TY - JOUR A1 - Bishop, Christopher Allen A1 - Schulze, Matthias Bernd A1 - Klaus, Susanne A1 - Weitkunat, Karolin T1 - The branched-chain amino acids valine and leucine have differential effects on hepatic lipid metabolism JF - The FASEB journal : the official journal of the Federation of American Societies for Experimental Biology N2 - Dairy intake, as a source of branched-chain amino acids (BCAA), has been linked to a lower incidence of type-2-diabetes and increased circulating odd-chain fatty acids (OCFA). To understand this connection, we aimed to investigate differences in BCAA metabolism of leucine and valine, a possible source of OCFA, and their role in hepatic metabolism. Male mice were fed a high-fat diet supplemented with leucine and valine for 1 week and phenotypically characterized with a focus on lipid metabolism. Mouse primary hepatocytes were treated with the BCAA or a Ppar alpha activator WY-14643 to systematically examine direct hepatic effects and their mechanisms. Here, we show that only valine supplementation was able to increase hepatic and circulating OCFA levels via two pathways; a PPAR alpha-dependent induction of alpha-oxidation and an increased supply of propionyl-CoA for de novo lipogenesis. Meanwhile, we were able to confirm leucine-mediated effects on the inhibition of food intake and transport of fatty acids, as well as induction of S6 ribosomal protein phosphorylation. Taken together, these data illustrate differential roles of the BCAA in lipid metabolism and provide preliminary evidence that exclusively valine contributes to the endogenous formation of OCFA which is important for a better understanding of these metabolites in metabolic health. KW - fatty acid metabolism KW - leucine KW - liver KW - OCFA KW - valine Y1 - 2020 U6 - https://doi.org/10.1096/fj.202000195R SN - 0892-6638 SN - 1530-6860 VL - 34 IS - 7 SP - 9727 EP - 9739 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Fedders, Ronja A1 - Muenzner, Matthias A1 - Weber, Pamela A1 - Sommerfeld, Manuela A1 - Knauer, Miriam A1 - Kedziora, Sarah A1 - Kast, Naomi A1 - Heidenreich, Steffi A1 - Raila, Jens A1 - Weger, Stefan A1 - Henze, Andrea A1 - Schupp, Michael T1 - Liver-secreted RBP4 does not impair glucose homeostasis in mice JF - The journal of biological chemistry N2 - Retinol-binding protein 4 (RBP4) is the major transport protein for retinol in blood. Recent evidence from genetic mouse models shows that circulating RBP4 derives exclusively from hepatocytes. Because RBP4 is elevated in obesity and associates with the development of glucose intolerance and insulin resistance, we tested whether a liver-specific overexpression of RBP4 in mice impairs glucose homeostasis. We used adeno-associated viruses (AAV) that contain a highly liver-specific promoter to drive expression of murine RBP4 in livers of adult mice. The resulting increase in serum RBP4 levels in these mice was comparable with elevated levels that were reported in obesity. Surprisingly, we found that increasing circulating RBP4 had no effect on glucose homeostasis. Also during a high-fat diet challenge, elevated levels of RBP4 in the circulation failed to aggravate the worsening of systemic parameters of glucose and energy homeostasis. These findings show that liver-secreted RBP4 does not impair glucose homeostasis. We conclude that a modest increase of its circulating levels in mice, as observed in the obese, insulin-resistant state, is unlikely to be a causative factor for impaired glucose homeostasis. KW - liver KW - retinoid-binding protein KW - glucose metabolism KW - insulin resistance KW - mouse KW - TTR Y1 - 2018 U6 - https://doi.org/10.1074/jbc.RA118.004294 SN - 1083-351X VL - 293 IS - 39 SP - 15269 EP - 15276 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - THES A1 - Henkel-Oberländer, Janin T1 - Einfluss von Prostaglandin E2 auf die Entstehung von Insulinresistenz und die Regulation der Entzündungsantwort bei der Diät-induzierten nicht-alkoholischen Fettlebererkrankung N2 - Weltweit sind fast 40 % der Bevölkerung übergewichtig und die Prävalenz von Adipositas, Insulinresistenz und den resultierenden Folgeerkrankungen wie dem Metabolischen Syndrom und Typ-2-Diabetes steigt rapide an. Als häufigste Ursachen werden diätetisches Fehlverhalten und mangelnde Bewegung angesehen. Die nicht-alkoholische Fettlebererkrankung (NAFLD), deren Hauptcharakteristikum die exzessive Akkumulation von Lipiden in der Leber ist, korreliert mit dem Body Mass Index (BMI). NAFLD wird als hepatische Manifestation des Metabolischen Syndroms angesehen und ist inzwischen die häufigste Ursache für Leberfunktionsstörungen. Die Erkrankung umfasst sowohl die benigne hepatische Steatose (Fettleber) als auch die progressive Form der nicht-alkoholischen Steatohepatitis (NASH), bei der die Steatose von Entzündung und Fibrose begleitet ist. Die Ausbildung einer NASH erhöht das Risiko, ein hepatozelluläres Karzinom (HCC) zu entwickeln und kann zu irreversibler Leberzirrhose und terminalem Organversagen führen. Nahrungsbestandteile wie Cholesterol und Fett-reiche Diäten werden als mögliche Faktoren diskutiert, die den Übergang einer einfachen Fettleber zur schweren Verlaufsform der Steatohepatitis / NASH begünstigen. Eine Ausdehnung des Fettgewebes wird von Insulinresistenz und einer niedrig-gradigen chronischen Entzündung des Fettgewebes begleitet. Neben Endotoxinen aus dem Darm gelangen Entzündungsmediatoren aus dem Fettgewebe zur Leber. Als Folge werden residente Makrophagen der Leber, die Kupfferzellen, aktiviert, die eine Entzündungsantwort initiieren und weitere pro-inflammatorische Mediatoren freisetzen, zu denen Chemokine, Cytokine und Prostanoide wie Prostaglandin E2 (PGE2) gehören. In dieser Arbeit soll aufgeklärt werden, welchen Beitrag PGE2 an der Ausbildung von Insulinresistenz, hepatischer Steatose und Entzündung im Rahmen von Diät-induzierter NASH im komplexen Zusammenspiel mit der Regulation der Cytokin-Produktion und anderen Co-Faktoren wie Hyperinsulinämie und Hyperlipidämie hat. In murinen und humanen Makrophagen-Populationen wurde untersucht, welche Faktoren die Bildung von PGE2 fördern und wie PGE2 die Entzündungsantwort aktivierter Makrophagen reguliert. In primären Hepatozyten der Ratte sowie in isolierten humanen Hepatozyten und Zelllinien wurde der Einfluss von PGE2 allein und in Kombination mit Cytokinen, deren Bildung durch PGE2 beeinflusst werden kann, auf die Insulin-abhängige Regulation des Glucose- und Lipid-stoffwechsels untersucht. Um den Einfluss von PGE2 im komplexen Zusammenspiel der Zelltypen in der Leber und im Gesamtorganismus zu erfassen, wurden Mäuse, in denen die PGE2-Synthese durch die Deletion der mikrosomalen PGE-Synthase 1 (mPGES1) vermindert war, mit einer NASH-induzierenden Diät gefüttert. In Lebern von Patienten mit NASH oder in Mäusen mit Diät-induzierter NASH war die Expression der PGE2-synthetisierenden Enzyme Cyclooxygenase 2 (COX2) und mPGES1 sowie die Bildung von PGE2 im Vergleich zu gesunden Kontrollen gesteigert und korrelierte mit dem Schweregrad der Lebererkrankung. In primären Makrophagen aus den Spezies Mensch, Maus und Ratte sowie in humanen Makrophagen-Zelllinien war die Bildung pro-inflammatorischer Mediatoren wie Chemokinen, Cytokinen und Prostaglandinen wie PGE2 verstärkt, wenn die Zellen mit Endotoxinen wie Lipopolysaccharid (LPS), Fettsäuren wie Palmitinsäure, Cholesterol und Cholesterol-Kristallen oder Insulin, das als Folge der kompensatorischen Hyperinsulinämie bei Insulinresistenz verstärkt freigesetzt wird, inkubiert wurden. Insulin steigerte dabei synergistisch mit LPS oder Palmitinsäure die Synthese von PGE2 sowie der anderen Entzündungsmediatoren wie Interleukin (IL) 8 und IL-1β. PGE2 reguliert die Entzündungsantwort: Neben der Induktion der eigenen Synthese-Enzyme verstärkte PGE2 die Expression der Immunzell-rekrutierenden Chemokine IL-8 und (C-C-Motiv)-Ligand 2 (CCL2) sowie die der pro-inflammatorischen Cytokine IL-1β und IL-6 in Makrophagen und kann so zur Verstärkung der Entzündungsreaktion beitragen. Außerdem förderte PGE2 die Bildung von Oncostatin M (OSM) und OSM induzierte in einer positiven Rückkopplungsschleife die Expression der PGE2-synthetisierenden Enzyme. Andererseits hemmte PGE2 die basale und LPS-vermittelte Bildung des potenten pro-inflammatorischen Cytokins Tumornekrosefaktor α (TNFα) und kann so die Entzündungsreaktion abschwächen. In primären Hepatozyten der Ratte und humanen Hepatozyten beeinträchtigte PGE2 direkt die Insulin-abhängige Aktivierung der Insulinrezeptor-Signalkette zur Steigerung der Glucose-Verwertung, in dem es durch Signalketten, die den verschiedenen PGE2-Rezeptoren nachgeschaltet sind, Kinasen wie ERK1/2 und