Publications
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Oldereid, Tine Simensen; Jiang, Xiaojun; Nordhus, Kathrine Sivertsen; Ponzetta, Andrea; Bjørnholt, Jørgen & Björkström, Niklas K.
[Show all 8 contributors for this article]
(2023).
Role of bacteria and microbial metabolites in immune modulation during early life.
Scandinavian Journal of Immunology.
ISSN 0300-9475.
doi:
10.1111/sji.13336.
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Abstract Host–microbiome interplay from birth is essential for immune imprinting and tuning. Live gut microbes and microbial‐derived metabolites regulate the development and modulation of the immune system, but whether microbial metabolites solely are sufficient to induce immune maturation remains unclear. Sterile faecal filtrates (FFT) were generated from murine gut contents. Newborn germ‐free (GF) mice were treated twice daily with FFT (GF‐FFT) or saline (GF‐NaCl) from post‐natal day 5 until 4 weeks of age. A third group of GF neonates were conventionalized by the transfer of caecal microbiota with live gut microbes. Host immune compartments were comprehensively immunophenotyped and systemically analysed in all available immune‐related organs using flow cytometry. Oral FFT was associated with reduced survival among neonates (n = 7/19; 36.8% mortality), while saline treatment was well tolerated (n = 1/17, 5.9% mortality). Four‐week‐old FFT‐treated pups were comparable in body weight to GF‐NaCl, and the major B‐cell, conventional T‐cell and unconventional T‐cell subsets were unchanged from saline‐treated mice. Live bacteria administered during early life induced clear changes in proportions of B cells, T cells and T‐cell subsets in all mucosal tissues and secondary lymphoid organs compared to GF‐FFT, including restoration of intestinal natural killer T (NKT) cells with characteristics similar to conventional pups. Our findings show that oral administration of a FFT made of microbial metabolites, antigens and bacteriophages alone is insufficient to induce normal immune development elicited by the presence of live bacteria. Reduced survival during neonatal FFT treatment suggests a potential bioactive attribute of sterile faecal filtrates.
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Harrison, Sean Philip; Siller, Richard; Tanaka, Yoshiaki; Dugarte, Maria Eugenia Chollet; Morena-Barrio, María Eugenia de la & Xiang, Yangfei
[Show all 36 contributors for this article]
(2023).
Scalable production of tissue-like vascularized liver organoids from human PSCs.
Experimental and Molecular Medicine.
ISSN 1226-3613.
55,
p. 2005–2024.
doi:
10.1038/s12276-023-01074-1.
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Abstract The lack of physiological parity between 2D cell culture and in vivo culture has led to the development of more organotypic models, such as organoids. Organoid models have been developed for a number of tissues, including the liver. Current organoid protocols are characterized by a reliance on extracellular matrices (ECMs), patterning in 2D culture, costly growth factors and a lack of cellular diversity, structure, and organization. Current hepatic organoid models are generally simplistic and composed of hepatocytes or cholangiocytes, rendering them less physiologically relevant compared to native tissue. We have developed an approach that does not require 2D patterning, is ECM independent, and employs small molecules to mimic embryonic liver development that produces large quantities of liver-like organoids. Using single-cell RNA sequencing and immunofluorescence, we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, and a population of resident macrophages: Kupffer cells. The organoids exhibit key liver functions, including drug metabolism, serum protein production, urea synthesis and coagulation factor production, with preserved post-translational modifications such as N-glycosylation and functionality. The organoids can be transplanted and maintained long term in mice producing human albumin. The organoids exhibit a complex cellular repertoire reflective of the organ and have de novo vascularization and liver-like function. These characteristics are a prerequisite for many applications from cellular therapy, tissue engineering, drug toxicity assessment, and disease modeling to basic developmental biology.
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Åsrud, Kathrine Sivertsen; Bjørnstad, Ronja; Kopperud, Reidun Kristin; Pedersen, Line; Hoeven, Barbara van der & Karlsen, Tine Veronica
[Show all 12 contributors for this article]
(2020).
Epac1 null mice have nephrogenic diabetes insipidus with deficient corticopapillary osmotic gradient and weaker collecting duct tight junctions.
Acta Physiologica.
ISSN 1748-1708.
229(1).
doi:
10.1111/apha.13442.
