Research ArticleMetabolism

The kinase PKD3 provides negative feedback on cholesterol and triglyceride synthesis by suppressing insulin signaling

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Science Signaling  06 Aug 2019:
Vol. 12, Issue 593, eaav9150
DOI: 10.1126/scisignal.aav9150
  • Fig. 1 PKD3 is the predominant PKD isoform expressed and activated in the liver.

    (A) Quantification of the four most abundant DAG classes in the livers of normal diet (ND)– or high-fat diet (HFD)–fed mice determined by LC-MS (n = 7 mice per diet). (B and C) Western blot analysis of PKD-pSer731/735 in isolated primary hepatocytes stimulated with cell-permeable 1,2-dioctanoyl-sn-glycerol (DAG) (100 μM) (B) or oleic acid (750 μM) (C) for the indicated time points, respectively (n = 3 independent experiments). (D) Western blot analysis of the indicated proteins from the livers of ND- or HFD-fed mice (n = 3 mice per diet). (E) Absolute quantification of PKD isoform expression in the liver by quantitative polymerase chain reaction (QPCR) with in-exon primers and normalizing values to a genomic DNA standard (n = 8 biological replicates per group, combined data from three independent experiments). In (A) and (E), data are presented as means ± SEM. *P > 0.05 [unpaired two-tailed Student’s t test (A) or one way ANOVA with post hoc Tukey’s test (E)].

  • Fig. 2 PKD3 promotes insulin resistance and glucose intolerance.

    (A) Body weight evolution of control and PKD3liverΔ/Δ mice fed an HFD for 24 weeks. (B) Organ weight of control and PKD3liverΔ/Δ mice fed an HFD for 24 weeks. (C to E) Glucose (2 g/kg BW) (C) and insulin (1.5 U/kg BW) (D) tolerance tests and serum insulin levels (E) in PKD3liverΔ/Δ and control mice after HFD feeding for 16, 18, and 24 weeks, respectively. (F and G) Western blot analysis for the indicated proteins in the livers of mice of the indicated genotypes given insulin (8 U/kg body weight) for 15 min after 24 weeks of HFD feeding and (F) corresponding densitometric quantification (G) (n = 5 mice per group). In (A) to (E), n = 8 mice [wild type (WT)] and n = 15 mice [knockout (KO)] were used, and in (A) to (E) and (G), data are presented as means ± SEM. *P > 0.05, **P > 0.01, and ***P > 0.001 [unpaired two-tailed Student’s t test (B and E), one-way ANOVA with post hoc Tukey’s test (G), or two-way ANOVA with post hoc Tukey’s test (A, C, and D)]. i.p., intraperitoneal; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

  • Fig. 3 Deletion of PKD3 promotes hepatic accumulation of TGs and cholesterol.

    (A) Representative microscopy pictures of H&E-stained liver sections from control and PKD3liverΔ/Δ mice fed an HFD for 24 weeks (scale bar, 50 μm; representative of six mice per group). (B and C) Quantification of TG (B) and cholesterol (C) content in extracted lipid phases normalized to total protein levels in livers from control and PKD3liverΔ/Δ mice fed an HFD for 24 weeks (n = 10 mice per group). (D to F) Thin-layer chromatography (TLC) separation of extracted hepatic lipids from mice of the indicated genotypes fed an HFD and the corresponding densitometric quantification using standards of known concentrations for cholesteryl ester (CE), TG, free fatty acids (FFAs), free cholesterol (FC), phosphatidylethanolamine (PE), and phospholipids (PLs) (n = 8 mice per group). (G and H) Quantification of serum cholesterol (G) and serum TG (H) concentrations in control and PKD3liverΔ/Δ mice fed an HFD for 20 weeks (n = 8 mice per group). In (B), (C), and (E) to (H), data are presented as means ± SEM. *P > 0.05, **P > 0.01, and ***P > 0.001 (unpaired two-tailed Student’s t test). a.u., arbitrary unit.

