Metabolic Profiling Reveals Aggravated Non-Alcoholic Steatohepatitis in High-Fat High-Cholesterol Diet-Fed Apolipoprotein E-Deficient Mice Lacking Ron Receptor Signaling

Figures

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  Figure 1. Metabolic phenotype of high-fat high-cholesterol diet (HFHCD)-fed ApoE knockout (KO) and double knockout (DKO) mice. (A) Scheme of experimental design shown. Six-week-old mice were maintained on a HFHCD for 18 weeks. (B) Effects of impaired Ron receptor signaling on total body weight (representative image shown) and (C) epidydimal white adipose tissue mass (representative image shown), n = 12–16 per group. (D) Correlation plots between measured body weight and epididymal white adipose tissue weights (eWAT) for HFHCD-fed animals (representative image shown), n = 12–16 per group. (E) Blood glucose concentration measured in mice fasted for 6 h (morning fast), n = 12–16 per group. (F) Serum insulin levels in HFHCD-fed ApoE KO and DKO animals (n = 12–16). (G) Intraperitoneal glucose tolerance test (IPGTT) using a fixed glucose dose of 50 mg/mouse following 6 h of fasting in ApoE KO and DKO mice fed a high-fat high-cholesterol diet for 18 weeks (n = 6 per group). Glucose levels were measured at 15, 30, 60, and 120 min. following glucose intraperitoneal (I.P) injection. The data are presented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 2. Ron receptor signaling alters the expression profile of epididymal white adipose tissue from high fat high cholesterol diet (HFHCD)-fed mice. (A) The number, diameter (μm), and area (μm2) of mature adipocytes in eWAT harvested from ApoE KO, and DKO animals (representative image shown to far right). (B) The frequency distribution of adipocyte area in adipose tissue from ApoE KO (n = 6) and DKO (n = 6) mice maintained on a HFHC diet. (C) The expression of genes that are responsive to hypoxia or mediate angiogenesis (n = 12–16 per group). (D) Quantitative RT-PCR analysis of pro-inflammatory and anti-inflammation adipokine expression in eWAT of HFHCD-fed animals. (E) Expression of lipogenic, fatty acid oxidation, (F) lipolysis and glucose uptake-related genes in epididymal white adipose tissue extracts, determined by quantitative real-time PCR (n = 12–16 per group). The data are presented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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  Figure 3. Protective role of Ron receptor signaling in steatohepatitis development and progression. (A) Representative images of liver gross morphology, hematoxylin and eosin (H&E) and Oil Red O (ORO) stained liver sections. (B) Relative liver weights of HFHCD-fed mice (n = 12–16 per group). (C) Quantitative analysis of Oil Red O stained area (%), n = 10 per group. (D) Orthogonal projection to latent structures with discriminant analysis (OPLS-DA) score plot of metabolites profiles on 1H NMR spectra of liver derived from HFHCD-fed mice. Each red and black circle represents one 1H CPMG NMR of liver of a DKO and an ApoE KO mouse, respectively. OPLS-DA coefficient plot of sera obtained from ApoE KO and DKO mice maintained on a HFHCD for 18 weeks. The upward orientation of the peaks denotes higher concentration of the metabolite in the corresponding group of HFHCD-fed mice. The color of the signals signifies the strength of relationship between the metabolite and corresponding HFHCD-fed mouse group with red representing highest significance and black representing least significance. (E) Quantitative representation of significant metabolites in the livers derived from ApoE KO and DKO mice. (F)…

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  Figure 4. Loss of MSP-dependent Ron receptor signaling augments the hepatic lipogenic program. (A) Expression of hepatic genes that are involved in fatty acid, triglyceride and cholesterol synthesis in HFHCD-fed mice (n = 12–16 per group). (B) Expression of hepatic genes involved in fatty acid uptake and E. hepatic lipolysis (n = 12–16 per group). (C) OPLS-DA score plot of metabolites profiles and (D) validation plot based on 1H NMR spectra of serum samples obtained from diet-fed ApoE and DKO mice. (E) OPLS-DA coefficient plot on 1H NMR spectra of blood sera derived from ApoE KO and DKO mice maintained on a HFHCD for 18 weeks (n = 12–16 per group). (F) Quantitative representation of triglyceride levels in blood serum from ApoE KO and DKO animals. The data are presented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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  Figure 5. Impaired Ron receptor signaling alters bile acid synthesis and metabolism. (A,C) Bile acid concentrations in liver and (B,D) feces from mice fed HFHCD for 18 weeks (n = six per group). The data are presented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0. 0001. Quantitative RT-PCR analysis of genes involved in (E) bile acid synthesis (n = five per group), (F) cholesterol uptake/bile acid metabolism regulatory and (G) bile acid regulation livers of HFHCD-fed mice (n = 12–16 per group).

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  Figure 6. Schematic summary of the metabolic outcome of impaired Ron receptor signaling.