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Aim The cAMP‐mediator Epac1 (RapGef3) has high renal expression. Preliminary observations revealed increased diuresis in Epac1−/− mice. We hypothesized that Epac1 could restrict diuresis by promoting transcellular collecting duct (CD) water and urea transport or by stabilizing CD paracellular junctions to reduce osmolyte loss from the renal papillary interstitium. Methods In Epac1−/− and Wt C57BL/6J mice, renal papillae, dissected from snap‐frozen kidneys, were assayed for the content of key osmolytes. Cell junctions were analysed by transmission electron microscopy. Urea transport integrity was evaluated by urea loading with 40% protein diet, endogenous vasopressin production was manipulated by intragastric water loading and moderate dehydration and vasopressin type 2 receptors were stimulated selectively by i.p.‐injected desmopressin (dDAVP). Glomerular filtration rate (GFR) was estimated as [14C]inulin clearance. The glomerular filtration barrier was evaluated by urinary albumin excretion and microvascular leakage by the renal content of time‐spaced intravenously injected 125I‐ and 131I‐labelled albumin. Results Epac1−/− mice had increased diuresis and increased free water clearance under antidiuretic conditions. They had shorter and less dense CD tight junction (TJs) and attenuated corticomedullary osmotic gradient. Epac1−/− mice had no increased protein diet‐induced urea‐dependent osmotic diuresis, and expressed Wt levels of aquaporin‐2 (AQP‐2) and urea transporter A1/3 (UT‐A1/3). Epac1−/− mice had no urinary albumin leakage and unaltered renal microvascular albumin extravasation. Their GFR was moderately increased, unless when treated with furosemide. Conclusion Our results conform to the hypothesis that Epac1‐dependent mechanisms protect against diabetes insipidus by maintaining renal papillary osmolarity and the integrity of CD TJs.
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Åsrud, Kathrine Sivertsen; Pedersen, Line; Aesoy, Reidun; Muwonge, Haruna; Aasebø, Elise & Pettersen, Ina Katrine Nitschke
[Show all 14 contributors for this article]
(2019).
Mice depleted for Exchange Proteins Directly Activated by cAMP (Epac) exhibit irregular liver regeneration in response to partial hepatectomy.
Scientific Reports.
9:13789,
p. 1–17.
doi:
10.1038/s41598-019-50219-8.
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The exchange proteins directly activated by cAMP 1 and 2 (Epac1 and Epac2) are expressed in a cell specific manner in the liver, but their biological functions in this tissue are poorly understood. The current study was undertaken to begin to determine the potential roles of Epac1 and Epac2 in liver physiology and disease. Male C57BL/6J mice in which expression of Epac1 and/or Epac2 are deleted, were subjected to partial hepatectomy and the regenerating liver was analyzed with regard to lipid accumulation, cell replication and protein expression. In response to partial hepatectomy, deletion of Epac1 and/or Epac2 led to increased hepatocyte proliferation 36 h post surgery, and the transient steatosis observed in wild type mice was virtually absent in mice lacking both Epac1 and Epac2. The expression of the protein cytochrome P4504a14, which is implicated in hepatic steatosis and fibrosis, was substantially reduced upon deletion of Epac1/2, while a number of factors involved in lipid metabolism were significantly decreased. Moreover, the number of Küpffer cells was affected, and Epac2 expression was increased in the liver of wild type mice in response to partial hepatectomy, further supporting a role for these proteins in liver function. This study establishes hepatic phenotypic abnormalities in mice deleted for Epac1/2 for the first time, and introduces Epac1/2 as regulators of hepatocyte proliferation and lipid accumulation in the regenerative process.
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Aesoy, Reidun; Muwonge, Haruna; Åsrud, Kathrine Sivertsen; Sabir, Misbah; Witsø, Solveig Lund & Bjørnstad, Ronja
[Show all 10 contributors for this article]
(2018).
Deletion of exchange proteins directly activated by cAMP (Epac) causes defects in hippocampal signaling in female mice.
PLOS ONE.
13(7).
doi:
10.1371/journal.pone.0200935.
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Previous studies demonstrate essential roles for the exchange proteins directly activated by cAMP 1 and 2 (Epac1 and Epac2; here collectively referred to as Epac) in the brain. In the hippocampus, Epac contributes to the control of neuronal growth and differentiation and has been implicated in memory and learning as well as in anxiety and depression. In the present study we address the hypothesis that Epac affects hippocampal cellular responses to acute restraint stress. Stress causes activation of the hypothalamus-pituitary-adrenal (HPA)-axis, and glucocorticoid receptor (GR) signaling is essential for proper feedback regulation of the stress response, both in the brain and along the HPA axis. In the hippocampus, GR expression is regulated by cAMP and the brain enriched micro RNA miR-124. Epac has been associated with miR-124 expression in hippocampal neurons, but not in regulation of GR. We report that hippocampal expression of Epac1 and Epac2 increased in response to acute stress in female wild type mice. In female mice genetically deleted for Epac, nuclear translocation of GR in response to restraint stress was significantly delayed, and moreover, miR-124 expression was decreased in these mice. Male mice lacking Epac also showed abnormalities in miR-124 expression, but the phenotype was less profound than in females. Serum corticosterone levels were slightly altered immediately after stress in both male and female mice deleted for Epac. The presented data indicate that Epac1 and Epac2 are involved in controlling cellular responses to acute stress in the mouse hippocampus and provide novel insights into the underlying transcriptional and signaling networks. Interestingly, we observe sex specific differences when Epac is deleted. As the incidence and prevalence of stress-related diseases are higher in women than in men, the Epac knockout models might serve as genetic tools to further elucidate the cellular mechanisms underlying differences between male and female with regard to regulation of stress.
