Influence of Genistein on Hepatic Lipid Metabolism in an In Vitro Model of Hepatic Steatosis

Nonalcoholic fatty liver disease (NAFLD) is among the leading causes of end-stage liver disease. The impaired hepatic lipid metabolism in NAFLD is exhibited by dysregulated PPARα and SREBP-1c signaling pathways, which are central transcription factors associated with lipid degradation and de novo lipogenesis. Despite the growing prevalence of this disease, current pharmacological treatment options are unsatisfactory. Genistein, a soy isoflavone, has beneficial effects on lipid metabolism and may be a candidate for NAFLD treatment. In an in vitro model of hepatic steatosis, primary human hepatocytes (PHHs) were incubated with free fatty acids (FFAs) and different doses of genistein. Lipid accumulation and the cytotoxic effects of FFAs and genistein treatment were evaluated by colorimetric and enzymatic assays. Changes in lipid homeostasis were examined by RT-qPCR and Western blot analyses. PPARα protein expression was induced in steatotic PHHs, accompanied by an increase in CPT1L and ACSL1 mRNA. Genistein treatment increased PPARα protein expression only in control PHHs, while CPTL1 and ACSL1 were unchanged and PPARα mRNA was reduced. In steatotic PHHs, genistein reversed the increase in activated SREBP-1c protein. The model realistically reflected the molecular changes in hepatic steatosis. Genistein suppressed the activation of SREBP-1c in steatotic hepatocytes, but the genistein-mediated effects on PPARα were abolished by high hepatic lipid levels

Subtoxic Concentrations of Hepatotoxic Drugs Lead to Kupffer Cell Activation in a Human In Vitro Liver Model

Drug induced liver injury (DILI) is an idiosyncratic adverse drug reaction leading to severe liver damage. Kupffer cells (KC) sense hepatic tissue stress/damage and therefore could be a tool for the estimation of consequent effects associated with DILI. Aim of the present study was to establish a human in vitro liver model for the investigation of immune-mediated signaling in the pathogenesis of DILI. Hepatocytes and KC were isolated from human liver specimens. The isolated KC yield was cells/g liver tissue with a purity of >80%. KC activation was investigated by the measurement of reactive oxygen intermediates (ROI, DCF assay) and cell activity (XTT assay). The initial KC activation levels showed broad donor variability. Additional activation of KC using supernatants of hepatocytes treated with hepatotoxic drugs increased KC activity and led to donor-dependent changes in the formation of ROI compared to KC incubated with supernatants from untreated hepatocytes. Additionally, a compound- and donor-dependent increase in proinflammatory cytokines or in anti-inflammatory cytokines was detected. In conclusion, KC related immune signaling in hepatotoxicity was successfully determined in a newly established in vitro liver model. KC were able to detect hepatocyte stress/damage and to transmit a donor- and compound-dependent immune response via cytokine production

In Vivo and In Vitro Characterization of Primary Human Liver Macrophages and Their Inflammatory State

Liver macrophages (LMs) play a central role in acute and chronic liver pathologies. Investigation of these processes in humans as well as the development of diagnostic tools and new therapeutic strategies require in vitro models that closely resemble the in vivo situation. In our study, we sought to gain further insight into the role of LMs in different liver pathologies and into their characteristics after isolation from liver tissue. For this purpose, LMs were characterized in human liver tissue sections using immunohistochemistry and bioinformatic image analysis. Isolated cells were characterized in suspension using FACS analyses and in culture using immunofluorescence staining and laser scanning microscopy as well as functional assays. The majority of our investigated liver tissues were characterized by anti-inflammatory LMs which showed a homogeneous distribution and increased cell numbers in correlation with chronic liver injuries. In contrast, pro-inflammatory LMs appeared as temporary and locally restricted reactions. Detailed characterization of isolated macrophages revealed a complex disease dependent pattern of LMs consisting of pro- and anti-inflammatory macrophages of different origins, regulatory macrophages and monocytes. Our study showed that in most cases the macrophage pattern can be transferred in adherent cultures. The observed exceptions were restricted to LMs with pro-inflammatory characteristics

Effect of glucose and insulin supplementation on the isolation of primary human hepatocytes

