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. 2018 Jan:27:138-150.
doi: 10.1016/j.ebiom.2017.11.029. Epub 2017 Dec 6.

Mice with an Oncogenic HRAS Mutation are Resistant to High-Fat Diet-Induced Obesity and Exhibit Impaired Hepatic Energy Homeostasis

Daiju Oba  1 , Shin-Ichi Inoue  2 , Sachiko Miyagawa-Tomita  3 , Yasumi Nakashima  4 , Tetsuya Niihori  1 , Seiji Yamaguchi  5 , Yoichi Matsubara  6 , Yoko Aoki  7
Affiliations

Mice with an Oncogenic HRAS Mutation are Resistant to High-Fat Diet-Induced Obesity and Exhibit Impaired Hepatic Energy Homeostasis

Daiju Oba et al. EBioMedicine. 2018 Jan.

Abstract

Costello syndrome is a "RASopathy" that is characterized by growth retardation, dysmorphic facial appearance, hypertrophic cardiomyopathy and tumor predisposition. >80% of patients with Costello syndrome harbor a heterozygous germline G12S mutation in HRAS. Altered metabolic regulation has been suspected because patients with Costello syndrome exhibit hypoketotic hypoglycemia and increased resting energy expenditure, and their growth is severely retarded. To examine the mechanisms of energy reprogramming by HRAS activation in vivo, we generated knock-in mice expressing a heterozygous Hras G12S mutation (HrasG12S/+ mice) as a mouse model of Costello syndrome. On a high-fat diet, HrasG12S/+ mice developed a lean phenotype with microvesicular hepatic steatosis, resulting in early death compared with wild-type mice. Under starvation conditions, hypoketosis and elevated blood levels of long-chain fatty acylcarnitines were observed, suggesting impaired mitochondrial fatty acid oxidation. Our findings suggest that the oncogenic Hras mutation modulates energy homeostasis in vivo.

Keywords: Cancer metabolism; Costello syndrome; Diet-induced obesity; ERK; Hras G12S; Mitochondrial fatty acid oxidation.