IKKβ aktivierte und eine inhibierende Serin-Phosphorylierung der Insulinrezeptorsubstrate bewirkte. PGE2 verstärkte außerdem die IL-6- oder OSM-vermittelte Insulinresistenz und Steatose in primären Hepatozyten der Ratte. Die Wirkung von PGE2 im Gesamtorganismus sollte in Mäusen mit Diät-induzierter NASH untersucht werden. Die Fütterung einer Hochfett-Diät mit Schmalz als Fettquelle, das vor allem gesättigte Fettsäuren enthält, verursachte Fettleibigkeit, Insulinresistenz und eine hepatische Steatose in Wildtyp-Mäusen. In Tieren, die eine Hochfett-Diät mit Sojaöl als Fettquelle, das vor allem (ω-6)-mehrfach-ungesättigte Fettsäuren (PUFAs) enthält, oder eine Niedrigfett-Diät mit Cholesterol erhielten, war lediglich eine hepatische Steatose nachweisbar, jedoch keine verstärkte Gewichtszunahme im Vergleich zu Geschwistertieren, die eine Standard-Diät bekamen. Im Gegensatz dazu verursachte die Fütterung einer Hochfett-Diät mit PUFA-reichem Sojaöl als Fettquelle in Kombination mit Cholesterol sowohl Fettleibigkeit und Insulinresistenz als auch hepatische Steatose mit Hepatozyten-Hypertrophie, lobulärer Entzündung und beginnender Fibrose in Wildtyp-Mäusen. Diese Tiere spiegelten alle klinischen und histologischen Parameter der humanen NASH im Metabolischen Syndrom wider. Nur die Kombination von hohen Mengen ungesättigter Fettsäuren aus Sojaöl und Cholesterol in der Nahrung führte zu einer exzessiven Akkumulation des Cholesterols und der Bildung von Cholesterol-Kristallen in den Hepatozyten, die zur Schädigung der Mitochondrien, schwerem oxidativem Stress und schließlich zum Absterben der Zellen führten. Als Konsequenz phagozytieren Kupfferzellen die Zelltrümmer der Cholesterol-überladenen Hepatozyten, werden dadurch aktiviert, setzen Chemokine, Cytokine und PGE2 frei, die die Entzündungsreaktion verstärken und die Infiltration von weiteren Immunzellen initiieren können und verursachen so eine Progression zur Steatohepatitis (NASH). Die Deletion der mikrosomalen PGE-Synthase 1 (mPGES1), dem induzierbaren Enzym der PGE2-Synthese aus Cyclooxygenase-abhängigen Vorstufen, reduzierte die Diät-abhängige Bildung von PGE2 in der Leber. Die Fütterung der NASH-induzierenden Diät verursachte in Wildtyp- und mPGES1-defizienten Mäusen eine ähnliche Fettleibigkeit und Zunahme der Fettmasse sowie die Ausbildung von hepatischer Steatose mit Entzündung und Fibrose (NASH) im histologischen Bild. In mPGES1-defizienten Mäusen waren jedoch Parameter für die Infiltration von Entzündungszellen und die Diät-abhängige Schädigung der Leber im Vergleich zu Wildtyp-Tieren erhöht, was sich auch in einer stärkeren Diät-induzierten systemischen Insulinresistenz widerspiegelte. Die Bildung des pro-inflammatorischen und pro-apoptotischen Cytokins TNFα war in mPGES1-defizienten Mäusen durch die Aufhebung der negativen Rückkopplungshemmung verstärkt, was einen gesteigerten Diät-induzierten Zelluntergang gestresster Lipid-überladener Hepatozyten und eine nach-geschaltete Entzündungsantwort zur Folge hatte. Zusammenfassend wurde unter den gewählten Versuchsbedingungen in vivo eine anti-inflammatorische Rolle von PGE2 verifiziert, da das Prostanoid vor allem indirekt durch die Hemmung der TNFα-vermittelten Entzündungsreaktion die Schädigung der Leber, die Verstärkung der Entzündung und die Ausbildung von Insulinresistenz im Rahmen der Diät-abhängigen Fettlebererkrankung abschwächte. N2 - Obesity is a worldwide problem affecting almost 40 % of the population. The prevalence of obesity, insulin resistance and the consequent diseases such as type-2-diabetes and metabolic syndrome is increasing rapidly. The main underlying reasons are high caloric diets and reduced physical exercise. The incidence of non-alcoholic fatty liver disease (NAFLD), characterized by hepatic lipid accumulation, is correlated with the body mass index. NAFLD is generally considered to be the hepatic manifestation of metabolic syndrome and is the most frequent cause of functional disorders of the liver. NAFLD comprises both the mild form of benign hepatic steatosis (fatty liver) as well as the progressive form of non-alcoholic steatohepatitis (NASH), in which hepatic steatosis is accompanied by inflammation and fibrosis. The development of NASH may result in hepatocellular carcinoma, liver cirrhosis and terminal organ failure. High fat diets and dietary