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Hedgehog signaling keeps liver clock in check

  • Gianluigi Mazzoccoli
    Affiliations
    Division of Internal Medicine and Laboratory of Chronobiology, Department of Medical Sciences, Fondazione IRCCS “Casa Sollievo Della Sofferenza”, San Giovanni Rotondo, Foggia, Italy
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  • Ali Keshavarzian
    Affiliations
    Department of Internal Medicine, Division of Digestive Disease and Nutrition, Rush University Medical Center, Chicago, IL, USA
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  • Manlio Vinciguerra
    Correspondence
    Corresponding author. Address: International Clinical Research Center, St’Anne University Hospital, Pekarska, 53, Brno, Czech Republic. Tel.: +39 3518886355.
    Affiliations
    International Clinical Research Center, St’Anne University Hospital, Brno, Czech Republic

    Institute for Liver and Digestive Health, Division of Medicine, University College London, London, United Kingdom
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Published:February 27, 2019DOI:https://doi.org/10.1016/j.jhep.2019.02.009

      Linked Article

      • Tick-tock hedgehog-mutual crosstalk with liver circadian clock promotes liver steatosis
        Journal of HepatologyVol. 70Issue 6
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          Circadian rhythm plays an important role in regulating physiology and behavior. In mammals, the natural light–dark cycle synchronizes the central circadian pacemaker in the suprachiasmatic nucleus (SCN), which, in turn, coordinates the rhythms of autonomous clocks in peripheral tissues such as the liver;1 these tissues adapt their functions to the rhythmic cycles of feeding and activity. Indeed, most metabolic and secretory functions of the liver show pronounced circadian rhythms.2,3 In particular, liver carbohydrate and lipid metabolism, which are crucial for energy supply of the entire organism, oscillate throughout the day.
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      Most organisms, including humans, have developed an autonomous time-keeping system hard-wired by biological clocks that generate daily fluctuations in physiological and cellular processes. These biological clocks are reset daily by light to adjust physiology to the day/night cycle generated by the rotation of the Earth around its axis. In organs involved in glucose and lipid metabolism, such as the liver, biological clocks are also reset by feeding cues, which allow the integration of systemic and nutritional signals to adapt fuel production and utilization according to the feeding/fasting cycle. In a ground-breaking report, published in 2019, Chinese researchers reported the generation of a monkey harboring deletion of the BMAL1 circadian gene, using CRISPR/Cas9 editing of monkey embryos.
      • Peiyuan Qiu J.J.
      • Zhen Liu
      • Yijun Cai
      • Tao Huang
      • Yan Wang
      • Qiming Liu
      • et al.
      Bmal1 knockout macaque monkeys display reduced sleep and psychiatric disorders.
      Not surprisingly, these monkeys exhibit insomnia and psychiatric disorders, producing a horrifying ethical minefield and widespread protests. In fact, it is already well known that circadian disruption is a risk factor for metabolic, psychiatric and age-related disorders, in an intertwined fashion.
      • Tevy M.F.
      • Giebultowicz J.
      • Pincus Z.
      • Mazzoccoli G.
      • Vinciguerra M.
      Aging signaling pathways and circadian clock-dependent metabolic derangements.
      • Maugeri A.
      • Medina-Inojosa J.R.
      • Kunzova S.
      • Agodi A.
      • Barchitta M.
      • Sochor O.
      • et al.
      Sleep duration and excessive daytime sleepiness are associated with obesity independent of diet and physical activity.
      • Asarnow L.D.
      • Soehner A.M.
      • Harvey A.G.
      Circadian rhythms and psychiatric illness.
      At the molecular level, in every single cell circadian rhythmicity is generated by an oscillator that comprises a feedback loop in which the expression of so-called clock genes is suppressed periodically by their protein products and completes one cycle each day. The core clock genes form interacting positive (BMAL1, CLOCK) and negative (PERIOD, CRYPTOCHROME) limbs: a heterodimer of the CLOCK and BMAL1 proteins rhythmically activates the transcription of clock genes PER1, PER2, PER3, CRY1, CRY2. The PER and CRY proteins form a complex that translocates back into the nucleus, interacts directly with the CLOCK:BMAL1 heterodimer and represses its transcriptional activity.