  • Fig. 4 Hepatic PKD3 suppresses de novo lipogenesis in a SREBP-dependent manner.

    (A and B) Basal and insulin-induced total de novo lipogenesis rate (measured under lipogenic conditions: serum deprived, 25 mM glucose, 0.5 mM sodium acetate, and 100 nM insulin) in primary hepatocytes isolated from mice of the indicated genotypes (a.u.) (A) and quantification of TLC-separated FC, TG, CE, and other lipids (FFA, PE, and PL) (B) (n = 3 biological replicates per group). (C) In vivo de novo lipogenesis rate (a.u.) in livers from control and PKD3liverΔ/Δ mice fed an HFD for 8 weeks, fasted overnight, and refed for 4 hours before analysis (n = 5 mice per group). (D) Western blot analysis of mature SREBP1 in primary hepatocytes of the indicated genotypes under the same lipogenic conditions as in (B) (n = 3 mice per group; representative of three individual experiments). (E) QPCR analysis of Srebp gene and target gene expression in primary hepatocytes of the indicated genotypes under the same lipogenic conditions as in (B) (n = 3 mice per group). (F) QPCR analysis of Srebp gene and target gene expression in livers from control and PKD3liverΔ/Δ mice fed for 20 weeks with HFD, subjected to fasting overnight and refeeding for 4 hours (n = 6 mice per group). (G) Western blot analysis for indicated proteins in livers from the mice in (F) (n = 3 mice per group). (H) QPCR analysis of Srebp gene and target gene expression in primary hepatocytes of the indicated genotypes stimulated with insulin for 4 hours that were either transfected with siNonTargeting (siNT) or the combination of siSrebp1 and siSrebp2 as indicated (n = 3 biological replicates per condition). In (A) to (C), (E), (F), and (H), data are presented as means ± SEM. *P > 0.05 and **P > 0.01 [unpaired two-tailed Student’s t test (C, E, and F) or one-way ANOVA with post hoc Tukey’s test (A, B, and H)].

  • Fig. 5 The effect of the PKD inhibitor CRT0066101 and refeeding on lipogenic gene expression and hepatic lipid accumulation.

    (A) QPCR analysis of insulin-stimulated Srebp1c expression in DMSO or CRT0066101 (0.1 μM)–treated primary hepatocytes of the indicated genotypes (n = 3 biological replicates per condition). (B) QPCR analysis of the expression of Srebp and target genes in the livers from HFD-fed C57BL/6JRj mice (for 8 weeks) that either received an intraperitoneal injection of vehicle or CRT0066101 (10 mg/kg BW) inhibitor for five consecutive days. Mice were fasted overnight and refed for 4 hours before the livers were excised (n = 8 mice per group). (C) QPCR analysis of the indicated genes in the livers from control and PKD3liverΔ/Δ mice after overnight fasting and refeeding for 4 hours (n = 4 mice per group; combined data from two individual experiments). (D and E) Quantification of serum TG (D) and serum cholesterol (E) concentrations at the indicated time points in control and PKD3liverΔ/Δ mice fed an ND for 7 weeks that were fasted and refed for the indicated time points (n = 12 mice per group). In (A) to (E), data are presented as means ± SEM. *P > 0.05 and **P > 0.01 [unpaired two-tailed Student’s t test (B and C), one-way ANOVA with post hoc Tukey’s test (A), or two-way ANOVA with post hoc Tukey’s test (D and E)].

  • Fig. 6 PKD3 suppresses de novo lipogenesis in an AKT- and mTORC1/2-dependent manner.