cholesterol might impact the transistion from fatty liver to NASH. The diet induced expansion of the white adipose tissue is associated with the development of insulin resistance as well as low-grade chronic inflammation. Inflammatory mediators from the adipose tissue in combination with dietary components from the gut reach the liver and activate Kupffer cells, the resident liver macrophages. As a consequence, macrophages initiate an inflammatory response that involves secretion of immune cell recruiting chemokines, pro-inflammatory cytokines and prostanoids like prostaglandin E2 (PGE2). The aim of the study was to elucidate the impact of PGE2 in the development of insulin resistance, hepatic steatosis and inflammation in diet-induced NASH. These processes implicate a complex interplay of various cell types in the liver, the PGE2-mediated regulation of cytokine synthesis, as well as factors like hyperinsulinemia and hyperlipidemia. In vitro studies with murine and human macrophage populations characterise the generation of PGE2 and the PGE2-mediated regulation of the inflammatory response. Primary rat and human hepatocytes, in addition to immortal cell lines, were incubated with PGE2 alone and in combination with PGE2-dependent generated cytokines. The intent of this experimental series was to clarify the impact of these mediators on the activation of the insulin signaling chain and resulting metabolic processes in glucose and lipid metabolism. The role of PGE2 in vivo was examined in mice with reduced PGE2 synthesis due to the genetic deletion of microsomal PGE synthase 1 (mPGES1), which were additionally fed a NASH-inducing diet. The hepatic expression of the PGE2-generating enzymes cyclooxygenase 2 (COX2) and mPGES1 was increased in mice with diet-induced NASH as well as in liver biopsies of patients with NASH compared to patients with simple hepatic steatosis or non-steatotic controls, indicating an enlarged capacity for PGE2 synthesis in NASH. Furthermore, the expression of COX2 and mPGES1 in the human study cohort correlated with the severity of the hepatic disease.. Treatment of macrophages with endotoxins like lipopolysaccharide (LPS), fatty acids like palmitic acid, cholesterol and cholesterol crystals, or insulin, which is released as a consequence of insulin resistance in the context of a compensatory hyperinsulinemia, resulted in an enhanced production of pro-inflammatory mediators such as chemokines, cytokines and PGE2. A combinatory treatment of human macrophages with insulin and LPS or palmitic acid induced a synergistic increase in PGE2 synthesis and production of interleukins (IL) like IL-8 and IL-1β. PGE2 itself modulates the inflammatory response: The prostanoid induced the enzymes involved in its own synthesis, in addition to immune cell recruiting chemokines such as IL-8 and (C-C-motiv) ligand 2 (CCL2), and pro-inflammatory cytokines such as IL-1β and IL-6 in macrophages. This may result in an amplification of the inflammatory response. Furthermore, PGE2 induced the production of oncostatin M (OSM), which in turn enhanced the expression of the enzymes generating PGE2 in a positive feedback loop. On the other hand, PGE2 inhibited the basal and LPS-mediated synthesis of the potent pro-inflammatory cytokine tumor necrosis factor α (TNFα). This may result in a reduced inflammatory response. In primary rat and human hepatocytes PGE2 directly interfered with the insulin mediated activation of the insulin receptor signaling chain and impaired glucose utilisation. Mechanistically, through interaction with different PGE2 receptors, PGE2 activated serine kinases including ERK1/2 and IKKβ, which cause inhibitory phosphorylations at serine residues of the insulin receptor substrates and force their degradation. PGE2 enhanced the insulin resistance and increased hepatic steatosis induced by IL-6 or OSM in primary rat hepatocytes. A murine model of diet-induced NASH was established to elucidate the impact of PGE2 in the complex in vivo regulation. Lard-based high fat diets containing mainly saturated fatty acids initiated a strong body weight gain, obesity, insulin resistance and hepatic steatosis without further damage of the liver in mice. Furthermore, mice fed a high fat diet based on soybean oil with high amounts of (ω-6)-poly-unsaturated fatty acids (PUFAs) or a low fat