      • Mazzoccoli G.
      • Vinciguerra M.
      • Oben J.
      • Tarquini R.
      • De Cosmo S.
      Non-alcoholic fatty liver disease: the role of nuclear receptors and circadian rhythmicity.
      The CLOCK:BMAL1 heterodimer activates an auxiliary loop promoting the expression of the nuclear receptors RORα, REV-ERBα (encoded by NR1D1) and REV-ERBβ (encoded by NR1D2), that in turn, control the rhythmic transcription of BMAL1, as the REV-ERBα/β protein binds to ROR/REV-ERB-response elements (RORE) of the BMAL1 promoter and represses BMAL1 transcription, preventing the binding and activating action of RORα. This stabilizing loop is important for precise control of the cell-autonomous biological clock, as well as hepatic lipid metabolism, and ultimately for the harmonization of metabolism with circadian behavioral and physiological rhythms.
      • Bugge A.
      • Feng D.
      • Everett L.J.
      • Briggs E.R.
      • Mullican S.E.
      • Wang F.
      • et al.
      Rev-erbalpha and rev-erbbeta coordinately protect the circadian clock and normal metabolic function.
      • Cho H.
      • Zhao X.
      • Hatori M.
      • Yu R.T.
      • Barish G.D.
      • Lam M.T.
      • et al.
      Regulation of circadian behaviour and metabolism by rev-erb-alpha and rev-erb-beta.
      Excessive lipid accumulation in the liver, otherwise termed hepatic steatosis or non-alcoholic fatty liver disease (NAFLD), represents a pandemic whose prevalence (25% in the general population) parallels the worldwide increase of obesity.
      • Younossi Z.M.
      Non-alcoholic fatty liver disease – a global public health perspective.
      NAFLD can often evolve into non-alcoholic steatohepatitis (NASH), a potentially progressive liver disease that can lead to cirrhosis, hepatocellular carcinoma and death. NASH is characterized by steatosis, hepatocellular ballooning and inflammation, and has a global prevalence ranging from 3% to 5%.
      • Younossi Z.M.
      Non-alcoholic fatty liver disease – a global public health perspective.
      Of concern, NAFLD can occur in several cases even in the absence of obesity, so-called “lean” NAFLD.
      • Younossi Z.M.
      Non-alcoholic fatty liver disease – a global public health perspective.
      The pathogenic mechanisms and the contribution of environmental and genetic factors to the risk of developing a progressive course of NAFLD/NASH are not totally understood. In the liver, the clock machinery is essential for the maintenance of metabolic homeostasis. All metabolic processes, ranging from carbohydrates, to lipid, to amino acid metabolism, to drug metabolism are tightly dependent on a functioning biological clock in hepatocytes.
      • Tahara Y.
      • Shibata S.
      Circadian rhythms of liver physiology and disease: Experimental and clinical evidence.
      It is now clear that biological clock dysfunction accelerates the development of NAFLD, cirrhosis and liver cancer, and in turn, these disorders also disrupt clock function.
      • Tahara Y.
      • Shibata S.
      Circadian rhythms of liver physiology and disease: Experimental and clinical evidence.
      • Mazzoccoli G.
      • De Cosmo S.
      • Mazza T.
      The biological clock: a pivotal hub in non-alcoholic fatty liver disease pathogenesis.
      The hepatic clock strictly regulates the pharmacokinetics of xenobiotics through each of its phases, including absorption and uptake, distribution, metabolism and elimination. Most of these studies took advantage of valuable mice models, such as global clock-gene-knockout mice, or mice with liver-specific knockout of clock genes or clock-controlled genes. Moreover, targeted reporter mutant mice expressing PER2::LUC bioluminescence similar to endogenous PER2 expression, became widely useful in chronobiology research as a real-time reporter of circadian dynamics in different tissues, particularly in the liver.
      • Yoo S.H.
      • Yamazaki S.
      • Lowrey P.L.