Primary human hepatocytes (PHHs) remain the gold standard for in vitro investigations of xenobiotic metabolism and hepatotoxicity. However, scarcity of liver tissue and novel developments in liver surgery has limited the availability and quality of tissue samples. In particular, warm ischemia shifts the intracellular metabolism from aerobic to anaerobic conditions, which increases glycogenolysis, glucose depletion and energy deficiency. Therefore, the aim of the present study was to investigate whether supplementation with glucose and insulin during PHH isolation could reconstitute intracellular glycogen storage and beneficially affect viability and functionality. Furthermore, the study elucidated whether the susceptibility of the tissue’s energy status correlates with body mass index (BMI). PHHs from 12 donors were isolated from human liver tissue obtained from partial liver resections using a two-step EDTA/collagenase perfusion technique. For a direct comparison of the influence of glucose/insulin supplementation, we modified the setup, enabling the parallel isolation of two pieces of one tissue sample with varying perfusate. Independent of the BMI of the patient, the glycogen content in liver tissue was notably low in the majority of samples. Furthermore, supplementation with glucose and insulin had no beneficial effect on the glycogen concentration of isolated PHHs. However, an indirect improvement of the availability of energy was shown by increased viability, plating efficiency and partial cellular activity after supplementation. The plating efficiency showed a striking inverse correlation with increasing lipid content of PHHs. However, 60 h of cultivation time revealed no significant impact on the maintenance of albumin and urea synthesis or xenobiotic metabolism after supplementation. In conclusion, surgical procedures and tissue handling may decrease hepatic energy resources and lead to cell stress and death. Consequently, PHHs with low energy resources die during the isolation process without supplementation of glucose/insulin or early cell culture, while their survival rates are improved with glucose/insulin supplementation

Isolation and characterization of primary human livercells and their applikation in in vivo like in vitro liver models

Diese Publikationspromotion umfasst drei in internationalen Fachzeitschriften publizierte Artikel, die sich mit der Isolation und Charakterisierung von primären humanen parenchymalen und nicht-parenchymalen Leberzellen (NPC) sowie deren Anwendung in in vitro Lebermodellen beschäftigen. Die klassische 2-dimensionale (2D) Kultur primärer humaner Hepatozyten (PHH) bildet den Goldstandard für die Untersuchung der in vitro Hepatotoxizität. Allerdings fehlt in diesen Kulturen die Anwesenheit NPC. Die NPC, zu denen Kupffer Zellen (KC), Leberendothelzellen (LEC) und hepatischen Stellat-Zellen (HSC) zählen, spielen eine zentrale Rolle bei physiologischen und pathophysiologischen Prozessen. Im Rahmen der Medikamenten-induzierten Leberschädigung (engl. Drug induced liver injury - DILI) kann es durch immunmodulierende Reaktionen von NPC sowohl zu einer vermehrten Schädigung des Lebergewebes, aber auch zur Induktion von immunologischer Toleranz kommen. Durch die Entwicklung innovativer Kokulturmodelle könnte die möglicherweise die immunologische Modulation von Hepatotoxiziät erfasst werden. Im Rahme dieser Publikationspromotion wurde eine Methode etabliert, um PHH und NPC aus demselben Lebergewebe zu isolieren. Nach erfolgreicher Identifizierung und Charakterisierung bildeten diese die Grundlage für die Etablierung eines in vitro Lebermodels zur Untersuchung immunologischer Reaktionen im Rahmen von DILI. PHH und NPC wurden mit Hilfe einer zweistufigen EDTA/Kollagenase- Perfusionstechnik aus einem Stück humanem Lebergewebe isoliert. Die in der NPC-Fraktion enthaltenen KC, LEC und HSC wurden mittels Adhärenz-Trennung und magnetischer Zellsortierung voneinander getrennt. Nach Identifikation der NPC mittels spezifischer Antikörper und immunfluoreszenz Mikroskopie konnten Ausbeuten von 1,9x106 KC, 2,7x105 LEC und 4,7x105 HSC pro g Lebergewebe mit Viabilitäten > 90% und Reinheiten > 90% verzeichnet werden. Die anschließende Charakterisierung funktioneller Parameter in Kultur über 5 Tage zeigte, dass KC über eine limitierte Lebenszeit verfügen, LEC sich aus einer heterogenen Mischpopulation zusammensetzen und HSC zur Transdifferenzierung in Myofibroblasten neigen. Um DILI zu simulieren wurden KC mit den Überständen von substanzbehandelten PHH stimuliert. Die KC Aktivierung wurde durch Messung der reaktiven Sauerstoffspezies (ROS, DCF-Assay) und der Zellaktivität (XTT- Test) sowie deren immunologische Reaktion mit Hilfe von verschiedenen Zytokin- ELISA evaluiert. In den KC konnte sowohl ein Anstieg der Zellaktivität, als auch eine donor-spezifische ROS-Bildung beobachtet werden. Zusätzlich zeigte sich eine donor- und medikamentenabhängige Ausschüttung von pro- und anti- inflammatorischen Zytokinen. Das beschriebene Isolationsprotokoll ermöglicht eine simultane Isolation von PHH und NPC in guter Qualität und Quantität aus einem Stück Lebergewebe. Die Charakterisierung zeigte, dass sich KC am besten für den Einsatz in einem Lebermodell eignen. Die Detektion sowohl donor- als auch medikamentenspezifischer immunologischer Reaktionen macht das etablierte Modell zu einem vielversprechenden Ansatz für die Untersuchung der Medikamenten induzierten Hepatotoxizität.This thesis comprises three peer reviewed publications dealing with the isolation, and characterization of primary human parenchymal and non- parenchymal liver cells (NPC) and their application in in vitro liver models. 2 dimensional monocultures of primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of hepatotoxicity. However, these models miss the presence of NPC. NPC consist of Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate-cells (HSC) and play a central role in physiological and pathophysiological processes. Regarding drug-induced liver injury (DILI), NPC can modulate immunologic reactions leading to an augmented damage of the liver tissue but also to induction of immunologic tolerance. The development of innovative co-culture models could possibly help to understand the immunologic modulation of hepatotoxicity. This thesis outlines the establishment of a method to isolate PHH and NPC from the same liver tissue specimen. The successful identification and characterization paved the way for the establishment of an in vitro liver model for the investigation of immunologic reactions caused by DILI. PHH and NPC were isolated from human tissue samples using a two-step EDTA/collagenase perfusion technique. KC, LEC, and HSC were separated using specific adherence properties and magnetic activated cell sorting. The NPC were identified using specific antibodies and immunofluorescent microscopy. The quantifications revealed a yield of 1.9x106 KC, 2.7x105 LEC and 4.7x105 HSC per gram liver tissue, showing viabilities >90% and purities >90%. Subsequently, the characterization of functional parameters during a culture time of 5 days showed that KC dispose a limited life span, LEC consist of a heterogeneous population and HSC tend to transdifferentiate in myofibroblasts. For the simulation of DILI, KC were stimulated with supernatants from drug treated PHH. KC activation was investigated by the measurement of reactive oxygen species (ROS, DCF-assay) and cell activity (XTT-assay) and the immunologic reactions by analysis of cytokine production (ELISA). In KC an increase of cell activity as well as a donor specific ROS-formation was observable. Additionally, donor and drug dependent releases of pro- and anti-inflammatory cytokines were detected. The isolation protocol described, enables the isolation of PHH and NPC in high quality and quantity from one piece of liver tissue. The characterization showed that KC were the most suitable cell type for usage in in vitro models. The detection of donor- as well as drug specific immunologic reactions makes the established model to a promising tool for the investigation of DILI

