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

Bif-1 deficiency impairs lipid homeostasis and causes obesity accompanied by insulin resistance Ying Liu 1 , Yoshinori Takahashi 2, 4 , Neelam Desai 2 , Jun Zhang 3 , Jacob M. Serfass 1 , Yu-Guang Shi 3 , Christopher J. Lynch 3 , and Hong-Gang Wang 1, 2, 4 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 Supplementary Information

a b Bif-1 KO HFD 60 *** WT HFD 25 Bif-1 KO CD 50 WT CD 20 Body weight (g) *** Fat mass (grams) 15 * 40 10 30 5 20 0 0 2 4 6 8 10 12 14 16 WT Bif-1 KO WT Bif-1 KO Weeks on CD/HFD CD HFD c d ** 40 50 40 ** 30 *** Lean mass (grams) 30 Fat% 20 20 10 10 0 0 WT Bif-1 KO WT Bif-1 KO WT Bif-1 KO WT Bif-1 KO CD HFD 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.

a b 3 Insulin (ng/ml) Leptin (ng/ml) 2 .0 2 1 .5 1 1 .0 0 WT Bif-1-KO WT Bif-1-KO 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.

a 1.2 Male WT Bif-1 KO 0.8 RER 0.4 0.0 y t h a g D i N b 1.2 Female WT Bif-1 KO 0.8 RER 0.4 0.0 y t h a g D i N 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 V CO2 and V O2 measured during a 48 h- cycle (n=4). All values are mean ± SEM.

a b Body weight (gram) 40 30 35 20 Fat (%) 30 25 10 20 0 15 O O T T K K W W 1 1 - - f f i i B 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.

a b c ** (Relative expression) (Relative expression) (Relative expression) * * 1.5 * 1.5 Lamp1 1.0 Atg9a 1.0 1.0 Bif-1 0.5 0.5 0.5 0.0 0.0 0.0 CD HFD c h c h c h c h WT Bif-1 KO WT Bif-1 KO WT d e (Relative expression) (Relative expression) * 3 15 ns 2 10 P62 Plin1 1 5 0 0 b f b f b f b f Bif-1 KO WT WT Bif-1 KO f g h (Relative expression) (Relative expression) *** ** (Relative expression) ns ns 30 ns *** 20 2.0 15 20 1.5 Poly-ubi Plin1 P62 10 1.0 10 5 0.5 0 0 0.0 - + - + insulin - + - + insulin - + - + insulin WT Bif-1 KO WT Bif-1 KO WT Bif-1 KO i j ns ns (Relative expression) ns (Relative expression) ns 2.5 2.0 2.0 Atg9a Lamp1 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 c h c h c h c h WT Bif-1 KO WT Bif-1 KO 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.