      • Shimomura K.
      • Ko C.H.
      • Buhr E.D.
      • et al.
      Period2: luciferase real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues.
      Compelling recent evidence demonstrates that, in addition to the circadian clock, dysregulation of the hedgehog (Hh) signaling pathway accelerates the development of NAFLD, cirrhosis and liver cancer.
      • Verdelho Machado M.
      • Diehl A.M.
      The hedgehog pathway in nonalcoholic fatty liver disease.
      Hh is a morphogenetic factor that is critical for embryonic development and limb patterning. Re-activation of this pathway occurs during adult tissue injury, as part of the regeneration and repair processes in various tissues, including the liver. Hh ligands (i.e. Sonic Hh, Indian Hh, Desert Hh) bind to the cognate cell surface receptor Patched (Ptch), which causes the release of its inhibitory action and de-represses the co-receptor Smoothened (Smo). Active Smo then transactivates downstream transcription factors, Glioma-associated oncogene (Gli1, 2, 3), which lead to the expression or repression of various target genes.
      • Verdelho Machado M.
      • Diehl A.M.
      The hedgehog pathway in nonalcoholic fatty liver disease.
      Healthy adult hepatocytes do not produce Hh ligands but hepatic synthesis of Hh ligands drastically increased during NAFLD and NASH.
      • Verdelho Machado M.
      • Diehl A.M.
      The hedgehog pathway in nonalcoholic fatty liver disease.
      • Sicklick J.K.
      • Li Y.X.
      • Melhem A.
      • Schmelzer E.
      • Zdanowicz M.
      • Huang J.
      • et al.
      Hedgehog signaling maintains resident hepatic progenitors throughout life.
      Although excessive Hh pathway activation is believed to promote the progression of NAFLD to NASH and beyond, some basal level of Hh activity may be necessary to prevent NAFLD. Therefore, transient Hh signaling activation might constitute a compensatory response to hepatic fat accumulation. This conceptual framework has been validated in a large number of genetic and dietary mice models for the disease.
      • Verdelho Machado M.
      • Diehl A.M.
      The hedgehog pathway in nonalcoholic fatty liver disease.
      For instance, conditional liver-specific deletion of Smo in adult mice downregulated the Hh pathway and induced NAFLD,
      • Matz-Soja M.
      • Rennert C.
      • Schonefeld K.
      • Aleithe S.
      • Boettger J.
      • Schmidt-Heck W.
      • et al.
      Hedgehog signaling is a potent regulator of liver lipid metabolism and reveals a gli-code associated with steatosis.
      through the induction of lipogenic factors. Vice versa, activation of the Hh pathway (through SUFU knockdown, small molecule Shh agonists or Gli overexpression) reversed NAFLD in the liver of genetic models of obesity-related NAFLD.
      • Matz-Soja M.
      • Rennert C.
      • Schonefeld K.
      • Aleithe S.
      • Boettger J.
      • Schmidt-Heck W.
      • et al.
      Hedgehog signaling is a potent regulator of liver lipid metabolism and reveals a gli-code associated with steatosis.
      In humans, the level of activation of the Hh pathway correlates with the severity of liver damage.
      • Verdelho Machado M.
      • Diehl A.M.
      The hedgehog pathway in nonalcoholic fatty liver disease.
      Even if the role of the Hh pathway in the initial stages of NAFLD – simple steatosis - is not completely understood, Hh appears consistently activated and is one of the best-studied pathways for regulating NASH-related fibrosis progression.
      In the present issue, Marbach-Breitrück et al.
      • Marbach-Breitruck E.
      • Matz-Soja M.
      • Abraham U.
      • Schmidt-Heck W.
      • Sales S.
      • Rennert C.
      • et al.
      Tick-tock hedgehog-mutual crosstalk with liver circadian clock promotes liver steatosis.
      report the results of an original study investigating the interplay between Hh signaling and the biological clock in the regulation of lipid metabolism in the liver. They assessed time-related patterns of expression of Hh signaling pathway and core clock components in liver and hepatocytes from controls and mice with ablation of Smoothened (SAC-KO) and crossbreeds with PER2::LUC reporter mice. Several Hh pathway genes (Ihh, Ptch1, Smo, Hhip1, Fu, SuFu) showed circadian fluctuations in the liver and hepatocytes in vitro, the genes encoding Gli transcription factors (Gli1, Gli3) exhibited only tiny fluctuations and the serum levels of Indian, the ligand encoded by Ihh, oscillated with 24-hour periodicity.
