Interleukin‐6 initiates muscle‐ and adipose tissue wasting in a novel C57BL/6 model of cancer‐associated cachexia

BACKGROUND: Cancer‐associated cachexia (CAC) is a wasting syndrome drastically reducing efficacy of chemotherapy and life expectancy of patients. CAC affects up to 80% of cancer patients, yet the mechanisms underlying the disease are not well understood and no approved disease‐specific medication exists. As a multiorgan disorder, CAC can only be studied on an organismal level. To cover the diverse aetiologies of CAC, researchers rely on the availability of a multifaceted pool of cancer models with varying degrees of cachexia symptoms. So far, no tumour model syngeneic to C57BL/6 mice exists that allows direct comparison between cachexigenic‐ and non‐cachexigenic tumours. METHODS: MCA207 and CHX207 fibrosarcoma cells were intramuscularly implanted into male or female, 10–11‐week‐old C57BL/6J mice. Tumour tissues were subjected to magnetic resonance imaging, immunohistochemical‐, and transcriptomic analysis. Mice were analysed for tumour growth, body weight and ‐composition, food‐ and water intake, locomotor activity, O(2) consumption, CO(2) production, circulating blood cells, metabolites, and tumourkines. Mice were sacrificed with same tumour weights in all groups. Adipose tissues were examined using high‐resolution respirometry, lipolysis measurements in vitro and ex vivo, and radioactive tracer studies in vivo. Gene expression was determined in adipose‐ and muscle tissues by quantitative PCR and Western blotting analyses. Muscles and cultured myotubes were analysed histologically and by immunofluorescence microscopy for myofibre cross sectional area and myofibre diameter, respectively. Interleukin‐6 (Il‐6) was deleted from cancer cells using CRISPR/Cas9 mediated gene editing. RESULTS: CHX207, but not MCA207‐tumour‐bearing mice exhibited major clinical features of CAC, including systemic inflammation, increased plasma IL‐6 concentrations (190 pg/mL, P ≤ 0.0001), increased energy expenditure (+28%, P ≤ 0.01), adipose tissue loss (−47%, P ≤ 0.0001), skeletal muscle wasting (−18%, P ≤ 0.001), and body weight reduction (−13%, P ≤ 0.01) 13 days after cancer cell inoculation. Adipose tissue loss resulted from reduced lipid uptake and ‐synthesis combined with increased lipolysis but was not associated with elevated beta‐adrenergic signalling or adipose tissue browning. Muscle atrophy was evident by reduced myofibre cross sectional area (−21.8%, P ≤ 0.001), increased catabolic‐ and reduced anabolic signalling. Deletion of IL‐6 from CHX207 cancer cells completely protected CHX207(IL6KO)‐tumour‐bearing mice from CAC. CONCLUSIONS: In this study, we present CHX207 fibrosarcoma cells as a novel tool to investigate the mediators and metabolic consequences of CAC in C57BL/6 mice in comparison to non‐cachectic MCA207‐tumour‐bearing mice. IL‐6 represents an essential trigger for CAC development in CHX207‐tumour‐bearing mice

Adiponutrin Functions as a Nutritionally Regulated Lysophosphatidic Acid Acyltransferase

SummaryNumerous studies in humans link a nonsynonymous genetic polymorphism (I148M) in adiponutrin (ADPN) to various forms of fatty liver disease and liver cirrhosis. Despite its high clinical relevance, the molecular function of ADPN and the mechanism by which I148M variant affects hepatic metabolism are unclear. Here we show that ADPN promotes cellular lipid synthesis by converting lysophosphatidic acid (LPA) into phosphatidic acid. The ADPN-catalyzed LPA acyltransferase (LPAAT) reaction is specific for LPA and long-chain acyl-CoAs. Wild-type mice receiving a high-sucrose diet exhibit substantial upregulation of Adpn in the liver and a concomitant increase in LPAAT activity. In Adpn-deficient mice, this diet-induced increase in hepatic LPAAT activity is reduced. Notably, the I148M variant of human ADPN exhibits increased LPAAT activity leading to increased cellular lipid accumulation. This gain of function provides a plausible biochemical mechanism for the development of liver steatosis in subjects carrying the I148M variant

