The role and therapeutic potential of transcription factor CREBH in metabolic homeostasis of the liver and intestine

  • Henry Wade

Student thesis: Doctoral ThesisDoctor of Philosophy


Metabolic stress regulating transcription factor cyclic adenosine monophosphate responsive element binding protein H(CREBH/CREB3L3) is a key regulator of metabolic homeostasis. CREBH is specifically expressed in the liver and intestine. Hepatic CREBH regulates metabolism of glucose, lipids, and iron. Consequently, dysfunction or loss of CREBH causes major metabolic distress in the form of atherosclerosis, hepatic steatosis, and insulin resistance. Research emerged showing that induction of hepatic CREBH in such metabolic syndromes functioned therapeutically. In contrast, function of intestinal CREBH is less investigated but has been associated with amelioration of hyperlipidaemia due to induction of apolipoprotein B (APOB). Knowing the significance of CREBH in maintenance of hepatic homeostasis, we suspected intestinal CREBH may function similarly to attenuate intestinal metabolic distress as is experienced in inflammatory bowel disease (IBD). IBD is a chronic inflammatory disease of the gastrointestinal system with a heterogeneous aetiology comprising a complex interaction between genetic dysfunction and environmental stimuli. Known environmental stimuli include metabolic syndrome-inducing high-fat and high-fructose diets, dysbiosis of the gut microbiota, and infection. Treatment of IBD relies on lifelong immune suppression, predisposing individuals to infection and cancer. Research into alternative therapies has proven probiotic supplementation to restore gut biosis to be a promising supplement to current therapies. Here, using murine models of experimental colitis, and ApoB KO hepatocytes, we explored the role and therapeutic potential of CREBH in IBD and expanded the understanding of APOB in metabolic homeostasis. Moreover, we assessed the relationship between the controversial probiotic bacterium Akkermansia muciniphila and CREBH in intestinal restitution.

Aim-1: Delineate the function of intestinal CREBH in pathogenesis of IBD
Aim-2: Investigate the metabolic function by which A. muciniphila interacts with the intestinal epithelium and how it protects against IBD
Aim-3: Study the role of APOB in regulating metabolic homeostasis in response to insulin and palmitic acid-induced metabolic challenge

WT and Crebh knockout (KO) C57/BL mice were provided drinking water with or without 1.5% dextran sodium sulphate (DSS) for a period of 14 days to induce IBD. A further cohort of such mice were pre-treated with200 μL 1*109 CFU/mL A. muciniphila every 2 days; after 3 doses, DSS treatment began. Intestinal tissue was collected for analysis of gene and protein expression by real-time quantitative polymerase chain reaction (RT-QPCR) and western blotting (WB) respectively. Parallel in vitro experiments were conducted on human colonic adenocarcinoma (CACO2) cells; CREBH overexpression was induced by transfection of plasmid expressing the constitutively active CREBH N-terminal. Additionally, CACO2 cells were transfected with expression vector expressing prominent A. muciniphila outer membrane protein AMUC_1100 implicated as a regulator of intestinal integrity. For study of APOB, we generated a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 mediated ApoB KO hepatocyte (AML12) cell line and imposed metabolic challenge upon them by exposure to insulin, palmitic acid, and hedgehog signalling inhibitor,GDC0449. Total protein and RNA were extracted from samples for analysis by immunoblotting and qRT-PCR, respectively.

Chapter 3: CREBH deficiency promoted the pathogenesis of IBD in murine models of DSS-induced experimental colitis and tumour necrosis factor (TNF)α-induced inflammation. Loss of CREBH exacerbated loss of intestinal epithelial barrier function associated with IBD. This manifested as a dysregulation of tight-junction proteins in the intestinal epithelium. Furthermore, Crebh KO impeded intestinal wound healing via dysregulation of intestinal insulin-like growth factor signalling (IGF) sensitivity. Induction of CREBH overexpression in colon (CACO2) and primary intestinal epithelial (IPEC-2J)cells induced expression of protective intestinal barrier forming tight-junction proteins and restored sensitivity to IGF signalling. Moreover, CREBH overexpression was sufficient to ameliorate inflammatory cytokine-induced dysregulation of these genes.
Chapter 4: Akkermansia muciniphila induced expression of intestinal CREBH in vivo and consequently reduced ER-stress. We found that A. muciniphila ameliorated experimental colitis-induced weight loss and morbidly increased intestinal barrier loss in a CREBH-dependent manner. Moreover, we found that A. muciniphila promoted intestinal IGF-1 sensitivity by upregulation of Igf-1 receptor and downregulation of Igfbp5 in via miR-143 andmiR-145. The means by which A. muciniphila exerted influence over the intestinal epithelium remains unknown. Abundant A. muciniphila outer membrane protein AMUC_1100 has been highlighted as a potential mediator of intestine-A. muciniphila interaction. Inducing AMUC_1100 expression in CACO2 cells, we found that that it recapitulated many of the beneficial effects of live A. muciniphila in vivo including upregulation of CREBH, IGF-1R, and downregulation of IGFBP5. Additionally, AMUC_1100 upregulated expression of protective CLDN5and downregulated claudin-2 expression.
Chapter 5: Homozygous KO of APOB is lethal to mammals, thus we utilised a CRISPR-Cas9 model of ApoB KO in AML12 hepatocytes. Under standard growth conditions ApoB KO reduced Crebh expression andHDL-associated lipoprotein genes implying a reduction of ER-stress and lipoprotein synthesis under basal conditions. However, ApoB KO impaired cellular response to insulin- and palmitic acid (PA)-induced stress. In particular, we saw that ApoB KO reduced serine/threonine kinase protein kinaseB (AKT) expression and activation in response to insulin. This was associated with a reduction in cell survival with ApoB KO cells proliferating slower compared to WT counterparts. Finally, we saw that ApoB KO dysregulated hepatic hedgehog (HH) signalling, a pathway that in adult livers is essential for hepatic regeneration in response to injury, but when chronically active, promotes hepatic fibrogenesis. ApoB KO reduced expression of major HH homologue sonic hedgehog (Shh); inhibition of SHH signalling ameliorated many ApoBKO-induced changes in gene expression.

CREBH played a protective role in intestinal homeostasis, promoting intestinal integrity by modulation of tight junction proteins and promotion of IGF-1 signalling. Consequently, the loss or dysfunction of CREBH exacerbates IBD, perpetuating leakage of luminal macromolecules across the intestinal barrier. Probiotic supplementation of A.muciniphila exerted a therapeutic effect in IBD, the protective effect of A. muciniphilaon intestinal barrier function and weight loss are CREBH-dependent. Moreover, we found evidence that these effects of A. muciniphila are mediated by A.muciniphila outer membrane protein AMUC_1100. Additionally, we found that ApoB deficiency reduces Crebh expression under basal conditions but impairs cellular response to metabolic challenge. Finally, we found that ApoB KO impairs cellular survival and replication in association with dysregulated HH signalling.

Thesis is embargoed until 31 July 2026.

Date of AwardJul 2023
Original languageEnglish
Awarding Institution
  • Queen's University Belfast
SponsorsNorthern Ireland Department for the Economy
SupervisorQiaozhu Su (Supervisor) & Jayne Woodside (Supervisor)


  • IBD
  • inflammatory
  • bowel
  • disease
  • Akkermansia
  • Muciniphila
  • APOB
  • metabolic
  • syndrome

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