM)
p=0.065
c
Gpnmb-/-(HFHS) Gpnmb+/+ (HFHS)
Gpnmb Tg (HFHS) WT (HFHS)
Liver MDA concentration (mM)
d
0.0 2.0 4.0 6.0 8.0
Gpnmb-/- (HFHS)
Gpnmb+/+
(HFHS)
0 2000 4000 6000 8000 10000 12000
WT Epi (HFHS)
WT Liver (HFHS)
Gpnmb Tg Epi (HFHS)
Gpnmb Tg Liver (HFHS) Relative GpnmbmRNA expression
Calnexin
GAPDH
Gpnmb-/- (HFHS)
Gpnmb Tg (HFHS)
Gpnmb-/- (HFHS)
Gpnmb Tg (HFHS)
Plasma Membrane Cytosol
Gpnmb
Supplementary Figure 7 a
b
Na+,K+-ATPase 100 kDa
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Supplementary Figure 8 a
Relative ｍRNA expression
LI90 cell (mGpnmb) LI90 cell (Vector)
** **
mGpnmb Vector
b
c
50μm
FLAG DAPI Merge
200μm mGpnmb
Vector
0 1000 2000 3000 4000 5000 6000
mGpnmb
0 0.2 0.4 0.6 0.8 1 1.2 1.4
LI90 cell (Vector) LI90 cell
(mGpnmb)
Fluorescence intensity
COL1A1 MMP3 ACTA1
Supplementary Figure 9
a b
0 10 20 30 40
grade1
(n=36) grade2
(n=18) grade3 (n=6)
Serum GPNMB levels (ng/ml)
Serum GPNMB levels (ng/ml)
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0
NAS:1-2
(n=13) NAS:3-4
(n=31) NAS:5-8 (n=16)