The role of adipose triglyceride lipase in liver and skeletal muscle

ATGL repräsentiert die Geschwindigkeitsbestimmende Triglyzerid (TG) Hydrolase im weißen Fettgewebe von Mäusen und Menschen. Das Fehlen einer funktionstüchtigen ATGL resultiert in TG Akkumulation in verschiedenen Geweben, einschließlich Leber und Skelettmuskel. Diese Ergebnisse deuten daraufhin, dass ATGL in verschiedenen Geweben eine Rolle spielt.Obwohl ATGL nur in sehr geringen Mengen in der Leber exprimiert wird, resultiert die Abwesenheit von ATGL in der Leber in einer reduzierten Lipolyse. Ferner weisen ATGL defiziente Leberzellen einen erhöhten FS Einbau in TG, gesteigerten TG Gehalt und eine erniedrigte Freisetzung von TG und FS im Vergleich zu Kontrollzellen auf. Dementsprechend führt eine Überexpression von ATGL in Leberzellen zu einem erniedrigten TG Gehalt. Spezifische Überexpression von ATGL in der Leber in vivo resultiert in einen reduzierten TG Gehalt und in eine erniedrigte VLDL-Synthese. Die mRNA-Expression von verschieden Genen, die in der Energieproduktion beteiligt sind, zeigen keine Veränderung im Vergleich zu Wildtypmäusen. Die gewonnen Ergebnisse zeigten deutlich, dass ATGL einen wichtigen Beitrag im Lipidstoffwechsel der Leber leistet.Um die Wichtigkeit von ATGL im Skelettmuskel und bei der Bereitstellung von Energie während des Trainings zu untersuchen, wurden Laufexperimente mit ATGL defizienten (ATGL-ko) Mäusen durchgeführt. Es wurden Blutparameter und Leberglykogen trainierter und untrainierter Mäuse bestimmt. Da ATGL-ko Mäuse vermehrt TG im Herzen speichern, wurden ATGL-ko Mäuse untersucht, die spezifisch ATGL im Herzen überexprimieren (ATGL-ko/CM). Untersuchungen von ATGL-ko und ATGL-ko/CM Mäusen zeigen, dass zirkulierende FS im Blut während des Trainings nicht erhöht werden können und sie daher bevorzugt Kohlenhydrate verwerten. Diese Resultate weisen daraufhin, dass ATGL für eine angemessene Energieversorgung während des Trainings benötigt wird.In mice and humans, deficiency of ATGL, the rate-limiting TG hydrolase, revealed massive TG accumulation in multiple tissues, including liver and skeletal muscle. Although the expression of ATGL is very low compared to white adipose tissue, ATGL deficiency causes reduced hepatic TG hydrolysis in the liver. Hepatocytes lacking ATGL exhibited an increase of fatty acid (FA) incorporation into TG and reduced release of FA and TG compared to wild-type cells. Conversely, overexpression of ATGL in hepatocytes led to depletion of TG stores. A specific overexpression of ATGL in the liver (liv-ATGL) in vivo resulted in reduced liver TG content. Liv-ATGL mice exhibited decreased VLDL synthesis, indicating that ATGL affects liver TG mobilisation, but showed no changes on mRNA levels of specific genes involved in energy production. Furthermore, ATGL expression and activity is strongly regulated by nutritional state, resulting in a massive upregulation of ATGL protein expression during fasting.Furthermore the influence of ATGL deficiency on energy availability and substrate utilization in working muscle was studied by using a treadmill. Blood energy metabolites and liver glycogen stores were determined. Since ATGL-deficient mice exhibit massive TG accumulation in the heart, we studied ATGL-deficient mice specifically overexpressing ATGL in the cardiac muscle (ATGL-deficient/CM). In contrast to ATGL-deficient mice, these mice did not accumulate TG in the heart. Exercise experiments revealed that ATGL-deficient and ATGL-deficient/CM mice are unable to increase circulating FA levels during exercise. The reduced availability of FA for energy conversion led to rapid depletion of liver glycogen stores and hypoglycemia. These data suggested that the increased energy requirements of the working muscle result in an increased use of carbohydrates for energy conversion. Thus, ATGL activity is required for proper energy supply of the skeletal muscle during exercise.Gabriele M. SchoiswohlAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersGraz, Univ., Diss., 2009(VLID)20800

Notch intracellular domain overexpression in adipocytes confers lipodystrophy in mice

