Normalization of hepatic ChREBP activity does not protect against liver disease progression in a mouse model for Glycogen Storage Disease type Ia.

BACKGROUND: Glycogen storage disease type 1a (GSD Ia) is an inborn error of metabolism caused by a defect in glucose-6-phosphatase (G6PC1) activity, which induces severe hepatomegaly and increases the risk for liver cancer. Hepatic GSD Ia is characterized by constitutive activation of Carbohydrate Response Element Binding Protein (ChREBP), a glucose-sensitive transcription factor. Previously, we showed that ChREBP activation limits non-alcoholic fatty liver disease (NAFLD) in hepatic GSD Ia. As ChREBP has been proposed as a pro-oncogenic molecular switch that supports tumour progression, we hypothesized that ChREBP normalization protects against liver disease progression in hepatic GSD Ia. METHODS: Hepatocyte-specific G6pc knockout (L-G6pc-/-) mice were treated with AAV-shChREBP to normalize hepatic ChREBP activity. RESULTS: Hepatic ChREBP normalization in GSD Ia mice induced dysplastic liver growth, massively increased hepatocyte size, and was associated with increased hepatic inflammation. Furthermore, nuclear levels of the oncoprotein Yes Associated Protein (YAP) were increased and its transcriptional targets were induced in ChREBP-normalized GSD Ia mice. Hepatic ChREBP normalization furthermore induced DNA damage and mitotic activity in GSD Ia mice, while gene signatures of chromosomal instability, the cytosolic DNA-sensing cGAS-STING pathway, senescence, and hepatocyte dedifferentiation emerged. CONCLUSIONS: In conclusion, our findings indicate that ChREBP activity limits hepatomegaly while decelerating liver disease progression and protecting against chromosomal instability in hepatic GSD Ia. These results disqualify ChREBP as a therapeutic target for treatment of liver disease in GSD Ia. In addition, they underline the importance of establishing the context-specific roles of hepatic ChREBP to define its therapeutic potential to prevent or treat advanced liver disease.

The chemotherapeutic drug doxorubicin does not exacerbate p16Ink4a-positive senescent cell accumulation and cardiometabolic disease development in young adult female LDLR-deficient mice.

Cancer survivors who received chemotherapy, such as the anthracycline doxorubicin (DOX), have an increased risk of developing complications later in life, including the development of chronic metabolic diseases. Although the etiology of this increased risk for late metabolic complications in cancer survivors is poorly understood, a causal role of therapy-induced senescent cells has been suggested. To study the role of cellular senescence in chemotherapy-induced metabolic complications, young adult female low-density lipoprotein receptor-deficient (Ldlr-/-)-p16-3MR mice, in which p16Ink4a-positive (p16Ink4a+) senescent cells can be genetically eliminated, were treated with four weekly injections of DOX (2.5 mg/kg) followed by a high-fat high-cholesterol diet for 12 weeks. While DOX treatment induced known short-term effects, such as reduction in body weight, gonadal fat mass, and adipose tissue inflammation, it was not associated with significant long-term effects on glucose homeostasis, hepatic steatosis, or atherosclerosis. We further found no evidence of DOX-induced accumulation of p16Ink4a+-senescent cells at 1 or 12 weeks after DOX treatment. Neither did we observe an effect of elimination of p16Ink4a+-senescent cells on the development of diet-induced cardiometabolic complications in DOX-treated mice. Other markers for senescence were generally also not affected except for an increase in p21 and Cxcl10 in gonadal white adipose tissue long-term after DOX treatment. Together, our study does not support a significant role for p16Ink4a+-senescent cells in the development of diet-induced cardiometabolic disease in young adult DOX-treated female Ldlr-/- mice. These findings illustrate the need of further studies to understand the link between cancer therapy and cardiometabolic disease development in cancer survivors.

Enhanced C/EBPß function promotes hypertrophic versus hyperplastic fat tissue growth and prevents steatosis in response to high-fat diet feeding.