      • Marbach-Breitruck E.
      • Matz-Soja M.
      • Abraham U.
      • Schmidt-Heck W.
      • Sales S.
      • Rennert C.
      • et al.
      Tick-tock hedgehog-mutual crosstalk with liver circadian clock promotes liver steatosis.
      Bmal1 downregulation in hepatocytes provoked important alterations in the expression of Hh pathway genes (upregulation of Gli1 and Ihh, downregulation of Ptch1) and, in turn, altered expression of clock genes in SAC-KO mice was demonstrated and corroborated in vitro using RNA interference against Gli1 and Gli3. Rhythmicity of many metabolic pathways, including hepatic lipid metabolism, was affected by Smo knockout in SAC-KO hepatocytes, as evidenced using genome-wide screening.
      • Marbach-Breitruck E.
      • Matz-Soja M.
      • Abraham U.
      • Schmidt-Heck W.
      • Sales S.
      • Rennert C.
      • et al.
      Tick-tock hedgehog-mutual crosstalk with liver circadian clock promotes liver steatosis.
      Interestingly, nutritional challenge represented by high-fat diet or starvation applied at diverse zeitgeber time differently impacted the biological clock-Hh signaling interplay and these results could supply a mechanistic explanation for the dissimilar effect of differently timed eating/fasting on the liver.
      This point could be of particular importance considering, on one side, the relevance of feeding time as a risk factor for dysmetabolism in humans, independently of the amount or content of food intake, and, on the other side, the positive metabolic effects of prolonged fasting or very low energy intake and time restricted feeding. In particular, the relationship between food intake and melatonin secretion onset impacts body composition, with augmented body fat and reduced glucose tolerance linked to eating at a later circadian time.
      • Mazzoccoli G.
      • De Cosmo S.
      • Mazza T.
      The biological clock: a pivotal hub in non-alcoholic fatty liver disease pathogenesis.
      • Mattson M.P.
      • Allison D.B.
      • Fontana L.
      • Harvie M.
      • Longo V.D.
      • Malaisse W.J.
      • et al.
      Meal frequency and timing in health and disease.
      • Longo V.D.
      • Panda S.
      Fasting, circadian rhythms, and time-restricted feeding in healthy lifespan.
      Furthermore as the Hh signaling pathway inhibits autophagy in mammalian cells,
      • Jimenez-Sanchez M.
      • Menzies F.M.
      • Chang Y.Y.
      • Simecek N.
      • Neufeld T.P.
      • Rubinsztein D.C.
      The hedgehog signalling pathway regulates autophagy.
      established Hh inhibitors, such as the FDA-approved Vismodegib (a potent Smo antagonist) may be studied in the future in combination with chronomodulated nutritional approaches to treat obesity and NAFLD/NASH.
      Overall, the results of this interesting study point to a tight bi-directional interplay between Hh signaling and the biological clock, mainly operated by the Gli transcription factors, as well as to a pivotal role of this interplay in the homeostasis of lipid metabolism and NAFLD pathogenesis. A future perspective could be represented by the investigation of the role played by Hh signaling for the modulation of cellular processes controlled by the biological clock, such as the pathways involved in bile acid turnover, autophagy, inflammation in the liver as well as gut microbiota dynamics, which are lead actors in the onset of hepatic steatosis-progression to NASH, and its impact on the metabolic effects of timed eating/fasting patterns, i.e. chrononutrition.

      Financial support

      We acknowledge funding support from the 5 × 1000 voluntary contribution and from the Italian Ministry of Health (No. RC1803ME40 ) (G.M.), and from the European Social Fund and European Regional Development Fund – Project MAGNET (No. CZ.02.1.01/0.0/0.0/15_003/0000492 ) (M.V.).

      Conflict of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following are the Supplementary data to this article:

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