diet with high cholesterol did not result in increased body weight gain compaired to mice fed a chow (low fat) diet, but did cause mild hepatic steatosis. In contrast, mice fed a high fat diet based on PUFA-rich soybean oil in combination with high dietary cholesterol caused body weight gain, obesity, insulin resistance and hepatic steatosis accompanied by hepatocyte hypertrophy, lobular inflammation and fibrosis in wildtype mice. This dietary model displayed all clinical and histological parameters of human NASH in the metabolic syndrome. Only the combination of soybean oil derived fatty acids and dietary cholesterol provoked an excessive accumulation of cholesterol in hepatocytes and the generation of cholesterol crystals that caused mitochondrial damage, severe oxidative stress, and subsequently hepatocyte death. Hepatic macrophages phagocytose hepatocyte debris, lipids and cholesterol crystals and thereby were activated to produce pro-inflammatory mediators like chemokines, cytokines and prostanoids like PGE2 that initiate an inflammatory response. This included immune cell infiltration, inflammation and fibrogenic processes that determine the progression to steatohepatitis (NASH). The deletion of microsomal PGE synthase 1 (mPGES1), the inducible enzyme generating PGE2 from COX2 derived PGH2, reduced the diet-dependent increase in hepatic PGE2 production in mice fed a NASH-inducing diet. While body weight gain, obesity and histological parameters of NASH including steatosis, inflammation and fibrosis were comparable in wild type and mPGES1-deficient mice fed a NASH inducing diet, parameters of immune cell infiltration and hepatic damage were augmented only in mPGES1-deficient mice. This results in a more pronounced diet-induced glucose intolerance and insulin resistance index in mPGES1-deficient mice compared to wild type littermates. In parallel, hepatic production of the potent pro-inflammatory and pro-apoptotic cytokine TNFα was enhanced in mice with the deletion of mPGES1 due to the abolished PGE2-mediated negative feedback loop. This was accompanied by increased diet induced cell death of lipid loaded stressed hepatocytes and could result in an intensified inflammatory response. In summary, in vivo studies verify an anti-inflammatory role of PGE2. The prostanoid PGE2 acts mainly indirectly and could attenuate the TNFα-mediated liver damage, immune response and the resulting insulin resistance in the context of diet induced fatty liver diseases. KW - Prostaglandine KW - Entzündung KW - Insulin KW - Leber KW - Fettleibigkeit KW - prostaglandins KW - inflammation KW - insulin KW - liver KW - obesity Y1 - 2021 ER - TY - JOUR A1 - Winkelbeiner, Nicola Lisa A1 - Wandt, Viktoria Klara Veronika A1 - Ebert, Franziska A1 - Lossow, Kristina A1 - Bankoglu, Ezgi E. A1 - Martin, Maximilian A1 - Mangerich, Aswin A1 - Stopper, Helga A1 - Bornhorst, Julia A1 - Kipp, Anna Patricia A1 - Schwerdtle, Tanja T1 - A Multi-Endpoint Approach to Base Excision Repair Incision Activity Augmented by PARylation and DNA Damage Levels in Mice BT - Impact of Sex and Age JF - International Journal of Molecular Sciences N2 - Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2’-deoxyguanosine (8-oxodG), 5-hydroxy-2’-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery. KW - maintenance of genomic integrity KW - ageing KW - sex KW - DNA damage KW - base excision repair (incision activity) KW - DNA damage response KW - poly(ADP-ribosyl)ation KW - liver Y1 - 2020 U6 - https://doi.org/10.3390/ijms21186600 SN - 1422-0067 VL - 21 IS - 18 PB - Molecular Diversity Preservation International CY - Basel ER - TY - GEN A1 - Winkelbeiner, Nicola Lisa A1 - Wandt, Viktoria Klara Veronika A1 - Ebert, Franziska A1 - Lossow, Kristina A1 - Bankoglu, Ezgi E. A1 - Martin, Maximilian A1 - Mangerich, Aswin A1 - Stopper, Helga A1 - Bornhorst, Julia A1 - Kipp, Anna Patricia A1 - Schwerdtle, Tanja T1 - A Multi-Endpoint Approach to Base Excision Repair Incision Activity Augmented by PARylation and DNA Damage Levels in Mice BT - Impact of Sex and Age T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2’-deoxyguanosine (8-oxodG), 5-hydroxy-2’-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1021 KW - maintenance of genomic integrity KW - ageing KW - sex KW - DNA damage KW - base excision repair (incision activity) KW - DNA