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Lipid transport and ATP synthesis are critical for the progression of non-alcoholic fatty liver disease (NAFLD), but the underlying mechanisms are largely unknown. Here, we report that the RNA-binding protein HuR (ELAVL1) forms complexes with NAFLD-relevant transcripts. It associates with intron 24 of Apob pre-mRNA, with the 3′UTR of Uqcrb, and with the 5′UTR of Ndufb6 mRNA, thereby regulating the splicing of Apob mRNA and the translation of UQCRB and NDUFB6. Hepatocyte-specific HuR knockout reduces the expression of APOB, UQCRB, and NDUFB6 in mice, reducing liver lipid transport and ATP synthesis, and aggravating high-fat diet (HFD)-induced NAFLD. Adenovirus-mediated re-expression of HuR in hepatocytes rescues the effect of HuR knockout in HFD-induced NAFLD. Our findings highlight a critical role of HuR in regulating lipid transport and ATP synthesis.
Non-alcoholic fatty liver disease (NAFLD) is associated with a variety of disease conditions including obesity, insulin resistance, diabetes, hypertension, hyperlipidemia, and metabolic syndrome1. Characterized by the accumulation of lipids in liver, NAFLD results from the imbalance between lipid acquisition and disposal2,3,4. Excessive intake of dietary fat, abnormal lipid synthesis, and liver oxidation increase the levels of liver lipids2,3,4, which can then be eliminated by two major paths: β-oxidation of fatty acids into acetyl-coenzyme A and to enter the Krebs cycle and generate ATP, and export of liver lipids to other tissues. Failure to dispose excess liver lipids by one of these mechanisms leads to NAFLD5,6.
Several different factors are involved in regulating hepatic lipid transport and ATP synthesis. PPARα (peroxisome proliferator-activated receptor, PPARα) is a key transcriptional regulator of fatty acid oxidation in mitochondria7,8. Oxidation of fatty acids occurs mainly in the mitochondria, generating ATP through oxidative phosphorylation through the action of proteins encoded by both mitochondrial and nuclear DNA6. Alterations in these factors leading to aberrant ATP production in NAFLD have been documented in patients9,10, as well as in mice and rats with NAFLD11,12. Impaired ATP synthesis in NAFLD arises from the reduced activity or levels of factors in complexes I–V9; for complex II, since there are no factors encoded by mitochondria DNA, deficiencies in the activity of this complex reflect aberrant expression of factors encoded from nuclear DNA. On the other hand, when liver lipids are in excess, they can be packaged into VLDL particles, transported to serum and distributed to other tissues5,13.
Apolipoprotein B-100 (APOB-100) and apolipoprotein E (APOE) are critical for the packaging and secretion of VLDL to maintain hepatic lipid homoeostasis14,15. APOB abundance is altered in NAFLD, suggesting a role for APOB in this disease. APOB production is regulated at multiple levels16,17,18,19. Transcription of the Apob gene is governed by transcription factors HNF-4, HNF-3β, ARP-1, and C/EBPβ16,17, while APOB translation is controlled by RNA-binding proteins (RBPs) and microRNAs interacting with Apob mRNA18. While the splicing of Apob pre-mRNA has been targeted therapeutically for lowering blood cholesterol levels19, the mediators of this regulation are unknown. HuR [‘human antigen R’, also known as ELAVL1 (embryonic lethal abnormal vision-like 1)], is a ubiquitous member of Hu/ELAV RBP family. HuR regulates the post-transcriptional fate of many coding and noncoding RNAs20,21,22, in turn regulating many cell functions (proliferation, survival, apoptosis, senescence, and differentiation) and affecting processes such as cancer and aging. HuR was also reported to promote ATP synthesis in cells by regulating the translation of cytochrome c (CYCS) and coenzyme Q7 (COQ7)23,24. A recent study describes HuR as a regulator for ABCA1 translation, influencing macrophage cholesterol metabolism in vivo25. However, the role of HuR in lipid metabolism and the underlying mechanisms remains to be studied.
In the present study, a conditional hepatocyte-specific HuR knockout mouse (cKO) is created to evaluate the role of HuR in high-fat diet (HFD)-induced NAFLD. We find evidence that HuR associated with mouse cytochrome c (Cycs), Uqcrb, and Ndufb6 mRNAs, as well as with Apob pre-mRNA, thereby regulating the translation of CYCS, UQCRB, and NDUFB6, as well as the production of Apob mRNA. These processes impact upon HFD-induced NAFLD and point to a mechanism whereby HuR controls liver lipid homeostasis.
HuR regulates lipid transport and ATP synthesis in NAFLD
To evaluate the role of HuR in NAFLD, we generated a conditional hepatocyte-specific HuR knockout (cKO) mouse by crossing a HuR Flox/Flox mouse (C57BL/6 J background, Jackson Laboratories) with an albumin Cre mouse. After confirming the specific knockout of HuR in hepatocytes by real-time qPCR and western blot analyses (Supplementary Fig. 1), we examined liver function in HuR cKO mice and wild-type (WT) littermates fed regular chow. As shown in Fig. 1a, deletion of HuR did not lead to alterations of body weight, liver weight, or the ratio of liver weight relative to body weight. By staining with hematoxylin and eosin (H&E) as well as with Oil Red O, we observed that hepatocytes in cKO mice were comparable morphologically to those of WT littermates (Fig. 1b). Although the levels of liver triglyceride and cholesterol in HuR cKO mice were slightly higher than those observed in WT mice, the difference was not significant (Fig. 1c). Furthermore, hepatocytes in cKO mice displayed slightly reduced levels of ATP, although this reduction was not significant (Fig. 1d). Additional results showed that deletion of HuR reduced the levels of serum APOB (p < 0.05) and HDL-C (high-density lipoprotein cholesterol) (p < 0.01), but lowered only mildly the serum levels of triglyceride, cholesterol, APOE, LDL-C (low-density lipoprotein cholesterol) (Supplementary Fig. 2a 0.01),a>
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