ANGPTL4 silencing via antisense oligonucleotides reduces plasma triglycerides and glucose in mice without causing lymphadenopathy

Figures

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  Figure 1. Abundance of ANGPTL4 protein in human liver. A: ANGPTL4 protein levels in cell lysates of human liver and adipose tissue treated with protein deglycosylation mix (PDM), as determined by Western blot. About 15 μg of proteins were loaded per lane. B: ANGPTL4 protein levels in the liver of five different individuals, as determined by Western blot. HSP90 was used as a loading control. C: LPL protein levels in human liver and adipose tissue as determined by Western blot. About 10 μg of proteins were loaded per lane. HSP90, heat shock protein 90.

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  Figure 2. In vitro characterization of ANGPTL4-specific ASO. Measurement of the concentration response of ANGPTL4-specific ASO (A) and nontargeting control ASO (B). About 15,000 primary mouse hepatocytes/well were seeded in 96-well plates and treated with different concentrations of the respective oligonucleotide. After 3 days of treatment, cells were lysed and Angptl4 mRNA expression was measured using the QuantiGene RNA Singleplex assay. Angptl4 mRNA expression values were normalized to the expression of the housekeeping gene HPRT1. Residual Angptl4 mRNA expression relative to mock-treated cells (set as 1) is shown. C: HEK293 cells expressing a mouse TLR9 NF-κB luciferase reporter plasmid were treated with the indicated concentrations of ODN1668, Neg-Ctrl, or ANGPTL4 ASO. After 20 h, the cells were treated with ONE-Glo EX reagent, and luminescence was measured at 560 nm. Values were normalized to untreated cells. ANGPTL4 ASO is indicated in green. Data are represented as the mean of triplicate wells ± SD. HEK, human embryonic kidney cell.

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  Figure 3. ANGPTL4 ASO reduces ANGPTL4 protein levels in mouse liver and adipose tissue. A: Schematic overview of the set-up of the study. Male C57BL/6J mice fed chow were randomly assigned to either treatment with Neg-Ctrl ASO (1.25 mg/kg, n = 23), low-dose ANGPTL4 ASO (0.625 mg/kg, n = 24), or high-dose ANGPTL4 ASO (1.25 mg/kg, n = 21) via subcutaneous injections twice a week for a total duration of 2 weeks. In each group, half of the mice were euthanized in the ad libitum fed state and half after a 24 h fast. B: Angptl4, Angptl3, and Angptl8 mRNA levels in the liver as determined by quantitative PCR (qPCR). C: Lpl and Lipc mRNA levels in the liver as determined by qPCR. D: Hepatic ANGPTL4 protein levels as determined by Western blot. Right panel: Livers from wild-type, Angptl4−/−, and Angptl4-Tg mice were used as a reference. E: Hepatic LPL protein levels as determined by Western blot. Adipose tissue was used as a reference. L.E., long exposure; S.E., short exposure. F: Adipose tissue ANGPTL4 protein levels as determined by Western blot. Adipose tissue from wild-type, Angptl4−/− and Angptl4-Tg mice was used as a reference. G: Adipose tissue LPL activity using EnzChek lipase substrate. H:…

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  Figure 4. ANGPTL4 ASO reduces plasma TGs independent of feeding status. A: Bodyweight. B: Liver weight. C: Liver TGs. D: Plasma TGs. E: HPLC-based plasma lipoprotein profiling. F: Plasma levels of cholesterol, nonesterified fatty acids, glycerol, and glucose. In the graphs, the horizontal bar represents the mean and the error bars represent SEM. Asterisk indicates significantly different from Neg-Ctrl ASO according to Tukey’s post hoc test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

