Metabolic dysregulation in the Atp7b-/- Wilson's disease mouse model

Abstract

Inactivating mutations in the copper transporter Atp7b result in Wilson's disease. The Atp7b^-/- mouse develops hallmarks of Wilson's disease. The activity of several nuclear receptors decreased in Atp7b^-/- mice, and nuclear receptors are critical for maintaining metabolic homeostasis. Therefore, we anticipated that Atp7b^-/- mice would exhibit altered progression of diet-induced obesity, fatty liver, and insulin resistance. Following 10 wk on a chow or Western-type diet (40% kcal fat), parameters of glucose and lipid homeostasis were measured. Hepatic metabolites were measured by liquid chromatography-mass spectrometry and correlated with transcriptomic data. Atp7b^-/- mice fed a chow diet presented with blunted body-weight gain over time, had lower fat mass, and were more glucose tolerant than wild type (WT) littermate controls. On the Western diet, Atp7b^-/- mice exhibited reduced body weight, adiposity, and hepatic steatosis compared with WT controls. Atp7b^-/- mice fed either diet were more insulin sensitive than WT controls; however, fasted Atp7b^-/- mice exhibited hypoglycemia after administration of insulin due to an impaired glucose counterregulatory response, as evidenced by reduced hepatic glucose production. Coupling gene expression with metabolomic analyses, we observed striking changes in hepatic metabolic profiles in Atp7b^-/- mice, including increases in glycolytic intermediates and components of the tricarboxylic acid cycle. In addition, the active phosphorylated form of AMP kinase was significantly increased in Atp7b^-/- mice relative to WT controls. Alterations in hepatic metabolic profiles and nuclear receptor signaling were associated with improved glucose tolerance and insulin sensitivity as well as with impaired fasting glucose production in Atp7b^-/- mice.

Keywords: copper; liver; nuclear receptors.

Fig. 1.

Blunted weight gain and adiposity in Atp7b^−/− mice. (A) Body and liver weights, fat mass, and eWAT weights in Atp7b^−/− and WT mice on either chow or WD. (B) Plasma and liver tissue CHOL, TG, and FFA were measured. ^#P < 0.05 vs. WT chow diet; *P < 0.05 vs. WT WD; two-way ANOVA, and Sidak’s post hoc test. n = 4 to 11/group.

Fig. 2.

Glucose tolerance and insulin sensitivity in Atp7b^−/− mice. (A and B) GTT. (C) Insulin released during intraperitoneal (IP)-GTT time course. (D and E) ITT was performed on 6-mo-old Atp7b^−/− and WT mice fed either (A and D) chow or (B and E) WD. (F) HOMA-IR. (G–K) Hyperinsulinemic euglycemic clamp was performed with 4- to 7-mo-old chow-fed Atp7b^−/− and WT mice. (L) Western blot analysis of gastrocnemius muscle AMPK and p-AMPK levels in chow-fed WT and Atp7b^−/− mice. (A–F) *P < 0.05, WT vs. Atp7b^−/−, chow; ^#P < 0.05 WT vs. Atp7b^−/−, WD; two-way ANOVA, and Sidak’s post hoc test. n = 4 to 11/group. (G–K) *P < 0.05, Student’s t test for Atp7b^−/− mice vs. WT; n = 5 to 6 mice/group.

Fig. 3.

Fasted-state gluconeogenesis and AMPK activation. (A and B) PTT on Atp7b^−/− and WT mice. (B) Western blot analysis of hepatic AMPK and p-AMPK levels in chow-fed 6-mo-old WT and Atp7b^−/− mice. (C) Hepatic gluconeogenic and (D) lipogenic gene mRNA expression measured by real-time PCR analysis. (A and B) *P < 0.05, Atp7b^−/− vs. WT diet-matched control, two-way ANOVA, and Sidak’s post hoc test. n = 4 to 11/group. (D and E) *P < 0.05 vs. WT chow; ^#P < 0.05 vs. WT WD; Student’s t test. n = 5 mice/group.

Fig. 4.

Hepatic metabolic changes in Atp7b^−/− mice. (A) Hepatic glycolytic/TCA cycle metabolites significantly changed (B) hepatic Pepck2 mRNA, (C) hepatic regulators of the pyruvate dehydrogenase complex, and (D) hepatic TCA cycle genes. (A) Student’s t test followed by false discovery rate (FDR) correction; *q < 0.25. n = 4 to 7 mice per group. (B–D) n = 5 to 11 mice per group. Student’s t test was used to determine differences between Atp7b^−/− mice vs. WT controls; *P < 0.05 vs. WT.

Fig. 5.

Transcriptomic analysis in Atp7b^−/− mice livers. Pathway analysis of transcription factor binding was performed with the significantly changed microarray analysis genes (Fc > 1.5, FDR < 0.05) using the web-based EnrichR platform (27141961). Binding sites identified from ChIP-Seq data within the following down-regulated genes: RXR(NR2B1)_22158963_ChIP-Seq_LIVER_Mouse; LXR(NR1H3)__22158963_ChIP-Seq_LIVER_Mouse; STAT5_23275557_ChIP-Seq_MAMMARY-EPITHELIUM_Mouse; CEBPa_23403033_ChIPSeq_LIVER_Mouse; PPARa(NR1C1)_22158963_ChIP-Seq_LIVER_Mouse; PXR(NR1I2)_20693526_ChIP-Seq_LIVER_Mouse; GATA1_22025678_ChIP-Seq_K562_Human. *-log₁₀(P value).