NAG-1/GDF15 prevents obesity by increasing thermogenesis, lipolysis and oxidative
metabolism
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Kali Chrysovergis , Xingya Wang , Justin Kosak1,4, Seong-Ho Lee3, Jong Sik Kim1, Julie F.
Foley2, Greg Travlos2, Shubha Singh1, Seung Joon Baek3 and Thomas E. Eling1¶
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Supplemental Figure Legends
Supplemental Figure 1: Food consumption is similar between WT and hNAG-1 Tg mice
(A) No difference in food consumption was observed between male or female WT (black
bars) and hNAG-1 Tg mice (white bars). Data is presented as ± SE. (B-C) Relative WAT (B) and
BAT (C) weights of WT and hNAG-1 mice on LFD or HFD. Data is presented as ± SE.
**p<0.01 and ***p<0.001 as determined by Student’s t-test.
Supplemental Figure 2: hNAG-1 Tg mice are resistant to obesity by HFD
Female hNAG-1 Tg mice gain little or no weight on HFD compared to WT littermates.
(A) All body weights of hNAG-1 Tg mice on both control (hNAG-1 Tg 10% diet- open square
and WT 10% diet- closed diamond) and HFD (hNAG-1 Tg 60% diet- x mark and WT 60% dietgray triangle) are significant to at least p<0.05. (B) The lack of weight gain occurs despite
similar food intake. (C) Female hNAG-1 Tg mice on HFD have little percentage body weight
change compared to WT littermates. Serum analysis of NAG-1, IGF-1, insulin, and leptin levels
in female mice by ELISA. Data is presented as ± SE. *p<0.05 and **p<0.01 as determined by
Mann-Whitney t-test for ELISA analysis. All other data is presented as ± SE. ***p<0.001 as
determined by Student’s t-test.
Supplemental Table 1A-B: Analysis of serum from HFD WT and hNAG-1 Tg mice
Serum cholesterol, triglycerides, low density lipid protein (LDL), high density lipid
protein (HDL), free fatty acids (FFA), glucose, insulin, leptin, and IGF-1 levels in male
(Supplemental Table 2A) and female (Supplemental Table 2B) WT and hNAG-1 Tg mice. Data
is presented as ± S.E. *p<0.05, **p<0.01, and ***p<0.001 as determined by Mann-Whitney ttest.
Supplemental Figure 3: NAG-1 protects against obesity in a genetic model
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Female Ay and Ay/NAG-1 Tg mice data (n=5-7 mice per group). (A) Lower body weights
of Ay/NAG-1 Tg mice (black squares) compared to Ay littermates (white diamonds). (B) Levels
of BAT and the components of WAT (inguinal, retroperitoneal, and gonadal) are reduced in
Ay/NAG-1 Tg mice compared to Ay littermates. Data is presented as ± SE. *p<0.05, **p<0.01,
and ***p<0.001 as determined by Student’s t-test.
Figure 4: hNAG-1 mice have decreased deleterious effects of obesity
H&E slides of liver (A) and WAT (B) (n=4 mice per group). (A) hNAG-1Tg mice have
very little steatosis compared to WT mice on HFD (upper left panel- WT control diet, upper right
panel- hNAG-1 control diet, lower left panel- WT HFD, and lower right panel- hNAG-1 HFD).
Magnification 10x. (B) hNAG-1 mice develop fewer CLRs (upper left panel- hNAG-1 control
diet, upper right panel- hNAG-1 HFD) compared to WT littermates (lower left panel- WT
control, and lower right panel- WT HFD). Magnification 20x. (C) Graphical representation of
degree of hepatic steatosis and the development of CLR in HFD mice. (D) Real-time PCR
analysis of the relative gene expression of macrophage marker F4/80 in WAT of WT and
hNAG-1 HFD mice. Data are presented as ± SE. *p<0.05 and **p<0.01 as determined by
Student’s t-test.
Figure 5: hNAG-1 mice have higher glucose tolerance
Glucose tolerance test (A). hNAG-1 female mice utilize glucose more efficiently than
WT littermates over time (n=4 mice per group). (B) Ay/NAG-1 female mice utilize glucose more
efficiently compared to Ay mice (n=5-7 mice per group). (C-D) hNAG-1 mice have lower serum
levels of insulin, leptin, and IGF-1 (n= 4-6 mice per group). (E) Serum leptin and insulin levels
of Ay and Ay/NAG-1 mice (n=5-7 mice per group). Data are presented as ± SE. *p<0.05,
**p<0.01, and ***p<0.001 as determined by Student’s t-test.
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Supplemental Figure 6: hNAG-1 transgenic mice have smaller adipocytes
H&E sections of liver (A) and WAT (B) of male WT and hNAG-1 Tg mice (n=6 mice
per group). (A) H&E staining of WT and hNAG-1 Tg mice. Magnification 10x. (B) H&E
staining of WT and hNAG-1 Tg WAT. Adipocytes of hNAG-1 Tg mice (lower right panel) are
smaller compared to WT littermates (lower left panel). Magnification 20x. (C) Adipocyte
diameter counts. Data is presented as ± SE. ***p<0.001 as determined by Mann-Whitney t-test.
Supplemental Figure 7: Western analysis of BAT
Western analysis of UCP-1, PGC1-α, PGC1-β, and GAPDH in BAT of regular diet (A)
and LFD (B) WT and hNAG-1 mice (n=5 mice per group for regular diet WT and hNAG-1
mice; n=4 per group for LFD mice). Supplemental Figure 8: NAG-1 actions in obesity
Obese C57BL/6 mice xenografted with stably transduced with vector (Control) or
hNAG-1 B16/F10 melanoma cells. Lipolysis relative gene marker expression in the BAT is not
changed in obese hNAG-1 tumor bearing mice as compared to obese control tumor bearing mice
(n=6 mice per group).
Supplemental Materials and Methods
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Liver and fat analysis- Thirteen-week-old 1377 mice (12 WT males, 12 WT females, 12 NAG-1
Tg males, and 12 NAG-1 Tg females) were euthanized and liver and fat tissue was taken for
analysis. Tissues from 6 mice from each genotype and gender were collected for histological
analysis. The remaining tissues were collected, snap frozen, and stored at -80ºC until further
analysis.
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