damage response KW - poly(ADP-ribosyl)ation KW - liver Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-484831 SN - 1866-8372 IS - 1021 ER - TY - THES A1 - Gonzalez Camargo, Rodolfo T1 - Insulin resistance in cancer cachexia and metabolic syndrome BT - role of insulin activated macrophages and miRNA-21-5p N2 - The ever-increasing fat content in Western diet, combined with decreased levels of physical activity, greatly enhance the incidence of metabolic-related diseases. Cancer cachexia (CC) and Metabolic syndrome (MetS) are both multifactorial highly complex metabolism related syndromes, whose etiology is not fully understood, as the mechanisms underlying their development are not completely unveiled. Nevertheless, despite being considered “opposite sides”, MetS and CC share several common issues such as insulin resistance and low-grade inflammation. In these scenarios, tissue macrophages act as key players, due to their capacity to produce and release inflammatory mediators. One of the main features of MetS is hyperinsulinemia, which is generally associated with an attempt of the β-cell to compensate for diminished insulin sensitivity (insulin resistance). There is growing evidence that hyperinsulinemia per se may contribute to the development of insulin resistance, through the establishment of low grade inflammation in insulin responsive tissues, especially in the liver (as insulin is secreted by the pancreas into the portal circulation). The hypothesis of the present study was that insulin may itself provoke an inflammatory response culminating in diminished hepatic insulin sensitivity. To address this premise, firstly, human cell line U937 differentiated macrophages were exposed to insulin, LPS and PGE2. In these cells, insulin significantly augmented the gene expression of the pro-inflammatory mediators IL-1β, IL-8, CCL2, Oncostatin M (OSM) and microsomal prostaglandin E2 synthase (mPGES1), and of the anti-inflammatory mediator IL-10. Moreover, the synergism between insulin and LPS enhanced the induction provoked by LPS in IL-1β, IL-8, IL-6, CCL2 and TNF-α gene. When combined with PGE2, insulin enhanced the induction provoked by PGE2 in IL-1β, mPGES1 and COX2, and attenuated the inhibition induced by PGE2 in CCL2 and TNF-α gene expression contributing to an enhanced inflammatory response by both mechanisms. Supernatants of insulin-treated U937 macrophages reduced the insulin-dependent induction of glucokinase in hepatocytes by 50%. Cytokines contained in the supernatant of insulin-treated U937 macrophages also activated hepatocytes ERK1/2, resulting in inhibitory serine phosphorylation of the insulin receptor substrate. Additionally, the transcription factor STAT3 was activated by phosphorylation resulting in the induction of SOCS3, which is capable of interrupting the insulin receptor signal chain. MicroRNAs, non-coding RNAs linked to protein expression regulation, nowadays recognized as active players in the generation of several inflammatory disorders such as cancer and type II diabetes are also of interest. Considering that in cancer cachexia, patients are highly affected by insulin resistance and inflammation, control, non-cachectic and cachectic cancer patients were selected and the respective circulating levels of pro-inflammatory mediators and microRNA-21-5p, a posttranscriptional regulator of STAT3 expression, assessed and correlated. Cachectic patients circulating cytokines IL-6 and IL-8 levels were significantly higher than those of non-cachectic and controls, and the expression of microRNA-21-5p was significantly lower. Additionally, microRNA-21-5p reduced expression correlated negatively with IL-6 plasma levels. These results indicate that hyperinsulinemia per se might contribute to the low grade inflammation prevailing in MetS patients and thereby promote the development of insulin resistance particularly in the liver. Diminished MicroRNA-21-5p expression may enhance inflammation and STAT3 expression in cachectic patients, contributing to the development of insulin resistance. N2 - O teor de gordura cada vez maior na dieta ocidental, combinada com a diminuição dos níveis de atividade física têm marcadamente aumentado à incidência de doenças relacionas ao metabolismo. A caquexia associada ao câncer (CC) e a síndrome metabólica (SM) são síndromes de etiologia