Chronic obesity is correlated with severe metabolic and cardiovascular diseases as well as with an increased risk for developing cancers. Obesity is usually characterized by fat accumulation in enlarged - hypertrophic - adipocytes that are a source of inflammatory mediators, which promote the development and progression of metabolic disorders. Yet, in certain healthy obese individuals, fat is stored in metabolically more favorable hyperplastic fat tissue that contains an increased number of smaller adipocytes that are less inflamed. In a previous study, we demonstrated that loss of the inhibitory protein-isoform C/EBPß-LIP and the resulting augmented function of the transactivating isoform C/EBPß-LAP promotes fat metabolism under normal feeding conditions and expands health- and lifespan in mice. Here, we show that in mice on a high-fat diet, LIP-deficiency results in adipocyte hyperplasia associated with reduced inflammation and metabolic improvements. Furthermore, fat storage in subcutaneous depots is significantly enhanced specifically in LIP-deficient male mice. Our data identify C/EBPß as a regulator of adipocyte fate in response to increased fat intake, which has major implications for metabolic health and aging.

Modeling Phenotypic Heterogeneity of Glycogen Storage Disease Type 1a Liver Disease in Mice by Somatic CRISPR/CRISPR-associated protein 9-Mediated Gene Editing.

BACKGROUND AND AIMS: Patients with glycogen storage disease type 1a (GSD-1a) primarily present with life-threatening hypoglycemia and display severe liver disease characterized by hepatomegaly. Despite strict dietary management, long-term complications still occur, such as liver tumor development. Variations in residual glucose-6-phosphatase (G6PC1) activity likely contribute to phenotypic heterogeneity in biochemical symptoms and complications between patients. However, lack of insight into the relationship between G6PC1 activity and symptoms/complications and poor understanding of the underlying disease mechanisms pose major challenges to provide optimal health care and quality of life for GSD-1a patients. Currently available GSD-1a animal models are not suitable to systematically investigate the relationship between hepatic G6PC activity and phenotypic heterogeneity or the contribution of gene-gene interactions (GGIs) in the liver. APPROACH AND RESULTS: To meet these needs, we generated and characterized a hepatocyte-specific GSD-1a mouse model using somatic CRISPR/CRISPR-associated protein 9 (Cas9)-mediated gene editing. Hepatic G6pc editing reduced hepatic G6PC activity up to 98% and resulted in failure to thrive, fasting hypoglycemia, hypertriglyceridemia, hepatomegaly, hepatic steatosis (HS), and increased liver tumor incidence. This approach was furthermore successful in simultaneously modulating hepatic G6PC and carbohydrate response element-binding protein, a transcription factor that is activated in GSD-1a and protects against HS under these conditions. Importantly, it also allowed for the modeling of a spectrum of GSD-1a phenotypes in terms of hepatic G6PC activity, fasting hypoglycemia, hypertriglyceridemia, hepatomegaly and HS. CONCLUSIONS: In conclusion, we show that somatic CRISPR/Cas9-mediated gene editing allows for the modeling of a spectrum of hepatocyte-borne GSD-1a disease symptoms in mice and to efficiently study GGIs in the liver. This approach opens perspectives for translational research and will likely contribute to personalized treatments for GSD-1a and other genetic liver diseases.

Acute systemic loss of Mad2 leads to intestinal atrophy in adult mice.

Chromosomal instability (CIN) is a hallmark of cancer, leading to aneuploid cells. To study the role that CIN plays in tumor evolution, several mouse models have been engineered over the last 2 decades. These models have unequivocally shown that systemic high-grade CIN is embryonic lethal. We and others have previously shown that embryonic lethality can be circumvented by provoking CIN in a tissue-specific fashion. In this study, we provoke systemic high-grade CIN in adult mice as an alternative to circumvent embryonic lethality. For this, we disrupt the spindle assembly checkpoint (SAC) by alleviating Mad2 or truncating Mps1, both essential genes for SAC functioning, with or without p53 inactivation. We find that disruption of the SAC leads to rapid villous atrophy, atypia and apoptosis of the epithelia of the jejunum and ileum, substantial weight loss, and death within 2-3 weeks after the start of the CIN insult. Despite this severe intestinal phenotype, most other tissues are unaffected, except for minor abnormalities in spleen, presumably due to the lower proliferation rate in these tissues. We conclude that high-grade CIN in vivo in adult mice is most toxic to the high cell turnover intestinal epithelia.

E2F7 Is a Potent Inhibitor of Liver Tumor Growth in Adult Mice.