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Figures

Fig. 1
Fig. 1
Generation of Hras G12S knock-in mice and the Hras+/+ and HrasG12S/+ mouse phenotypes. (a) Gene-targeting strategy to generate the HrasG12S/+ knock-in mice. Exons (solid boxes), neomycin cassettes (hexagonal boxes), Hras cDNA (Exons 1–4)-poly A cassettes (open boxes), splice acceptor sites (SA, labeled gray boxes), loxP sites (open arrowheads) and Flp recombination target (FRT) sites (solid arrowheads) are indicated. The SA-HRAS cDNA-poly A and Neo cassettes were removed by crossing with CAG-Cre transgenic mice. (b, c, d) Representative facial appearance, tooth arrangement and anal images of Hras+/+ and HrasG12S/+ mice. (b) The panels indicate the round facial appearance (upper panel) and shortened nasal bridge (lower panel) in HrasG12S/+ mice compared with Hras+/+ mice. (c) Malocclusion in HrasG12S/+ mice. (d) Rectal prolapse in HrasG12S/+ mice. (e) Body weights of male (upper graph) and female (lower graph) Hras+/+ and HrasG12S/+ mice fed a control diet (CD). The data are presented as the mean ± SD (Hras+/+ (n = 17) and HrasG12S/+ (n = 16) for males; Hras+/+ (n = 17) and HrasG12S/+ (n = 18) for females). *p < 0.05, **p < 0.01 (Welch's t-test) compared with Hras+/+ mice. (f) Gross morphology of hearts from Hras+/+ and HrasG12S/+ mice at 1 year of age. The lower bar graph shows the heart weight (HW) to body weight (BW) ratios of Hras+/+ and HrasG12S/+ mice at 16 weeks and 1 year of age. The data are expressed as the mean ± SD (Hras+/+ (n = 8) and HrasG12S/+ (n = 8) at 16 weeks of age; Hras+/+ (n = 10) and HrasG12S/+ (n = 7) at 1 year of age). *p < 0.05, **p < 0.01 (Welch's t-test) compared with Hras+/+ mice. Scale bar = 5 mm. (g) Transverse sections of wheat germ agglutinin-stained hearts from Hras+/+ and HrasG12S/+ mice at 16 weeks and 1 year of age. The lower bar graph shows the average area of left ventricular (LV) cardiomyocytes in Hras+/+ and HrasG12S/+ mice at 16 weeks and 1 year of age. The data are expressed as the mean ± SD (Hras+/+ (n = 5) and HrasG12S/+ (n = 7) at 16 weeks of age; Hras+/+ (n = 5) and HrasG12S/+ (n = 7) at 1 year of age). **p < 0.01 (Tukey's test) compared with Hras+/+ mice at 16 weeks and 1 year of age. ##p < 0.01 (Tukey's test) compared with HrasG12S/+ mice at 16 weeks of age. Scale bar = 50 μm. (h) Gross morphology of kidneys from Hras+/+ and HrasG12S/+ mice at 1 year of age. The bar graph indicates the kidney weight (KW) to body weight (BW) ratios of Hras+/+ and HrasG12S/+ mice at 16 weeks and 1 year of age. The data are expressed as the mean ± SD (Hras+/+ (n = 8) and HrasG12S/+ (n = 8) at 16 weeks of age; Hras+/+ (n = 10) and HrasG12S/+ (n = 7) at 1 year of age). **p < 0.01 (Welch's t-test) compared with Hras+/+ mice. Scale bar = 5 mm. (i and j) Coronal sections of kidneys from Hras+/+ and HrasG12S/+ mice at 1 year of age stained with HE, PAS and Sirius red. Scale bar = 100 μm. (j) The box plot shows the proportion of fibrosis area in Sirius red-stained sections (Hras+/+ (n = 9) and HrasG12S/+ (n = 8)). The open circle (○) indicates an outlier. *p < 0.05 (Mann-Whitney U test) compared with Hras+/+ mice. (K) Gross morphology of a cystic kidney from a HrasG12S/+ mouse at 16 weeks of age.
Fig. 2
Fig. 2
Resistance to HFD-induced obesity in HrasG12S/+ mice. (a) Representative appearance of Hras+/+ and HrasG12S/+ mice fed a HFD at 16 weeks and 1 year of age. (b) Body weights of Hras+/+ and HrasG12S/+ mice fed a HFD. The data are expressed as the mean ± SD (Hras+/+ (n = 17) and HrasG12S/+ (n = 18)). *p < 0.05, **p < 0.01 (Welch's t-test) compared with Hras+/+ mice. (c and d) Food intake and body weight gain in Hras+/+ and HrasG12S/+ mice fed a HFD from 5 to 16 weeks of age. The data are expressed as the mean ± SD (Hras+/+ (n = 7) and HrasG12S/+ (n = 7)). *p < 0.05 (Welch's t-test) compared with Hras+/+ mice. (e) Representative appearance of inguinal white adipose tissue (IWAT) and IWAT weight in Hras+/+ and HrasG12S/+ mice fed a HFD at 16 weeks and 1 year of age (Hras+/+ (n = 8) and HrasG12S/+ (n = 5)). *p < 0.05 (Mann-Whitney U-test) compared with Hras+/+ mice. (f) Survival rates of Hras+/+ and HrasG12S/+ mice fed a CD or a HFD (Hras+/+ CD (n = 13) and HrasG12S/+ CD (n = 18); Hras+/+ HFD (n = 17) and HrasG12S/+ HFD (n = 18)). *p < 0.05, **p < 0.01 (log-rank test) compared with Hras+/+ CD mice; ##p < 0.01 (log-rank test) compared with Hras+/+ HFD mice.
Fig. 3
Fig. 3