    (A and C) Western blot analysis for the indicated proteins in extracts from control and PKD3-deficient primary hepatocytes stimulated with 100 nM insulin for the indicated time points (n = 3 independent experiments). (B and D) Western blot analysis of the indicated proteins in extracts from PKD3-deficient primary hepatocytes transduced with either adenovirus expressing EGFP control (Ad-EGFP) or constitutive active PKD3 (Ad-mycPKD3ca) and stimulated with 100 nM insulin for 15 min (n = 3 independent experiments). IB, immunoblot. (E) De novo lipogenesis rate in the presence of insulin and under lipogenic conditions in primary hepatocytes treated with DMSO, CRT0066101 (1 μM), Akti-1/2 (10 μM), KU0063794 (0.7 μM), and rapamycin (0.7 μM) as indicated for 5 hours (n = 3 biological replicates per condition). In (E), data are presented as means ± SEM. *P > 0.05 and **P > 0.01 (one-way ANOVA with post hoc Tukey’s test).

  • Fig. 7 Liver-specific expression of constitutively active PKD3 promotes insulin resistance.

    (A and B) Glucose (2 g/kg BW) (A) and insulin (1 U/kg BW) (B) tolerance test in TgPKD3caliver and control mice fed an ND for 12 and 10 weeks, respectively [n = 11 mice (control) and n = 13 mice (Tg)]. (C to E) After overnight fast, blood glucose (C) and serum insulin (D) were measured and used to determine HOMA-IR (E) in mice of the indicated genotypes [n = 11 mice (control) and n = 13 mice (Tg)]. (F and G) Western blot analysis of the indicated proteins from livers of mice of the indicated genotypes that were fasted overnight and refed 4 hours before analysis (F) and corresponding densitometric quantification (G) (n = 3 mice per group). (H) Summary of PKD3 signaling in the liver. In (A) to (E) and (G), data are presented as means ± SEM. *P > 0.05, **P > 0.01, and ***P > 0.001 [unpaired two-tailed Student’s t test (C to E and G) or two-way ANOVA with post hoc Tukey’s test (A and B)].

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/593/eaav9150/DC1

    Fig. S1. Stimulation of hepatocytes with DAG suppresses expression of lipogenic genes.

    Fig. S2. PKD3 deletion is restricted to the liver.

    Fig. S3. Liver-specific PKD3 deletion does not affect metabolism of mice fed an ND.

    Fig. S4. PKD3 does not affect proliferation, immune cell infiltration, or apoptosis in the liver.

    Fig. S5. TG accumulation in the livers of PKD3liverΔ/Δ mice does not depend on FA oxidation or VLDL secretion.

    Fig. S6. The abundance and/or phosphorylation of mTORC1/2 components are not affected by deletion or overexpression of PKD3 in hepatocytes.

    Fig. S7. Quantifications of Western blots of control and PKD3-deficient primary hepatocytes.

    Fig. S8. Quantifications of Western blots of EGFP- and PKD3ca-transduced primary hepatocytes.

    Fig. S9. Liver-specific expression of PKD3ca improves glucose tolerance and insulin sensitivity.

    Table S1. List of antibodies used for Western blotting and immunohistochemistry.

    Table S2. Sequence of primers used for QPCR and genotyping.

  • This PDF file includes:

    • Fig. S1. Stimulation of hepatocytes with DAG suppresses expression of lipogenic genes.
    • Fig. S2. PKD3 deletion is restricted to the liver.
    • Fig. S3. Liver-specific PKD3 deletion does not affect metabolism of mice fed an ND.
    • Fig. S4. PKD3 does not affect proliferation, immune cell infiltration, or apoptosis in the liver.
    • Fig. S5. TG accumulation in the livers of PKD3liver∆/∆ mice does not depend on FA oxidation or VLDL secretion.
    • Fig. S6. The abundance and/or phosphorylation of mTORC1/2 components are not affected by deletion or overexpression of PKD3 in hepatocytes.
    • Fig. S7. Quantifications of Western blots of control and PKD3-deficient primary hepatocytes.
    • Fig. S8. Quantifications of Western blots of EGFP- and PKD3ca-transduced primary hepatocytes.
    • Fig. S9. Liver-specific expression of PKD3ca improves glucose tolerance and insulin sensitivity.
    • Table S1. List of antibodies used for Western blotting and immunohistochemistry.
    • Table S2. Sequence of primers used for QPCR and genotyping.

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