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  Figure 5. Pilot study on the effect of ANGPTL4 ASO in diet-induced obese mice. A: Schematic overview of the set-up of the study. Male C57BL/6J mice were randomly assigned to either treatment with Neg-Ctrl ASO (1.25 mg/kg, n = 6) or ANGPTL4 ASO (1.25 mg/kg, n = 7) via subcutaneous injections twice a week for a total duration of 20 weeks. During the 20 weeks, mice were given a HFD (60 en% fat). B: Angptl4 and Angptl3 mRNA levels in the liver as determined by quantitative PCR (qPCR). C: Bodyweight change from the start of the ASO and HFD treatment. D: Relative fat and lean mass as determined by echo MRI. E: Average food intake determined per cage but expressed per mouse during the 20 weeks. F: Plasma TG concentration. All plasma measurements were after a 6 h fast. G: Plasma alanine aminotransferase activity. H: Serum amyloid A level in plasma. I: Plasma glucose concentration. J: Intraperitoneal glucose tolerance test after a 6 h fast. K: Area under the curve for the glucose tolerance test corrected for baseline glucose levels. The horizontal bar represents the mean, and the error bars represent SEM. Asterisk indicates significantly different from Neg-Ctrl ASO according to Student’s t-test. ∗P…

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  Figure 6. ANGPTL4 ASO reduces food intake and body weight gain in diet-induced obese mice. A: Schematic overview of the set-up of the study. Male C57BL/6J mice fed a HFD (45 en% fat) were randomly assigned to either treatment with Neg-Ctrl ASO (1.25 mg/kg, n = 10), low-dose ANGPTL4 ASO (0.625 mg/kg, n = 10), or high-dose ANGPTL4 ASO (1.25 mg/kg, n = 10) via subcutaneous injections twice a week for a total duration of 20 weeks. B: Angptl4, Angptl3, and Angptl8 mRNA levels in the liver as determined by quantitative PCR (qPCR). C: ANGPTL4 protein levels in the adipose tissue and heart as determined by Western blot. HSP90 was used as a loading control. D: Bodyweight change from the start of the ASO and HFD treatment. E: Average food intake determined per cage (two mice/cage) during the 20 weeks. Mean food intake per cage across 20 weeks was significantly lower in the low- and high-dose ANGPTL4 ASO groups compared with the Neg-Ctrl ASO group (P < 0.05 and P < 0.01, respectively, indicated by an asterisk). F: Weight of the epididymal fat pad (absolute weight) and the liver (percent of body weight). G: Representative hematoxylin and eosin staining of the mesenteric lymph nodes. In the graphs, the…

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  Figure 7. ANGPTL4 ASO reduces steatosis and increases liver LPL content in diet-induced obese mice. Male C57BL/6J mice fed a HFD (45 en% fat) were randomly assigned to either treatment with Neg-Ctrl ASO (1.25 mg/kg, n = 10), low-dose ANGPTL4 ASO (0.625 mg/kg, n = 10), or high-dose ANGPTL4 ASO (1.25 mg/kg, n = 10) via subcutaneous injections twice a week for a total duration of 20 weeks. A: Serum amyloid A level in plasma. B: Hepatic mRNA levels of inflammatory markers as determined by quantitative PCR (qPCR). C: Hepatic mRNA levels of Lpl and Lipc. D: Hepatic LPL protein levels as determined by Western blot. Adipose tissue was used as a reference. HSP90 was used as a loading control. E: LPL protein levels in the adipose tissue and heart as determined by Western blot. HSP90 was used as a loading control. F: Adipose tissue LPL activity using EnzChek lipase substrate. G: Liver TG levels. H: Representative hematoxylin and eosin staining of the liver. I: Liver glycogen levels. J: Plasma alanine aminotransferase activity. In the graphs, the horizontal bar represents the mean and the error bars represent SEM. Asterisk indicates significantly different from Neg-Ctrl ASO according to Tukey’s post…

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  Figure 8. ANGPTL4 ASO reduces plasma lipids in diet-induced obese mice. Male C57BL/6J mice fed a HFD (45 en% fat) were randomly assigned to either treatment with Neg-Ctrl ASO (1.25 mg/kg, n = 10), low-dose ANGPTL4 ASO (0.625 mg/kg, n = 10), or high-dose ANGPTL4 ASO (1.25 mg/kg, n = 10) via subcutaneous injections twice a week for a total duration of 20 weeks. A: Plasma TGs. B: Plasma total TG and VLDL-TG levels as determined by HPLC-based lipoprotein profiling. C: Plasma total cholesterol, LDL-cholesterol, and HDL-cholesterol as determined by HPLC-based lipoprotein profiling. D: Plasma levels of nonesterified fatty acids, glycerol, and glucose. The horizontal bar represents the mean, and the error bars represent SEM. Asterisk indicates significantly different from Neg-Ctrl ASO according to Tukey’s post hoc test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.