Nonalcoholic fatty liver disease in CLOCK mutant mice
Xiaoyue Pan, … , Joyce Queiroz, M. Mahmood Hussain
Xiaoyue Pan, … , Joyce Queiroz, M. Mahmood Hussain
Published May 12, 2020
Citation Information: J Clin Invest. 2020;130(8):4282-4300. https://doi.org/10.1172/JCI132765.
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Research Article Hepatology Metabolism

Nonalcoholic fatty liver disease in CLOCK mutant mice

Abstract

Nonalcoholic fatty liver disease (NAFLD) is becoming a major health issue as obesity increases around the world. We studied the effect of a circadian locomotor output cycles kaput (CLOCK) mutant (ClkΔ19/Δ19) protein on hepatic lipid metabolism in C57BL/6 Clkwt/wt and apolipoprotein E–deficient (Apoe−/−) mice. Both ClkΔ19/Δ19 and ClkΔ19/Δ19 Apoe−/− mice developed a full spectrum of liver diseases (steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma) recognized in human NAFLD when challenged with a Western diet, lipopolysaccharide, or CoCl2. We identified induction of CD36 and hypoxia-inducible factor 1α (HIF1α) proteins as contributing factors for NAFLD. Mechanistic studies showed that WT CLOCK protein interacted with the E-box enhancer elements in the promoters of the proline hydroxylase domain (PHD) proteins to increase expression. In ClkΔ19/Δ19 mice, PHD levels were low, and HIF1α protein levels were increased. When its levels were high, HIF1α interacted with the Cd36 promoter to augment expression and enhance fatty acid uptake. Thus, these studies establish a regulatory link among circadian rhythms, hypoxia response, fatty acid uptake, and NAFLD. The mouse models described here may be useful for further mechanistic studies in the progression of liver diseases and in the discovery of drugs for the treatment of these disorders.

Authors

Xiaoyue Pan, Joyce Queiroz, M. Mahmood Hussain

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Figure 3

Decreased hepatosteatosis after knockdown of HIF1α in ClkΔ19/Δ19 mice.

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Decreased hepatosteatosis after knockdown of HIF1α in ClkΔ19/Δ19 mice. C...
ClkΔ19/Δ19 mice (male, 10 months old, chow-fed, 5–6 per group) were injected once via tail vein with lentiviruses expressing either shCtrl or shHIF1α (1 × 109 PFU/mouse). After 2 weeks, plasma and livers were collected. Another set of animals was used for fatty acid uptake studies (n = 3 per group). (A) Liver samples were used to measure Hif1α, Cd36, Vegf, and Gapdh mRNA and protein levels of CD36 and Gapdh (inset). Mean ± SD; ***P < 0.001, multiple t tests. (B) For hepatic fatty acid uptake, mice were injected i.p. with 1.0 μCi/mouse of [3H]OA. After 2 hours, livers were collected to measure radioactivity. Mean ± SD; **P < 0.0001, Welch’s 2-tailed t test. (C) Liver triglyceride, cholesterol, and TBARS levels were lower in ClkΔ19/Δ19 mice 2 weeks after injection of shHif1α. Mean ± SD; *P < 0.01, **P < 0.001, Welch’s 2-tailed t test. (D) Plasma ALT and triglyceride levels were reduced in shHif1α-injected ClkΔ19/Δ19 mice, but plasma cholesterol and β-HB concentrations were normal. Mean ± SD; *P < 0.001, Welch’s 2-tailed t test. (E) Liver sections were stained with Oil Red O, H&E, and anti-macrophage and anti-HIF1α antibodies. Data are representative of 2 experiments. (F) Transmission electron microscopy (EM) revealed reduced lipid droplets in ClkΔ19/Δ19 mice injected with shHif1α (scale bar: 100 μm). (G) Quantification of different mRNAs in livers of ClkΔ19/Δ19 mice 2 weeks after injection of shHIF1α. Mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, multiple t tests.