Influence of Genistein on Hepatic Lipid Metabolism in an In Vitro Model of Hepatic Steatosis

Nonalcoholic fatty liver disease (NAFLD) is among the leading causes of end-stage liver disease. The impaired hepatic lipid metabolism in NAFLD is exhibited by dysregulated PPARα and SREBP-1c signaling pathways, which are central transcription factors associated with lipid degradation and de novo lipogenesis. Despite the growing prevalence of this disease, current pharmacological treatment options are unsatisfactory. Genistein, a soy isoflavone, has beneficial effects on lipid metabolism and may be a candidate for NAFLD treatment. In an in vitro model of hepatic steatosis, primary human hepatocytes (PHHs) were incubated with free fatty acids (FFAs) and different doses of genistein. Lipid accumulation and the cytotoxic effects of FFAs and genistein treatment were evaluated by colorimetric and enzymatic assays. Changes in lipid homeostasis were examined by RT-qPCR and Western blot analyses. PPARα protein expression was induced in steatotic PHHs, accompanied by an increase in CPT1L and ACSL1 mRNA. Genistein treatment increased PPARα protein expression only in control PHHs, while CPTL1 and ACSL1 were unchanged and PPARα mRNA was reduced. In steatotic PHHs, genistein reversed the increase in activated SREBP-1c protein. The model realistically reflected the molecular changes in hepatic steatosis. Genistein suppressed the activation of SREBP-1c in steatotic hepatocytes, but the genistein-mediated effects on PPARα were abolished by high hepatic lipid levels

Influence of Genistein on Hepatic Lipid Metabolism in an In Vitro Model of Hepatic Steatosis

Nonalcoholic fatty liver disease (NAFLD) is among the leading causes of end-stage liver disease. The impaired hepatic lipid metabolism in NAFLD is exhibited by dysregulated PPARα and SREBP-1c signaling pathways, which are central transcription factors associated with lipid degradation and de novo lipogenesis. Despite the growing prevalence of this disease, current pharmacological treatment options are unsatisfactory. Genistein, a soy isoflavone, has beneficial effects on lipid metabolism and may be a candidate for NAFLD treatment. In an in vitro model of hepatic steatosis, primary human hepatocytes (PHHs) were incubated with free fatty acids (FFAs) and different doses of genistein. Lipid accumulation and the cytotoxic effects of FFAs and genistein treatment were evaluated by colorimetric and enzymatic assays. Changes in lipid homeostasis were examined by RT-qPCR and Western blot analyses. PPARα protein expression was induced in steatotic PHHs, accompanied by an increase in CPT1L and ACSL1 mRNA. Genistein treatment increased PPARα protein expression only in control PHHs, while CPTL1 and ACSL1 were unchanged and PPARα mRNA was reduced. In steatotic PHHs, genistein reversed the increase in activated SREBP-1c protein. The model realistically reflected the molecular changes in hepatic steatosis. Genistein suppressed the activation of SREBP-1c in steatotic hepatocytes, but the genistein-mediated effects on PPARα were abolished by high hepatic lipid levels