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Nygaard, Gyrid; Herfindal, Lars; Åsrud, Kathrine Sivertsen; Bjørnstad, Ronja; Kopperud, Reidun Kristin & Oveland, Eystein
[Show all 13 contributors for this article]
(2017).
Epac1-deficient mice have bleeding phenotype and thrombocytes with decreased GPIbβ expression.
Scientific Reports.
7:8725,
p. 1–15.
doi:
10.1038/s41598-017-08975-y.
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Epac1 (Exchange protein directly activated by cAMP 1) limits fluid loss from the circulation by tightening the endothelial barrier. We show here that Epac1−/− mice, but not Epac2−/− mice, have prolonged bleeding time, suggesting that Epac1 may limit fluid loss also by restraining bleeding. The Epac1−/− mice had deficient in vitro secondary hemostasis. Quantitative comprehensive proteomics analysis revealed that Epac1−/− mouse platelets (thrombocytes) had unbalanced expression of key components of the glycoprotein Ib-IX-V (GPIb-IX-V) complex, with decrease of GP1bβ and no change of GP1bα. This complex is critical for platelet adhesion under arterial shear conditions. Furthermore, Epac1−/− mice have reduced levels of plasma coagulation factors and fibrinogen, increased size of circulating platelets, increased megakaryocytes (the GP1bβ level was decreased also in Epac1−/− bone marrow) and higher abundance of reticulated platelets. Viscoelastic measurement of clotting function revealed Epac1−/− mice with a dysfunction in the clotting process, which corresponds to reduced plasma levels of coagulation factors like factor XIII and fibrinogen. We propose that the observed platelet phenotype is due to deficient Epac1 activity during megakaryopoiesis and thrombopoiesis, and that the defects in blood clotting for Epac1−/− is connected to secondary hemostasis.
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Edland, Ravn Fritdtjov; Wergeland, Anita; Kopperud, Reidun Kristin; åsrud, Kathrine Sivertsen; Høivik, Erling Andre & Witsø, Solveig Lund
[Show all 12 contributors for this article]
(2016).
Long-term consumption of an obesogenic high fat diet prior to ischemia-reperfusion mediates cardioprotection via Epac1-dependent signaling.
Nutrition & Metabolism.
13(1),
p. 1–11.
doi:
10.1186/s12986-016-0147-1.
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Background: Obesity is still considered a risk factor for cardiovascular disease, although more recent knowledge also suggests obesity to be associated with reduced morbidity and mortality - the “obesity paradox”. This study explores if long-term feeding of an obesogenic high fat diet renders the myocardium less susceptible to ischemic-reperfusion induced injury via Epac-dependent signaling. Methods: Wild type (wt), Epac1 (Epac1−/−) and Epac2 (Epac2−/−) deficient mice were fed a high fat (HFD) or normal chow diet (ND) for 33 ± 1 weeks. Six experimental groups were included: (1) control wt ND (wt ND), (2) control wt HFD (wt HFD), (3) Epac1−/− mice on ND (Epac1−/− ND), (4) Epac1−/− mice on HFD (Epac1−/− HFD), (5) Epac2−/− mice on ND (Epac2−/− ND), and (6) Epac2−/− mice on HFD (Epac2−/− HFD). Isolated ex vivo mice hearts were perfused in a constant pressure Langendorff mode, and exposed to 30min of global ischemia (GI) and 60min of reperfusion. Endpoints were infarct size and functional recovery. Results: All groups fed a HFD presented with significantly enhanced body weight, visceral fat content and reduced glucose clearance compared to corresponding ND groups. Although the HFD cohorts presented with an overall comparable systemic capability to clear glucose, the Epac1−/− HFD group presented with glucose levels slightly above the human diabetes criteria at the end of the intraperitoneal glucose tolerance test (ipGTT). Moreover, the HFD significantly reduced infarct size in both wild type (wt HFD 41.3 ± 5.5% vs. wt ND 58.0 ± 9.8%, p < 0.05) and Epac2−/− cohorts (Epac2−/− HFD 34.4 ± 7.2% vs. Epac2−/− ND 56.5 ± 3.8%, p < 0.05). Interestingly, however, the HFD did not reduce infarct size in Epac1−/− deficient mice hearts (Epac1−/− HFD 65.1 ± 5.1% vs. Epac1−/− ND 56.1 ± 3.5%, ns.). Conclusion: Epac1-dependent signaling is involved in mediating the cardioprotection afforded by long-term feeding of an obesogenic high fat diet in mice hearts.
