Nonalcoholic fatty liver disease in CLOCK mutant mice
Xiaoyue Pan, Joyce Queiroz, M. Mahmood Hussain
Xiaoyue Pan, Joyce Queiroz, M. Mahmood Hussain
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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 1

CLOCK regulates PHD proteins and HIF1α to regulate CD36.

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CLOCK regulates PHD proteins and HIF1α to regulate CD36. (A–F) Primary h...
(A–F) Primary hepatocytes from WT chow-fed mice were used for these studies. (A and B) Hepatocytes were transfected in triplicate with indicated siRNAs, and Cd36 (top) and Gapdh (bottom) mRNA levels were measured after 72 hours. (B) After 72 hours, cells were incubated with 0.5 μCi of [3H]OA. After 1 hour, cells were washed, and radioactivity was quantified. (C and D) Primary hepatocytes were transfected in triplicate with siCtrl or siClk for 72 hours and treated with cycloheximide (20 μM) or actinomycin D (2 μM) for 6 or 12 hours. Cd36 (C) or Egln2 and Egln3 (D) mRNA levels were quantified. Data are representative of 3 experiments; mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; multiple-comparisons 1-way ANOVA. (E) Hepatocytes were transfected with siClk or siCtrl. After 72 hours, ChIP was performed using indicated antibodies to amplify E-boxes (Supplemental Figure 5B) in Egln1 and Egln3 promoters. Data are representative of 2 experiments. (F) Hepatocytes transfected with siClk or siCtrl for 48 hours were treated with or without CoCl2 for 12 hours and used for ChIP to study the binding of HIF1α (Supplemental Figure 5C) in the Cd36 promoter. Data are representative of 2 independent experiments. (G and H) Livers from chow-fed, male, 10-month-old ClkΔ19/Δ19 and Clkwt/wt mice (n = 5 per group) were used to measure mRNA (G) (mean ± SD; ***P < 0.001, ****P < 0.0001, multiple t tests compared with controls; representative of 2 experiments) and protein levels (H). (I) Livers from chow-fed, male, 10-month-old ClkΔ19/Δ19 and Clkwt/wt mice were used for ChIP; representative of 2 experiments. (J and K) Schematic diagram explaining the molecular mechanisms regulating CD36 expression and OA uptake in livers of ClkΔ19/Δ19 and Clkwt/wt mice. (J) In Clkwt/wt mice, CLOCK binds to the E-boxes in the promoters of Phd genes to increase transcription and protein levels. In the presence of high PHD levels, HIF1α protein is degraded. (K) In ClkΔ19/Δ19 mice, expression of PHD proteins is low and HIF1α protein levels are increased. Under these conditions, HIF1α binds to the Cd36 promoter to increase transcription and protein levels. This might be one mechanism for increased fatty acid uptake in these mice.