Hepatic microvesicular steatosis in HrasG12S/+ mice fed a HFD. (a) Representative gross morphology of livers from Hras+/+ and HrasG12S/+ mice fed a HFD at 1 year of age. (b) Crude liver weight (left bar graph) and liver weight (LW) to body weight (BW) ratio (right bar graph) of Hras+/+ and HrasG12S/+ mice fed a CD or HFD at 1 year of age. The data are expressed as the mean ± SD (Hras+/+ (n = 11) and HrasG12S/+ (n = 8) for CD-fed mice; Hras+/+ (n = 8) and HrasG12S/+ (n = 8) for HFD-fed mice). (c) HE-stained liver sections from Hras+/+ and HrasG12S/+ mice fed a HFD at 1 year of age. The arrows and arrowheads indicate macrovesicular and microvesicular lipid drops, respectively. Scale bars = 100 μm (upper panel) and 50 μm (lower panel). (d) HE-stained liver sections from Hras+/+ and HrasG12S/+ mice fed a HFD under fed or fasted conditions at 16 weeks of age. Samples were obtained using the described protocol (Supplementary Fig. 5). Scale bar = 50 μm. (e–h) Samples were obtained according to the described protocol (Supplementary Fig. 5). Total lipid (e), triglyceride (f), total cholesterol (g) and free fatty acid (h) levels in the liver from fed or fasted Hras+/+ and HrasG12S/+ mice. The data are expressed as the mean ± SD (Hras+/+ (n = 5) and HrasG12S/+ (n = 6) in the fed condition; Hras+/+ (n = 6) and HrasG12S/+ (n = 6) in the fasted condition). **p < 0.01 (Welch's t-test) compared with fed Hras+/+ and HrasG12S/+ mice. (i-k) Blood glucose (i), urinary β-hydroxybutyric acid (j) and blood β-hydroxybutyric acid (k) levels in Hras+/+ and HrasG12S/+ mice. Blood and urinary samples were obtained as described (Supplementary Fig. 5). The data are expressed as the mean ± SD (Hras+/+ (n = 7) and HrasG12S/+ (n = 6) in the blood samples; Hras+/+ (n = 7) and HrasG12S/+ (n = 7) in the urinary samples). *p < 0.05, **p < 0.01 (Welch's t-test) compared with Hras+/+ mice.
Fig. 4
Fig. 4
Changes in parameters related to mitochondrial fatty acid oxidation in liver tissues from fed or fasted Hras+/+ and HrasG12S/+ mice. (a and b) Relative blood acylcarnitine (a) and amino acid (b) levels in Hras+/+ and HrasG12S/+ mice after 24 h of fasting. Blood samples were obtained as described (Supplementary Fig. 5). The data are expressed as the mean ± SD (Hras+/+ (n = 7) and HrasG12S/+ (n = 7)). *p < 0.05, **p < 0.01 (Welch's t-test) compared with Hras+/+ mice. Val, valine; Leu, leucine; Ileu, Isoleucine; Met, methionine; Cit, citrulline; Phe, phenylalanine; Tyr, tyrosine; Arg, arginine; Ala, alanine. (c) Relative mRNA expression of genes related to mitochondrial β-oxidation in liver tissues from Hras+/+ and HrasG12S/+ mice (Hras+/+ (n = 6) and HrasG12S/+ (n = 7) in the fed condition; Hras+/+ (n = 6) and HrasG12S/+ (n = 6) in the fasted condition). Samples were obtained as described (Supplementary Fig. 5). mRNA levels of target genes were normalized to those of Gapdh. *p < 0.05,** p < 0.01 (Welch's t-test or Mann-Whitney U-test) compared with Hras+/+ mice.
Fig. 5
Fig. 5
Changes in hepatic gene expression related to glucose, organic acid and glutamine metabolism and glucose and pyruvic acid utilization in fed or fasted Hras+/+ and HrasG12S/+ mice. (a–c) Samples were obtained as described (Supplementary Fig. 5). Relative mRNA expression levels of genes related to glucose (a), glutamine (b) and organic acid (c) metabolism in liver tissues from fed or fasted Hras+/+ and HrasG12S/+ mice. (Hras+/+ (n = 6) and HrasG12S/+ (n = 7) in the fed condition; Hras+/+ (n = 6) and HrasG12S/+ (n = 6) in the fasted condition). mRNA levels of target genes were normalized to those of Gapdh. The open circle (○) indicates an outlier. *p < 0.05, **p < 0.01 (Welch's t-test or Mann-Whitney U-test) compared with Hras+/+ mice. (d) IPGTT analysis of blood glucose levels in Hras+/+ and HrasG12S/+ mice at 16 weeks of age. The data are expressed as the mean ± SD (Hras+/+ (n = 8) and HrasG12S/+ (n = 5)). *p < 0.05, **p < 0.01 (Welch's t-test) compared with Hras+/+ mice. (e) Urinary pyruvic acid levels in Hras+/+ and HrasG12S/+ mice (Hras+/+ (n = 7) and HrasG12S/+ (n = 7)). Urine samples were obtained as described (Supplementary Fig. 5). The bars indicate the mean. Statistical analysis was performed using the Mann-Whitney U-test. (f) Western blotting of liver tissues from fasted Hras+/+ and HrasG12S/+ mice. The lower bar graphs show the relative phospho-ERK and phospho-AKT levels. The band intensity was normalized to that of the non-phosphorylated protein. The data are expressed as the mean ± SD (Hras+/+ (n = 5) and HrasG12S/+ (n = 5)). **p < 0.01 (Welch's t-test) compared with Hras+/+ mice.
Fig. 6
Fig. 6
Schematic of the metabolic changes in the liver of fed and fasted HrasG12S/+ mice. (a and b) Changes in gene expression in the liver of HrasG12S/+ mice under fed (a) and fasted (b) conditions. Upregulated or activated proteins and genes are shown in red. Decreased gene expression is shown in blue. (a) In the fed condition, the pentose phosphate pathway is activated due to an increase in G6pd gene expression. A decrease in Pdha gene expression inhibits the conversion of pyruvate to acetyl-CoA. Low Glud1 and Got2 gene expression results in a decrease in alpha-ketoglutaric acid. In the fatty acid oxidation pathway, decreased Acsl1, Cpt2, Acadm and Hadhb gene expression leads to a lack of acetyl-CoA. (b) In the fasted condition, the Hras G12S mutation leads to increased Got1 expression and decreased Glud1 expression through ERK activation. The G6pd gene is also activated in the fed condition. Upregulation of Got1 and downregulation of Glud1 lead to diminished levels of alpha-ketoglutaric acid, which is used to produce ATP in the TCA cycle. The reduced gene expression of Acadm and Hadh leads to impaired fatty acid oxidation, which is the main energy supplier under conditions of starvation. Gln, glutamine; Glu, glutamate; Asp, aspartic acid; OAA, oxaloacetic acid; α-KG, alpha-ketoglutaric acid.
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