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Æsøy, Reidun; Gradin, Katarina; Åsrud, Kathrine Sivertsen; Høivik, Erling Andre; Ruas, Jorge Lira & Poellinger, Lorenz
[Show all 7 contributors for this article]
(2014).
Regulation of CDKN2B expression by interaction of Arnt with Miz-1 - a basis for functional integration between the HIF and Myc gene regulatory pathways.
Molecular Cancer.
13(1:54).
doi:
10.1186/1476-4598-13-54.
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Background: Hypoxia- and Myc-dependent transcriptional regulatory pathways are frequently deregulated in cancer cells. These pathways converge in many cellular responses, but the underlying molecular mechanisms are unclear. Methods: The ability of Miz-1 and Arnt to interact was identified in a yeast two-hybrid screen. The mode of interaction and the functional consequences of complex formation were analyzed by diverse molecular biology methods, in vitro. Statistical analyses were performed by Student’s t-test and ANOVA. Results: In the present study we demonstrate that the aryl hydrocarbon receptor nuclear translocator (Arnt), which is central in hypoxia-induced signaling, forms a complex with Miz-1, an important transcriptional regulator in Myc-mediated transcriptional repression. Overexpression of Arnt induced reporter gene activity driven by the proximal promoter of the cyclin-dependent kinase inhibitor 2B gene (CDKN2B), which is an established target for the Myc/Miz-1 complex. In contrast, mutated forms of Arnt, that were unable to interact with Miz-1, had reduced capability to activate transcription. Moreover, repression of Arnt reduced endogenous CDKN2B expression, and chromatin immunoprecipitation demonstrated that Arnt interacts with the CDKN2B promoter. The transcriptional activity of Arnt was counteracted by Myc, but not by a mutated variant of Myc that is unable to interact with Miz-1, suggesting mutually exclusive interaction of Arnt and Myc with Miz-1. Our results also establish CDKN2B as a hypoxia regulated gene, as endogenous CDKN2B mRNA and protein levels were reduced by hypoxic treatment of U2OS cells. Conclusions: Our data reveal a novel mode of regulation by protein-protein interaction that directly ties together, at the transcriptional level, the Myc- and hypoxia-dependent signaling pathways and expands our understanding of the roles of hypoxia and cell cycle alterations during tumorigenesis.
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Pedersen, Line; Bakke, Marit & Åsrud, Kathrine Sivertsen
(2014).
Characterization of liver functions in Epac knockout models.
Universitetet i Bergen.
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The discovery of the exchange proteins directly activated by cAMP 1 and 2 (Epac1 and
Epac2) opened up new avenues in cAMP-related research. Since their discovery in the late
1990’s, physiological roles have been described for Epac1/2 in the brain, heart, kidneys and
lungs. However, despite the fact that the liver expresses a unique Epac2 isoform, the
functions of the Epac proteins in this organ remains virtually unknown, and have not been
investigated in in vivo models. In this master project I used genetic mouse models deleted for
both Epac1 and Epac2 (Epac1/2-/- mice) to study whether loss of Epac proteins affects crucial
liver functions.
The present study is the first to suggest roles of Epac1/2 in liver regeneration after partial
hepatectomy. I demonstrate that deletion of Epac1/2 leads to a remarkably increase in
proliferating hepatocytes in the regenerating liver, as assessed by BrdU-incorporation. Of
great interest is also the finding that fat vacuoles, which normally accumulate in rodent livers
after partial hepatectomy, are nearly absent in regenerated livers of Epac1/2-/- mice.
Moreover, determination of oxygen consumption in liver extracts revealed increased rates of
mitochondrial respiration and fatty acid driven respiration in Epac1/2-/- mice, which might be
associated with the loss of fat vacuoles. The amount of bile acids in feces was not altered as a
consequence of deleting Epac1/2 when the mice were housed under standard conditions.
However, at the mRNA level, the expression of specific enzymes and transcription factors
involved in bile acid biosynthesis were differently expressed in mice deleted for Epac1/2.
This effect was especially pronounced in response to food deprivation. Taken together the
results presented in this thesis show that although deletion of Epac1/2 does not cause gross
morphological changes and liver dysfunction in mice kept at standard protected conditions in
the animal facility, various stressful conditions do lead to penetration of liver specific
phenotypes in Epac1/2-/- mice. The obtained data provide novel insights into liver physiology,
and moreover, will serve as a basis for future studies to more precisely describe the role of
the Epac proteins in the liver. A detailed understanding of the molecular mechanisms that
underlie liver function and hepatocyte proliferation is important and clinically relevant as
hepatectomy is a therapeutic option for patients with severe liver diseases.
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Published
Mar. 4, 2019 1:42 PM
- Last modified
Aug. 27, 2024 11:43 AM
