Non-alcoholic fatty liver disease, vascular inflammation and insulin resistance are exacerbated by TRAIL deletion in mice

Figures

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  Figure 1. Patients with NASH have reduced circulating TRAIL. (a) Circulating TRAIL levels in control, steatosis or NASH patients. (b) Circulating TRAIL levels in control and obese individuals. (c) Correlation between plasma TRAIL and ALT levels (Spearman rho −0.516, p = 0.003). Natural log (ln) of serum ALT presented graphically. n = 9–10/group; ANOVA and Mann Whitney U test.

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  Figure 2. TRAIL protects against lipid uptake in HepG2 cells. (a) Palmitate increases triglyceride uptake in HepG2 cells over 5 days as measured by Oil red O. (b) Inflammation and (c) fibrotic markers are increased in HepG2 cells in response to palmitate over 5 d. (d) mRNA expression for TRAIL is significantly reduced with lipid uptake over time. (e) DR4 and (f) DR5 expression in response to palmitate. (g) 1 ng/ml TRAIL inhibits palmitate-induced oil red O in HepG2 cells at 24 h compared to the vehicle PBS. Control, untreated cells; n = 3–4/treatment. mRNA expression was normalized to GAPDH. Results are expressed as mean ± SEM; ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

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  Figure 3. Trail −/− mice have impaired insulin sensitivity. (a) Insulin tolerance tests (ITT) in 12 w HFD mice (left panel); expressed as area under the curve (right panel; n = 10/genotype) or, (b) ITT at baseline (0 w HFD wildtype and Trail −/− mice; n = 3/genotype). (c) Glucose tolerance tests in 12 w HFD mice (left panel; n = 10/genotype) or, (d) at baseline (0 w HFD wildtype and Trail −/− mice; n = 3/genotype). (e) Western blotting showing that muscle from insulin-stimulated 12 w HFD Trail −/− mice have reduced p-Akt protein expression (3 independent mice/group). Total Akt protein expression is unaltered. (f) GLUT4 staining in muscle from insulin-stimulated (45 min) 0 w HFD Trail −/− and wildtype mice (n = 4/genotype) and (g) reduced glucose uptake ex vivo (n = 3–4/genotype). (h) Inflammatory marker expression is increased in muscle tissue of 12 w HFD Trail −/− vs. wildtype, but not at (i) baseline (n = 4–5/genotype). mRNA expression was normalized to β-actin. Results are expressed as mean ± SEM; ANOVA or Mann Whitney U test. *p < 0.05 and **p < 0.01.

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  Figure 4. Liver from Trail −/− mice exhibit altered glucose and lipid metabolism. (a) Hepatic glucose uptake ex vivo is reduced in baseline 0 w HFD Trail −/− vs. wildtype mice (n = 3–5/genotype). mRNA expression for (b) Glucose-6 phosphatase (G6Pase) or GLUT2 mRNA in baseline mice. (c) Insulin-stimulated PEPCK mRNA expression is increased in baseline Trail −/− mice (n = 6/genotype). (d) 12 w HFD Trail −/− mice exhibit increased PAS staining and have (e) elevated SREBP-1 and (f) HMGCoAR mRNA expression (n = 6/genotype). mRNA expression was normalized to HPRT. Results are expressed as mean ± SEM; Mann-Whitney U-test; *p < 0.05, and ***p < 0.001.

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  Figure 5. Liver from 12 w HFD Trail −/− mice have NAFLD. (a) Liver from 12 w HFD Trail −/− mice have elevated steatosis compared to 12 w HFD wildtype mice (40× magnification). (b) Oil red O is increased in hepatic tissue of 12 w HFD Trail −/− vs. wildtype. (c) Milligan’s trichrome quantification of fibrosis shows that 12 w HFD Trail −/− mice have increased fibrosis. (d) Hepatic tissue from 12 w HFD Trail −/− mice have increased apoptosis, measured using the Cell Death Detection ELISA. (e) mDR5 expression is unaltered in 12 w HFD Trail −/− vs. wildtype liver. mRNA expression was normalized to β-actin. (f) Vimentin staining is increased in liver from 12 w HFD wildtype mice (arrows), while (g) SMA staining is increased in 12 w HFD Trail −/− liver (arrows). Staining was quantified as described in the Materials and Methods. Results are expressed as mean ± SEM (n = 10/genotype); Mann-Whitney U-test; *p < 0.05.

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  Figure 6. Trail −/− vascular tissue have altered insulin signals. Concentration-response curves for ex vivo aortic relaxation induced by insulin at (a) baseline 0 w HFD or (b) 12 w HFD wildtype and Trail −/− mice; (n = 5/genotype). (c) Concentration-response curves for ex vivo aortic relaxation induced by Ach (left) and SNP (right) in 12 w HFD mice. (d) Aortas from 12 w HFD Trail −/− mice in response to an insulin challenge (45 min) have reduced p-Akt protein expression, with no change in total Akt levels. (e) Aortic mRNA expression of insulin receptor-β and GLUT-4 in 12 w HFD Trail −/− vs. wildtype; mRNA levels were normalised to β-actin (n = 3/genotype). Results are expressed as mean ± SEM; ANOVA or Mann-Whitney U-test; *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 7. Trail −/− mice display severe vascular tissue inflammation in response to a HFD. (a) Aortic mRNA expression for MCP-1, TNF-α, IL-6, IL-1β and TRAIL in 0 vs. 12 w HFD wildtype mice (n = 4–5/treatment). (b) Aortic mRNA expression for inflammatory markers in 12 w HFD or (c) baseline Trail −/− vs. wildtype mice. (d) Baseline Trail −/− have increased aortic mRNA for VCAM-1 but not ICAM-1. mRNA levels were normalized to β-actin or 18S and expressed as mean ± SEM; Mann-Whitney U-test (n = 4–5/genotype); *p < 0.05.