Volume 26, Issue 4, Pages 884-892.e4 (January 2019) Gain-of-Function Mutations of SLC16A11 Contribute to the Pathogenesis of Type 2 Diabetes  Yongxu Zhao, Zhuanghui Feng, Yongxian Zhang, Yingmin Sun, Yanhao Chen, Xiaojian Liu, Shuang Li, Tingting Zhou, Lanlan Chen, Yuda Wei, Danjun Ma, Kathy O. Lui, Hao Ying, Yan Chen, Qiurong Ding  Cell Reports  Volume 26, Issue 4, Pages 884-892.e4 (January 2019) DOI: 10.1016/j.celrep.2018.12.100 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2019 26, 884-892.e4DOI: (10.1016/j.celrep.2018.12.100) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 Constitutive Depletion of Slc16a11 in Mice Does Not Cause Significant Metabolic Defects under HFD (A) Body weight of mice fed with HFD starting at 7 weeks old. (B) Average food intake. (C) Body fat percentage. (D) Liver tissue triglyceride levels. (E) Serum triglyceride levels. (F) Serum total cholesterol levels. (G) Metabolite profiling of liver tissue. Shown are relative differences of indicated metabolites in Slc16a11 knockout livers as compared to wild-type livers. Green, significantly downregulated; red, significantly upregulated. (H) Responses to glucose and insulin tolerance tests. Experiments were carried out in male mice of different genotypes after treatment with HFD for around 3 months. n = 6 animals for each group in metabolites profiling in (G); n = 8–10 animals for each group in other analyses (A)–(F) and (H); one of at least two experiments. All data are represented as means with SEM. The unpaired, two-tailed Student’s t test (A)–(F) and one-way ANOVA, post hoc Bonferroni multiple-comparison test in (H) was used for statistical analysis. Cer, ceramide; CL, diphosphatidyl glycerols; DG, diacylglycerol; HexCer, Hex2Cer, hexosylceramide; N.S., not significant; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; TC, total cholesterol; TG, triglyceride. See also Figures S1 and S2. Cell Reports 2019 26, 884-892.e4DOI: (10.1016/j.celrep.2018.12.100) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 Reconstitution of Mutant Slc16a11 in Mouse Liver Causes Increased Blood Triglyceride Levels under Normal Diet (A) Slc16a11 mRNA expression levels in mouse livers. (B) Serum triglyceride levels. (C) Serum total cholesterol levels. (D) Liver tissue triglyceride levels. (E) Responses to glucose tolerance tests. (F) Responses to insulin tolerance tests. Mice were administrated with the indicated adeno-associated viruses and subjected to analysis 3 months after viral delivery. All mice were on normal diet. n = 8–10 animals for each group; one of two experiments. All data are represented as means with SEM. One-way ANOVA, post hoc Bonferroni multiple-comparison test was used for statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001. Cell Reports 2019 26, 884-892.e4DOI: (10.1016/j.celrep.2018.12.100) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 Reconstitution of Mutant Slc16a11 in Mouse Liver Causes Excessive Lipid Accumulation and Induction of Insulin Resistance under HFD (A) Body weight of mice fed with HFD after viral delivery starting at 7 weeks old. (B) Serum triglyceride levels. (C) Serum total cholesterol levels. (D) Liver tissue triglyceride levels. (E) Responses to glucose tolerance tests. (F) Responses to insulin tolerance tests. (G) Representative images of liver tissue H&E staining. Scale bars represent 500 μm. (H) Expression of SLC16A11 mRNA levels (left) and cellular triglyceride levels (right) in HepG2 cells transfected with empty vector, wild-type, or mutant SLC16A11 plasmids, treated with or without sodium stearate (1 mM). n = 3 biological replicates; one of three experiments. For analyses in mice, experiments were carried out in male mice administrated with the indicated adeno-associated viruses and subjected to analyses around 3 months after viral delivery and HFD feeding. n = 8–10 animals for each group in (A)–(F); one of two experiments. All data are represented as means with SEM. The one-way ANOVA, post hoc Bonferroni multiple-comparison test (A)–(F) and (H) was used for statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Cell Reports 2019 26, 884-892.e4DOI: (10.1016/j.celrep.2018.12.100) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 Mutant SLC16A11 Increases Liver Triglyceride Level by Upregulating Lipin 1 (A) Gene Ontology analysis of genes upregulated and downregulated in livers from Slc16a11 knockout mice reconstituted with mutant Slc16a11 (ko+AAV-mut) as compared to mice reconstituted with wild-type Slc16a11 in liver (ko+AAV-wt). n = 6 animals for each group. (B) Heatmap depicting upregulated genes involved in triglyceride metabolic process. (C) Gene expression analysis of liver tissues from Slc16a11 knockout mice reconstituted with mutant (KM) or wild-type Slc16a11 (KW). n = 10 or 11 animals for each group; one of two experiments. (D) Western analysis of liver tissues from Slc16a11 knockout mice reconstituted with mutant (ko+AAV mut) or wild-type Slc16a11 (ko+AAV wt). (E) Gene expression analysis of HepG2 cells transfected with empty vector, wild-type, or mutant SLC16A11 plasmids, treated with or without sodium stearate (1 mM). n = 3 biological replicates; one of three experiments. (F) Western analysis of lipin 1 expression in HepG2 cells infected with control or shLPIN1 viruses. (G) Cellular triglyceride levels in HepG2 cells transfected with empty vector, wild-type, or mutant SLC16A11 plasmids, treated with or without sodium stearate (1 mM), and infected with control or shLPIN1 viruses. n = 3 biological replicates; one of three experiments. All data are represented as means with SEM. The unpaired, two-tailed Student’s t test in (C), one-way ANOVA, and post hoc Bonferroni multiple-comparison test in (E) and (G) was used for statistical analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001. exp., exposure. See also Figure S3. Cell Reports 2019 26, 884-892.e4DOI: (10.1016/j.celrep.2018.12.100) Copyright © 2018 The Author(s) Terms and Conditions