Volume 26, Issue 1, Pages 1-10.e7 (January 2019) CerS1-Derived C18:0 Ceramide in Skeletal Muscle Promotes Obesity-Induced Insulin Resistance  Sarah M. Turpin-Nolan, Philipp Hammerschmidt, Weiyi Chen, Alexander Jais, Katharina Timper, Motoharu Awazawa, Susanne Brodesser, Jens C. Brüning  Cell Reports  Volume 26, Issue 1, Pages 1-10.e7 (January 2019) DOI: 10.1016/j.celrep.2018.12.031 Copyright © 2018 The Authors Terms and Conditions

Cell Reports 2019 26, 1-10.e7DOI: (10.1016/j.celrep.2018.12.031) Copyright © 2018 The Authors Terms and Conditions

Figure 1 C18:0 Ceramide Is Elevated in Skeletal Muscle of Diet-Induced Obese Mice (A) CerS mRNA expression levels in quadriceps (Quad) muscle of male C57BL/6N mice fed a normal chow (control diet) or a high-fat diet for 24 weeks relative to CerS1 mRNA expression in quadriceps muscle of control-diet-fed animals (8v8). (B–D) Content of (B) ceramide (6v8), (C) dihydroceramide (6v8), and (D) sphingomyelin species (8v8) in quadriceps muscle of control-diet- or high-fat-diet-fed male C57BL/6N mice. Data are represented as mean ± SEM. Statistical analyses were performed by unpaired Student’s t test. ∗p < 0.05 versus control-diet-fed mice. Cell Reports 2019 26, 1-10.e7DOI: (10.1016/j.celrep.2018.12.031) Copyright © 2018 The Authors Terms and Conditions

Figure 2 CerS1 Deficiency Reduces C18:0 Ceramide Content and Improves Glucose Metabolism (A–L) Analysis of control mice (white) and CerS1Δ/Δ littermates (black) fed a normal chow diet (A and B) or a high-fat diet (C–L). (A) Relative CerS1 mRNA expression in quadriceps (Quad) muscle, heart, liver, and perigonadal white adipose tissue (PGAT) (5-6v6). (B) Relative mRNA expression levels of CerS2, CerS4, CerS5, and CerS6 in quadriceps muscle (6v6). (C–F) Content of ceramide species in (C) quadriceps muscle (5v8), (D) heart (4v8), (E) liver (5v8), and (F) white adipose tissue (6v7). (G) Body weight curves from 4 to 14 weeks of age (6v8). (H) Total body fat mass relative to body weight (10v11) and relative fat pad masses of PGAT (10v11), subcutaneous white adipose tissue (SCAT) (10v11), and brown adipose tissue (BAT) (6v8). (I) Energy expenditure corrected for lean mass (8v8). Grey background: dark phase; white background: light phase. (J) Total food intake over 24 hr (7v8). (K) Insulin tolerance test in random-fed mice (11v11). (L) Glucose tolerance test in mice fasted for 6 hr (8v11). Data are represented as mean ± SEM. Statistical analyses were performed by unpaired Student’s t test (A–F, H, and J) or by two-way ANOVA followed by Bonferroni’s multiple comparison test (G, I, K, and L). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus control mice. See also Figure S1. Cell Reports 2019 26, 1-10.e7DOI: (10.1016/j.celrep.2018.12.031) Copyright © 2018 The Authors Terms and Conditions

Figure 3 Muscle-Specific CerS1 Deficiency Reduces Skeletal Muscle C18:0 Ceramide and Improves Glucose Metabolism (A–M) Analysis of control mice (white) and CerS1ΔSkM littermates (black) fed a high-fat diet. (A) Relative CerS1 mRNA expression in quadriceps (Quad) muscle, heart, liver, and PGAT (6–9v6–8). (B) Relative mRNA expression levels of CerS2, CerS4, CerS5, and CerS6 in quadriceps muscle (9v8). (C–F) Content of ceramide species in (C) quadriceps muscle (10v9), (D) heart (6v6), (E) liver (6v7), and (F) white adipose tissue (5v5). (G) Total diacylglycerol (DAG) (7v5) and total triacylglycerol (TAG) (6v6) lipid content in skeletal muscle as determined via thin-layer chromatography. (H) Body weight curves from 5 to 14 weeks of age (12v12). (I) Total body fat mass relative to body weight (7v11) and relative fat pad masses of PGAT (8v11), SCAT (8v11), and BAT (7v10). (J) Energy expenditure corrected for lean mass (7v11). Grey background: dark phase; white background: light phase. (K) Total food intake over 24 hr (5v8). (L) Insulin tolerance test in random-fed mice (12v9). (M) Glucose tolerance test in mice fasted for 6 hr (14v11). Data are represented as mean ± SEM. Statistical analyses were performed by unpaired Student’s t test (A–G, I, and K) or by two-way ANOVA followed by Bonferroni’s multiple comparison test (H, J, L, and M). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus control mice. See also Figure S2. Cell Reports 2019 26, 1-10.e7DOI: (10.1016/j.celrep.2018.12.031) Copyright © 2018 The Authors Terms and Conditions

Figure 4 Reducing C18:0 Ceramide in Skeletal Muscle Alleviates Systemic Insulin Resistance in a Fgf21-Dependent Manner (A–J) Analysis of control mice (white) and CerS1ΔSkM littermates (black) fed a high-fat diet. (A) Glucose infusion rate during a hyperinsulinemic-euglycemic clamp (CLAMP) experiment (20v20). (B) Glucose uptake into quadriceps (Quad) muscle, red and white gastrocnemius (Gast) muscles, heart, BAT, PGAT, and whole brain (14–19v16–20; CLAMP). (C) Hepatic glucose production at basal and steady state (18v18; CLAMP). (D) Immunoblot analysis and quantifications of phosphorylated AKT (pAKT), relative to total AKT levels, after intravenous saline treatment (n = 2) or insulin stimulation (n = 4) in quadriceps muscle. (E) Relative Il6, Il15, and Fgf21 mRNA expression levels in quadriceps muscle (8–9v14). (F) Relative Fgf21 mRNA expression levels in liver (6v7). (G and H) Immunoblot analysis and quantifications of Fgf21 protein levels normalized to the levels of calnexin in (G) quadriceps muscle and (H) liver from overnight fasted animals relative to controls (7v6). (I) Circulating serum Fgf21 levels (8v14). (J) Glucose tolerance test and respective areas under the curve (AUCs) in 6 hr fasted control and CerS1ΔSkM mice that had been treated with an IgG control antibody (10v11) or an Fgf21 neutralizing antibody (anti-Fgf21) (16v9). Data in (A), (B), (C), (E), (F), (I), and (J) are represented as mean ± SEM. Whisker-and-box plots in (D), (G), and (H) show min. to max. values. Statistical analyses were performed by unpaired Student’s t test (B, D, E, F, G, H, and I), one-way ANOVA followed by Tukey’s multiple comparison test (for AUCs in J) or by two-way ANOVA followed by Bonferroni’s multiple comparison test (A [during steady state only], C, and for longitudinal studies in J). Asterisks in (J) at 30 min and 60 min indicate statistically significant difference of the CerS1ΔSkM group to all other groups as determined by Bonferroni’s post hoc test. ∗p < 0.05; ∗∗p < 0.01 versus control mice. See also Figure S4. Cell Reports 2019 26, 1-10.e7DOI: (10.1016/j.celrep.2018.12.031) Copyright © 2018 The Authors Terms and Conditions