complexa e multifatorial, não totalmente compreendida, e com mecanismos subjacentes ao seu desenvolvimento não completamente revelados. No entanto, apesar de serem consideradas "lados opostos", a CC e a MetS apresentam várias características em comum, tais como resistência à insulina e inflamação de baixo grau, com macrófagos teciduais como importantes coadjuvantes, devido à sua capacidade de produzir e liberar mediadores inflamatórios, e microRNAs, descritos como RNAs não-codificantes ligados à regulação da expressão de proteínas e reconhecidos como participantes ativos na geração de várias doenças inflamatórias, tais como o câncer e diabetes tipo II. Uma das principais características da MetS é a hiperinsulinemia, que está geralmente associada com uma tentativa da célula β do pâncreas de compensar a diminuição da sensibilidade à insulina (resistência à insulina). Um número crescente de evidências sugere que a hiperinsulinemia “por si só”, pode contribuir com o desenvolvimento de resistência à insulina através do estabelecimento de um quadro inflamatório de baixo grau, em tecidos sensíveis a insulina, e em particular no fígado, devido ao fato da insulina ser secretada pelo pâncreas na circulação portal. A hipótese do presente estudo foi que a insulina pode induzir uma resposta inflamatória em macrófagos e culminar em diminuição da sensibilidade hepática à insulina. Para confirmar esta hipótese, primeiramente, macrófagos diferenciados da linhagem de células humanas U937 foram expostos à insulina, LPS e PGE2. Nestas células, a insulina aumentou significativamente a expressão gênica dos mediadores pró-inflamatórios IL-1β, IL- 8, CCL2, oncostatina M (OSM) e prostaglandina E2 sintase microssomal (mPGES1), e do mediador anti-inflamatório IL-10. Além disso, o sinergismo entre insulina e LPS aumentou a indução provocada por LPS nos genes da IL-1β, IL-8, IL-6, CCL2 e TNF-α. Quando combinado com PGE2, a insulina aumentou a indução provocada pela PGE2 nos genes da IL-1β, mPGES1 e COX2, e restaurou a inibição induzida pela PGE2 no gene CCL2 e TNF-α.Subsequentemente, sobrenadantes dos macrófagos U937 tratados com insulina modulou negativamente a sinalização da insulina em culturas primárias de hepatócitos de rato, como observado pela atenuação de 50% da indução dependente de insulina da enzima glicoquinase. Citocinas contidas no sobrenadante de macrófagos U937 tratados com insulina também ativaram em hepatócitos ERK1/2, resultando na fosforilação do resíduo de serina inibitório do substrato do receptor de insulina. Adicionalmente, o fator de transcrição STAT3 foi ativado por um elevado grau de fosforilação e a proteína SOCS3, capaz de interromper a via de sinalização do receptor de insulina, foi induzida. Considerando que na caquexia associada ao câncer, pacientes são altamente afetados pela resistência à insulina e inflamação, pacientes controle, não caquéticos e caquéticos foram seleccionados e os respectivos níveis circulantes de mediadores pró-inflamatórios e microRNA-21-5p, um regulador pós-transcricional da expressão de STAT3, avaliados e correlacionados. Pacientes caquéticos exibiram citocinas circulantes IL-6 e IL-8 significativamente maiores do que pacientes não caquéticos e controles, assim como a expressão de microRNA-21-5p significativamente diminuida. Além disso, a reduzida expressão de microRNA-21-5p correlaciona-se negativamente com níveis de IL-6 no plasma. Estes resultados indicam que a hiperinsulinemia pode, por si só contribuir para o desenvolvimento da inflamação de baixo grau prevalente em pacientes com excesso de peso e obesos e, assim, promover o desenvolvimento de resistência à insulina especialmente no fígado e o nível reduzido de miRNA-21-5p pode modular a inflamação e expressão de STAT3 em pacientes caquéticos, contribuindo para o desenvolvimento da resistência à insulina. N2 - Der stetig steigende Fettgehalt in westlicher Ernährung in Kombination mit reduzierter körperlicher Aktivität hat zu einem dramatischen Anstieg der Inzidenz metabolischer Erkrankungen geführt. Tumorkachexie (Cancer cachexia, CC) und Metabolisches Syndrom (MetS) sind sehr komplexe, multifaktorielle metabolische Erkrankungen, deren Ätiologie nicht vollständig verstanden ist. Die molekularen Ursachen, die zu diesen Symptomkomplexen führen, sind noch unzureichend aufgeklärt. Obwohl ihr äußeres