ANGPTL3 orchestrates hepatic fructose sensing and metabolism

Figures

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  Figure 1.. Fructose uptake is elevated in fatty livers (A) Body weight of male mice fed with chow diet or MASLD diet for 6 weeks (N = 4 mice per group, repeated in two independent cohorts). (B) Histological analyses using H&E and oil red O (ORO) in livers from male mice fed with chow diet or MASLD diet for 6 weeks (N = 5 mice per group, repeated in two independent cohorts). Scale bar: 100 μm. (C) Uptake of [3H]-fructose-derived carbons in the liver measured by [3H]-fructose incorporation via intraperitoneal injection at 100 μCi/kg in male mice fed with chow diet or MASLD diet for 6 weeks (N = 3 mice per group, repeated in two independent cohorts). (D) [3H]-fructose uptake measured by [3H]-fructose incorporation in primary hepatocytes isolated from male chow and MASLD mice (N = 3 mice per group). (E) Diagram of time-dependent in vivo measurements of [3H]-fructose or [3H]-glucose incorporation in male mice at 1, 2.5, 4, 5.5, and 7 min after i.p. injection. (F) Time-dependent [3H]-fructose or [3H]-glucose levels in circulation at 1, 2.5, 4, 5.5, and 7 min after i.p. injection (N = 3–5 male mice per group). (G) Time-dependent uptake of [3H]-fructose- or glucose-derived carbons in the liver…

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  Figure 2.. ANGPTL3 is elevated in response to dietary fructose and induces hepatocyte fructose uptake (A) Diagram of the experimental design. Male mice were fed with chow or MASLD diet for 6 weeks. Primary hepatocytes were isolated and cultured for 48 h. Conditioned media were collected, and AML12 cells were treated with conditioned media for 4 h, after which fructose uptake in AML12 cells was measured by [3H]-fructose incorporation. (B) Fructose uptake in AML12 cells treated with fractions of the conditioned medium separated by a 3K filter using >3 kDa (proteins) or <3 kDa (metabolites) (N = 3 samples per group, repeated in two independent experiments). (C) Fractions of MASLD media separated by fast protein liquid chromatography (FPLC) fractionation and activity in inducing fructose uptake in AML12 cells (N = 2 samples per fraction for the fructose uptake assays). (D) Silver stain of all fractions of MASLD media separated by FPLC fractionation. (E) Number of total proteins and secreted proteins identified by unbiased proteomics. (F) Venn diagram showing the number of proteins that overlap between datasets. (G) Fold change of differentially expressed genes in male chow and MASLD mouse…

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  Figure 3.. ANGPTL3 loss of function leads to reduced fructolysis enzyme levels (A) Relative gene expression analysis of angptl3 in male mouse organs (N = 5 mice per group). (B) Relative gene expression analysis of khk (khk-a and khk-c) in male mouse organs (N = 5 mice per group). (C) Relative gene expression analysis of aldob in male mouse organs (N = 5 mice per group). (D–F) Gene expression levels of angptl3, aldob, and khk (khk-a and khk-c) in male chow and MASLD mouse livers measured by single-cell RNA sequencing (N = 5 mice per group). (G) Relative gene expression analysis of angptl3 in livers from AAV8-Ctl- and AAV8-shAngptl3-treated male mice (N = 5 mice per group, repeated in two independent cohorts). (H) Representative western blot of the liver from AAV8-Ctl- and AAV8-shAngptl3-treated male mice using an anti-Angptl3 antibody (N = 4 mice per group). (I) Relative gene expression analysis of hepatic lipogenesis genes from AAV8-Ctl- and AAV8-shAngptl3-treated male mice (N = 5 mice per group). (J) Uptake of [3H]-fructose-derived carbons in the liver of male mice fed with MASLD diet for 1 week (N = 5 mice per group). (K) Relative gene expression levels of Glut8 in livers from AAV8-Ctl-…

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  Figure 4.. ANGPTL3 activates the AKT pathway independently of its interaction with LPL (A) [3H]-fructose uptake in AML12 cells after treatment with increasing concentrations of LPL in the presence of 100 nM human ANGPTL3 (N = 3 samples per group). (B) Schematic of generated point mutants of ANGPTL3. (C) [3H]-fructose uptake in AML12 cells after treatment with 100 nM wild-type or catalytically dead mutant of human ANGPTL3 in the presence or absence of LPL (N = 3 samples per group). (D) Representative western blot and quantification of p-AKT, p-AKT substrate, p-S6 in HepG2 cells treated with indicated doses of full-length human ANGPTL3, and positive controls showing the intracellular signaling pathways over time (N = 1 sample per group, repeated in two independent experiments). (E) Representative western blot and quantification of p-AKT, p-AKT substrate, p-S6 in HepG2 cells treated with indicated doses of C-terminal human ANGPTL3, and positive controls showing the intracellular signaling pathways over time (N = 1 sample per group, repeated in two independent experiments). (F) Representative western blot of p-AKT; p-AKT substrate; p-S6 induced by C-terminal human ANGPTL3 (100 nM); or positive…