Objective: The Notch family of intermembrane receptors is highly conserved across species and is involved in cell fate and lineage control. Previous in vitro studies have shown that Notch may inhibit adipogenesis. Here we describe the role of Notch in adipose tissue by employing an in vivo murine model which overexpresses Notch in adipose tissue. Methods: Albino C57BL/6J RosaNICD/NICD::Adipoq-Cre (Ad-NICD) male mice were generated to overexpress the Notch intracellular domain (NICD) specifically in adipocytes. Male RosaNICD/NICD mice were used as controls. Mice were evaluated metabolically at the ages of 1 and 3 months by assessing body weights, serum metabolites, body composition (EchoMRI), glucose tolerance and insulin tolerance. Histological sections of adipose tissue depots as well as of liver were examined. The mRNA expression profile of genes involved in adipogenesis was analyzed by quantitative real-time PCR. Results: The Ad-NICD mice were heavier with significantly lower body fat mass compared to the controls. Small amounts of white adipose tissue could be seen in the 1-month old Ad-NICD mice, but was almost absent in the 3-months old mice. The Ad-NICD mice also had higher serum levels of glucose, insulin, triglyceride and non-esterified fatty acids. These differences were more prominent in the older (3-months) than in the younger (1-month) mice. The Ad-NICD mice also showed severe insulin resistance along with a steatotic liver. Gene expression analysis in the adipose tissue depots showed a significant repression of lipogenic (Fasn, Acacb) and adipogenic pathways (C/ebpα, C/ebpβ, Pparγ2, Srebf1). Conclusions: Increased Notch signaling in adipocytes in mice results in blocked expansion of white adipose tissue which leads to ectopic accumulation of lipids and insulin resistance, thus to a lipodystrophic phenotype. These results suggest that further investigation of the role of Notch signaling in adipocytes could lead to the manipulation of this pathway for therapeutic interventions in metabolic disease

The Crystal Structure of Mouse Ces2c, a Potential Ortholog of Human CES2, Shows Structural Similarities in Substrate Regulation and Product Release to Human CES1

Members of the carboxylesterase 2 (Ces2/CES2) family have been studied intensively with respect to their hydrolytic function on (pro)drugs, whereas their physiological role in lipid and energy metabolism has been realized only within the last few years. Humans have one CES2 gene which is highly expressed in liver, intestine, and kidney. Interestingly, eight homologous Ces2 (Ces2a to Ces2h) genes exist in mice and the individual roles of the corresponding proteins are incompletely understood. Mouse Ces2c (mCes2c) is suggested as potential ortholog of human CES2. Therefore, we aimed at its structural and biophysical characterization. Here, we present the first crystal structure of mCes2c to 2.12 Å resolution. The overall structure of mCes2c resembles that of the human CES1 (hCES1). The core domain adopts an α/β hydrolase-fold with S230, E347, and H459 forming a catalytic triad. Access to the active site is restricted by the cap, the flexible lid, and the regulatory domain. The conserved gate (M417) and switch (F418) residues might have a function in product release similar as suggested for hCES1. Biophysical characterization confirms that mCes2c is a monomer in solution. Thus, this study broadens our understanding of the mammalian carboxylesterase family and assists in delineating the similarities and differences of the different family members

Rosiglitazone Reverses Inflammation in Epididymal White Adipose Tissue in Hormone-Sensitive Lipase-Knockout Mice

Hormone-sensitive lipase (HSL) plays a crucial role in intracellular lipolysis, and loss of HSL leads to diacylglycerol (DAG) accumulation, reduced FA mobilization, and impaired PPARγ signaling. Hsl knockout mice exhibit adipose tissue inflammation, but the underlying mechanisms are still not clear. Here, we investigated if and to what extent HSL loss contributes to endoplasmic reticulum (ER) stress and adipose tissue inflammation in Hsl knockout mice. Furthermore, we were interested in how impaired PPARγ signaling affects the development of inflammation in epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT) of Hsl knockout mice and if DAG and ceramide accumulation contribute to adipose tissue inflammation and ER stress. Ultrastructural analysis showed a markedly dilated ER in both eWAT and iWAT upon loss of HSL. In addition, Hsl knockout mice exhibited macrophage infiltration and increased F4/80 mRNA expression, a marker of macrophage activation, in eWAT, but not in iWAT. We show that treatment with rosiglitazone, a PPARγ agonist, attenuated macrophage infiltration and ameliorated inflammation of eWAT, but expression of ER stress markers remained unchanged, as did DAG and ceramide levels in eWAT. Taken together, we show that HSL loss promoted ER stress in both eWAT and iWAT of Hsl knockout mice, but inflammation and macrophage infiltration occurred mainly in eWAT. Also, PPARγ activation reversed inflammation but not ER stress and DAG accumulation. These data indicate that neither reduction of DAG levels nor ER stress contribute to the reversal of eWAT inflammation in Hsl knockout mice