Erscheinungsbild stark gegensätzlich ist, haben MetS und CC etliche Gemeinsamkeiten wie zum Beispiel Insulinresistenz und eine chronische unterschwellige Entzündung. Sowohl bei der Entstehung der Insulinresistenz als auch bei der chronischen Entzündung spielen Makrophagen eine Schlüsselrolle, weil sie in der Lage sind pro-inflammatorische Mediatoren zu produzieren und freizusetzen. Eine der hervorstechendsten Auffälligkeiten des MetS ist die Hyperinsulinämie, die durch den Versuch der β-Zelle, die verminderte Insulinsensitivität (Insulinresistenz) zu kompensieren, zustande kommt. Es gibt zunehmend Hinweise darauf, dass die Hyperinsulinämie selber an der Entzündungsentstehung in Insulin-abhängigen Geweben beteiligt ist und dadurch zur Entwicklung und Verstärkung der Insulinresistenz beitragen kann. Dies trifft besonders auf die Leber zu, weil hier die Insulinspiegel besonders hoch sind, da Insulin vom Pankreas direkt in den Pfortaderkeislauf gelangt. Daher wurde in dieser Arbeit die Hypothese geprüft, ob Insulin selber eine Entzündungsantwort auslösen und dadurch die hepatische Insulinsensitivität senken kann. Zu diesem Zweck wurde die humane Zelllinie U937 durch PMA-Behandlung zu Makrophagen differenziert und diese Makrophagen mit Insulin, LPS und PGE2 inkubiert. In diesen Zellen steigerte Insulin die Expression der pro-inflammatorischen Mediatoren IL-1β, IL-8, CCL2, Oncostatin M (OSM) signifikant und induzierte die mikrosomale PGE-Synthase 1 (mPGES1) ebenso wie das antiinflammatorische Cytokin IL-10. Ferner verstärkte Insulin die LPS-abhängige Induktion des IL-1β-, IL-8-, IL-6-, CCL2- und TNFα-Gens. Ebenso verstärkte Insulin die PGE2-abhängige Induktion von IL-1β, mPGES1 und COX2. Im Gegensatz dazu schwächte es die Hemmende Wirkung von PGE2 auf Expression von TNFα und CCL2 ab und trug so auf beide Weisen zu einer Verstärkung der Entzündungsantwort bei. Überstände von Insulin-behandelten U937 Makrophagen reduzierten die Insulin-abhängige Induktion der Glukokinase in Hepatocyten um 50%. Die Cytokine, die im Überstand Insulin-behandelter Makrophagen enthalten waren, aktivierten in Hepatocyten ERK1/2, was zu einer inhibitorischen Serin-Phosphorylierung der Insulin Rezeptor Substrats (IRS) führte. Zusätzlich führten die Cytokine zu einer Phosphorylierung und Aktivierung von STAT3 und einer dadurch bedingten Induktion von SOCS3, das seinerseits die Insulinrezeptor-Signalkette unterbrechen kann. MicroRNAs, nicht-codierende RNAs, die an der Regulation der Proteinexpression beteiligt sind und deren Beteiligung an der Regulation der Entzündungsantwort bei zahlreichen Erkrankungen, unter anderem Tumorerkrankungen und Typ II Diabetes gezeigt wurde, sind auch von Interesse. Unter dem Blickwinkel, dass Tumor-Kachexie Patienten sich durch eine Insulinresistenz und eine systemische Entzündung auszeichnen, wurden in nichtkachektische und tumorkachektische Patienten Plasmaspiegel von pro-inflammatorischen Mediatoren und der microRNA-21-5p bestimmt, von der bekannt ist, dass sie ein posttranskriptioneller Regulator der STAT3 Expression ist. Die Spiegel der proinflammatorischen Mediatoren und der miRNA-21-5p wurden korreliert. In kachektischen Patienten waren die Spiegel der Cytokine IL-6 und IL-8 signifikant höher, die der miRNA-21- 5p signifikant niedriger als in nicht-kachektischen Patienten. Die Plasma IL-6-Spiegel korrelierten negativ mit den miRNA21-5p Spiegeln. Insgesamt zeigen die Ergebnisse, dass eine Hyperinsulinämie selber zu der Entwicklung einer unterschwellingen Entzündung, wie sie in Patienten mit einem MetS vorherrscht, beitragen, und dadurch besonders in der Leber eine Insulinresistenz auslösen oder verstärken kann. Eine verringerte Expression der MicroRNA-21-5p kann in kachektischen Patienten die Entzündungsantwort, im Speziellen die STAT3 Expression, verstärken und dadurch zur Entwicklung einer Insulinresistenz beitragen KW - cachexia KW - metabolic syndrome KW - inflammation KW - insulin resistance KW - microRNAs KW - insulin KW - liver KW - macrophages KW - caquexia KW - síndrome metabólica KW - inflamação KW - resistência à insulina KW - microRNAs KW - insulina KW - fígado KW - macrófagos KW - Kachexie KW - metabolisches Syndrom KW - Entzündung KW - Insulinresistenz KW - MicroRNAs KW - Insulin KW - Leber KW - Makrophagen Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-100973 ER -