Bif-1 deficiency impairs lipid homeostasis and causes obesity - - PDF document

Ying Liu 1, Yoshinori Takahashi 2, 4, Neelam Desai 2, Jun Zhang 3, Jacob M. Serfass1, Yu-Guang Shi 3, Christopher J. Lynch 3, and Hong-Gang Wang 1, 2, 4 Supplementary Information

1 Department of Pharmacology, 2 Department of Pediatrics, 3 Department of Molecular

Physiology, 4 Penn State Cancer Institute, Penn State University College of Medicine, Hershey, PA 17033 Bif-1 deficiency impairs lipid homeostasis and causes obesity accompanied by insulin resistance

b a

***

Weeks on CD/HFD Body weight (g)

5 10 15 20 25

* ***

Fat mass (grams)

10 20 30 40

** **

10 20 30 40 50

***

Fat% Lean mass (grams)

c d

2 4 6 8 10 12 14 16 20 30 40 50 60

Bif-1 KO HFD WT HFD Bif-1 KO CD WT CD WT Bif-1 KO WT Bif-1 KO CD HFD WT Bif-1 KO WT Bif-1 KO CD HFD WT Bif-1 KO WT Bif-1 KO CD HFD

Figure S1. Metabolic characteristics of male mice fed with indicated diet. (a) Mice were fed with HFD or control CD starting at 6-week of age and body weight of mice was monitored for 15

• weeks. (b-d) Loss of Bif-1 leads to larger total (b) and percent (c) fat mass and has minimal

effect on lean mass (d). Body composition of mice fed with the indicated diet for 19 weeks was measured using TD-NMR. Statistical significance was determined using one-way ANOVA followed by Bonferroni’s multiple comparison test. Oral glucose tolerance test was performed in 16 h-fasted mice, n=9-11. All values are mean ± SEM. Differences with controls were significant for *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure S2. Plasma insulin and leptin levels in 10-week-old mice. Levels of insulin (a) and leptin (b) in plasma from 10-week-old mice fed with CD were measured by ELISA assays, n=1- 2.

Insulin (ng/ml) Leptin (ng/ml)

a b

1 2 3 1 .0 1 .5 2 .0

WT Bif-1-KO WT Bif-1-KO

a b

Figure S3. Respiratory exchange ratio (RER) of WT and Bif-1 KO mice. (a, b) RER values for male (a) and female (b) mice were calculated using VCO2 and VO2 measured during a 48 h- cycle (n=4). All values are mean ± SEM.

0.0 0.4 0.8 1.2 0.0 0.4 0.8 1.2

Male Female RER RER D a y D a y N i g h t N i g h t WT Bif-1 KO WT Bif-1 KO

Body weight (gram) Fat (%)

10 20 30 15 20 25 30 35 40

W T B i f

• 1

K O W T B i f

• 1

K O

a b

Figure S4. Body weight and fat percentage in 6-week-old mice. (a, b) Body weight (a) and fat percentage over body weight (b) in control diet-fed, 6-week-old mice.

** WT Bif-1 KO

• +
• +

insulin ns WT Bif-1 KO insulin

• +
• +

*** ns WT Bif-1 KO c h c h * ** * WT Bif-1 KO c h c h 5 10 15 20 10 20 30 Atg9a (Relative expression) Lamp1 (Relative expression) WT Bif-1 KO c h c h Atg9a (Relative expression) ns ns Lamp1 (Relative expression) WT Bif-1 KO c h c h ns ns 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 WT Bif-1 KO insulin

• +
• +

*** ns Poly-ubi (Relative expression) WT Bif-1 KO f b f b * ns WT Bif-1 KO f b f b 1 2 3 P62 (Relative expression) Plin1 (Relative expression) P62 (Relative expression) 5 10 15 Plin1 (Relative expression) 0.0 1.0 2.0 0.5 1.5 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5

a b c d e f g h i j

Figure S5. Quantifications for immunoblots in Figure 6. (a-c) Quantification for immunoblots in Figure 6a. (d-e) Quantification for immunoblots in Figure 6b. (f-h) Quantification for immunoblots in Figure 6c. (j, k) Quantification for immunoblots in Figure 6d. Image J software was used in quantifications and expression of indicated proteins was normalized to β-actin. Statistical significance was determined using Student’s t-test for single comparison and One-way ANOVA for multiple comparison followed by Bonferroni’s or Dunn’s multiple comparison test. n=3-4. All values are mean ± SEM. Differences with controls were significant for *p <0.05, **p <0.01, and ***p <0.001.

CD HFD Bif-1 (Relative expression) 0.5 0.0 1.0 * WT