Influence of Genistein on Hepatic Lipid Metabolism in an In Vitro Model of Hepatic Steatosis

Nonalcoholic fatty liver disease (NAFLD) is among the leading causes of end-stage liver disease. The impaired hepatic lipid metabolism in NAFLD is exhibited by dysregulated PPARα and SREBP-1c signaling pathways, which are central transcription factors associated with lipid degradation and de novo lipogenesis. Despite the growing prevalence of this disease, current pharmacological treatment options are unsatisfactory. Genistein, a soy isoflavone, has beneficial effects on lipid metabolism and may be a candidate for NAFLD treatment. In an in vitro model of hepatic steatosis, primary human hepatocytes (PHHs) were incubated with free fatty acids (FFAs) and different doses of genistein. Lipid accumulation and the cytotoxic effects of FFAs and genistein treatment were evaluated by colorimetric and enzymatic assays. Changes in lipid homeostasis were examined by RT-qPCR and Western blot analyses. PPARα protein expression was induced in steatotic PHHs, accompanied by an increase in CPT1L and ACSL1 mRNA. Genistein treatment increased PPARα protein expression only in control PHHs, while CPTL1 and ACSL1 were unchanged and PPARα mRNA was reduced. In steatotic PHHs, genistein reversed the increase in activated SREBP-1c protein. The model realistically reflected the molecular changes in hepatic steatosis. Genistein suppressed the activation of SREBP-1c in steatotic hepatocytes, but the genistein-mediated effects on PPARα were abolished by high hepatic lipid levels

Prolonged Lipid Accumulation in Cultured Primary Human Hepatocytes Rather Leads to ER Stress than Oxidative Stress

Overweight has become a major health care problem in Western societies and is accompanied by an increasing incidence and prevalence of non-alcoholic fatty liver disease (NAFLD). The progression from NAFLD to non-alcoholic steatohepatitis (NASH) marks a crucial tipping point in the progression of severe and irreversible liver diseases. This study aims to gain further insight into the molecular processes leading to the evolution from steatosis to steatohepatitis. Steatosis was induced in cultures of primary human hepatocytes by continuous five-day exposure to free fatty acids (FFAs). The kinetics of lipid accumulation, lipotoxicity, and oxidative stress were measured. Additionally, ER stress was evaluated by analyzing the protein expression profiles of its key players: PERK, IRE1a, and ATF6a. Our data revealed that hepatocytes are capable of storing enormous amounts of lipids without showing signs of lipotoxicity. Prolonged lipid accumulation did not create an imbalance in hepatocyte redox homeostasis or a reduction in antioxidative capacity. However, we observed an FFA-dependent increase in ER stress, revealing thresholds for triggering the activation of pathways associated with lipid stress, inhibition of protein translation, and apoptosis. Our study clearly showed that even severe lipid accumulation can be attenuated by cellular defenses, but regenerative capacities may be reduced

Prolonged Lipid Accumulation in Cultured Primary Human Hepatocytes Rather Leads to ER Stress than Oxidative Stress

Overweight has become a major health care problem in Western societies and is accompanied by an increasing incidence and prevalence of non-alcoholic fatty liver disease (NAFLD). The progression from NAFLD to non-alcoholic steatohepatitis (NASH) marks a crucial tipping point in the progression of severe and irreversible liver diseases. This study aims to gain further insight into the molecular processes leading to the evolution from steatosis to steatohepatitis. Steatosis was induced in cultures of primary human hepatocytes by continuous five-day exposure to free fatty acids (FFAs). The kinetics of lipid accumulation, lipotoxicity, and oxidative stress were measured. Additionally, ER stress was evaluated by analyzing the protein expression profiles of its key players: PERK, IRE1a, and ATF6a. Our data revealed that hepatocytes are capable of storing enormous amounts of lipids without showing signs of lipotoxicity. Prolonged lipid accumulation did not create an imbalance in hepatocyte redox homeostasis or a reduction in antioxidative capacity. However, we observed an FFA-dependent increase in ER stress, revealing thresholds for triggering the activation of pathways associated with lipid stress, inhibition of protein translation, and apoptosis. Our study clearly showed that even severe lipid accumulation can be attenuated by cellular defenses, but regenerative capacities may be reduced