BACKGROUND AND AIMS: Up-regulation of the E2F-dependent transcriptional network has been identified in nearly every human malignancy and is an important driver of tumorigenesis. Two members of the E2F family, E2F7 and E2F8, are potent repressors of E2F-dependent transcription. They are atypical in that they do not bind to dimerization partner proteins and are not controlled by retinoblastoma protein. The physiological relevance of E2F7 and E2F8 remains incompletely understood, largely because tools to manipulate their activity in vivo have been lacking. APPROACH AND RESULTS: Here, we generated transgenic mice with doxycycline-controlled transcriptional activation of E2f7 and E2f8 and induced their expression during postnatal development, in adulthood, and in the context of cancer. Systemic induction of E2f7 and, to lesser extent, E2f8 transgenes in juvenile mice impaired cell proliferation, caused replication stress, DNA damage, and apoptosis, and inhibited animal growth. In adult mice, however, E2F7 and E2F8 induction was well tolerated, yet profoundly interfered with DNA replication, DNA integrity, and cell proliferation in diethylnitrosamine-induced liver tumors. CONCLUSION: Collectively, our findings demonstrate that atypical E2Fs can override cell-cycle entry and progression governed by other E2F family members and suggest that this property can be exploited to inhibit proliferation of neoplastic hepatocytes when growth and development have subsided during adulthood.

Surgical resection and radiofrequency ablation initiate cancer in cytokeratin-19+- liver cells deficient for p53 and Rb.

The long term prognosis of liver cancer patients remains unsatisfactory because of cancer recurrence after surgical interventions, particularly in patients with viral infections. Since hepatitis B and C viral proteins lead to inactivation of the tumor suppressors p53 and Retinoblastoma (Rb), we hypothesize that surgery in the context of p53/Rb inactivation initiate de novo tumorigenesis.We, therefore, generated transgenic mice with hepatocyte and cholangiocyte/liver progenitor cell (LPC)-specific deletion of p53 and Rb, by interbreeding conditional p53/Rb knockout mice with either Albumin-cre or Cytokeratin-19-cre transgenic mice.We show that liver cancer develops at the necrotic injury site after surgical resection or radiofrequency ablation in p53/Rb deficient livers. Cancer initiation occurs as a result of specific migration, expansion and transformation of cytokeratin-19+-liver (CK-19+) cells. At the injury site migrating CK-19+ cells formed small bile ducts and adjacent cells strongly expressed the transforming growth factor ß (TGFß). Isolated cytokeratin-19+ cells deficient for p53/Rb were resistant against hypoxia and TGFß-mediated growth inhibition. CK-19+ specific deletion of p53/Rb verified that carcinomas at the injury site originates from cholangiocytes or liver progenitor cells.These findings suggest that human liver patients with hepatitis B and C viral infection or with mutations for p53 and Rb are at high risk to develop tumors at the surgical intervention site.

Regeneration of human extrahepatic biliary epithelium: the peribiliary glands as progenitor cell compartment.

BACKGROUND & AIMS: Although regeneration of intrahepatic bile ducts has been extensively studied and intrahepatic progenitor cells have been identified, few studies have focussed on the extrahepatic bile duct (EHBD). We hypothesized that local progenitor cells are present within the EHBD of humans. Human EHBD specimens (n = 17) were included in this study. METHODS: Specimens of normal EHBD tissue were obtained from healthy donor livers (n = 6), mildly injured EHBD from patients with cholangitis (n = 6) and severely injured EHBD from patients with ischaemic type biliary lesions (n = 5). Double immunostaining for K19 and the proliferation marker Ki-67 was performed to identify and localize proliferating cells. In addition, immunofluorescent doublestaining using antibodies against K19 and c-Kit was performed to identify and localize cholangiocytes co-expressing putative progenitor cell markers. RESULTS: In normal EHBD, few Ki-67(+) cells were detected, whereas large numbers of Ki-67(+) were found in the diseased EHBD. In EHBD affected by cholangitis, Ki-67(+) cells were mainly located in the basal layer of the lumen. EHBD specimens from patients with ischaemic type biliary lesions displayed histological signs of epithelial cell loss and large numbers of Ki-67(+) cells were observed in the peribiliary glands. C-Kit expression was localized throughout the EHBD wall and immunofluorescent doublestaining identified a few K19(+) /c-Kit(+) cells in the luminal epithelium of the EHBD as well as in the peribiliary glands. CONCLUSIONS: These findings support the hypothesis that progenitor cells exist in the EHBD and that the peribiliary glands can be considered a local progenitor cell niche in the human EHBD.