The Effect of Cytokine Adsorption on Leukocyte and Platelet Activation after Extracorporeal Cardiopulmonary Resuscitation.

BACKGROUND: Post-cardiac arrest syndrome (PCAS) is a frequent complication following successful cardiopulmonary resuscitation and correlates with poor outcome. PCAS is characterized by an excessive inflammatory response to whole-body ischemia and reperfusion. Cytokine adsorption was suggested as an adjunctive treatment option for the removal of cytokines from the patients' blood to restore the physiological equilibrium of pro- and anti-inflammatory activity and thus mitigate hemodynamic instability and end-organ complications. MATERIAL AND METHODS: To better understand the cellular effects of cytokine adsorption in patients receiving extracorporeal cardiopulmonary resuscitation (ECPR) after in- and out-of-hospital cardiac arrest, we compared the activation status of neutrophils, monocytes, and platelets as well as the formation of platelet-leukocyte complexes in intravenous whole blood samples from an exploratory subgroup (n = 24) from the randomized CYTER study. RESULT: At 48 hours after initiation of ECPR, flow cytometry analyses did neither reveal significant differences in neutrophil (CD11b, CD66b, L-selectin, and PSGL-1) and monocyte (CD11b, L-selectin, and PSGL-1) surface molecule expression nor in circulating platelet-monocyte complexes between patients receiving cytokine adsorption and those without. CONCLUSION: Data did not show a relevant effect of cytokine adsorption on neutrophil and monocyte activation during the first 48 hours after initiation of ECPR.

Serotonin transporter-deficient mice display enhanced adipose tissue inflammation after chronic high-fat diet feeding.

Introduction: Serotonin is involved in leukocyte recruitment during inflammation. Deficiency of the serotonin transporter (SERT) is associated with metabolic changes in humans and mice. A possible link and interaction between the inflammatory effects of serotonin and metabolic derangements in SERT-deficient mice has not been investigated so far. Methods: SERT-deficient (Sert -/-) and wild type (WT) mice were fed a high-fat diet, starting at 8 weeks of age. Metabolic phenotyping (metabolic caging, glucose and insulin tolerance testing, body and organ weight measurements, qPCR, histology) and assessment of adipose tissue inflammation (flow cytometry, histology, qPCR) were carried out at the end of the 19-week high-fat diet feeding period. In parallel, Sert -/- and WT mice received a control diet and were analyzed either at the time point equivalent to high-fat diet feeding or as early as 8-11 weeks of age for baseline characterization. Results: After 19 weeks of high-fat diet, Sert -/- and WT mice displayed similar whole-body and fat pad weights despite increased relative weight gain due to lower starting body weight in Sert -/-. In obese Sert -/- animals insulin resistance and liver steatosis were enhanced as compared to WT animals. Leukocyte accumulation and mRNA expression of cytokine signaling mediators were increased in epididymal adipose tissue of obese Sert -/- mice. These effects were associated with higher adipose tissue mRNA expression of the chemokine monocyte chemoattractant protein 1 and presence of monocytosis in blood with an increased proportion of pro-inflammatory Ly6C+ monocytes. By contrast, Sert -/- mice fed a control diet did not display adipose tissue inflammation. Discussion: Our observations suggest that SERT deficiency in mice is associated with inflammatory processes that manifest as increased adipose tissue inflammation upon chronic high-fat diet feeding due to enhanced leukocyte recruitment.

Platelets regulate ischemia-induced revascularization and angiogenesis by secretion of growth factor-modulating factors.

In ischemic tissue, platelets can modulate angiogenesis. The specific factors influencing this function, however, are poorly understood. Here, we characterized the complement anaphylatoxin C5a-mediated activation of C5a receptor 1 (C5aR1) expressed on platelets as a potent regulator of ischemia-driven revascularization. We assessed the relevance of the anaphylatoxin receptor C5aR1 on platelets in patients with coronary artery disease as well as those with peripheral artery disease and used genetic mouse models to characterize its significance for ischemia and growth factor-driven revascularization. The presence of C5aR1-expressing platelets was increased in the hindlimb ischemia model. Ischemia-driven angiogenesis was significantly improved in C5aR1-/- mice but not in C5-/- mice, suggesting a specific role of C5aR1. Experiments using the supernatant of C5a-stimulated platelets suggested a paracrine mechanism of angiogenesis inhibition by platelets by means of antiangiogenic CXC chemokine ligand 4 (CXCL4, PF4). Lineage-specific C5aR1 deletion verified that the secretion of CXCL4 depends on C5aR1 ligation on platelets. Using C5aR1-/-CXCL4-/- mice, we observed no additional effect in the revascularization response, underscoring a strong dependence of CXCL4 secretion on the C5a-C5aR1-axis. We identified a novel mechanism for inhibition of neovascularization via platelet C5aR1, which was mediated by the release of antiangiogenic CXCL4.

Platelets in Myocardial Ischemia/Reperfusion Injury.

Coronary artery disease, including myocardial infarction (MI), remains a leading cause of global mortality. Rapid reperfusion therapy is key to the improvement of patient outcome but contributes substantially to the final cardiac damage. This phenomenon is called "ischemia/reperfusion injury (IRI)." The underlying mechanisms of IRI are complex and not fully understood. Contributing cellular and molecular mechanisms involve the formation of microthrombi, alterations in ion concentrations, pH shifts, dysregulation of osmolality, and, importantly, inflammation. Beyond their known action as drivers of the development of coronary plaques leading to MI, platelets have been identified as important mediators in myocardial IRI. Circulating platelets are activated by the IRI-provoked damages in the vascular endothelium. This leads to platelet adherence to the reperfused endothelium, aggregation, and the formation of microthrombi. Furthermore, activated platelets release vasoconstrictive substances, act via surface molecules, and enhance leukocyte infiltration into post-IR tissue, that is, via platelet-leukocyte complexes. A better understanding of platelet contributions to myocardial IRI, including their interaction with other lesion-associated cells, is necessary to develop effective treatment strategies to prevent IRI and further improve the condition of the reperfused myocardium. In this review, we briefly summarize platelet properties that modulate IRI. We also describe the beneficial impacts of antiplatelet agents as well as their mechanisms of action in IRI beyond classic effects.

Platelet Subtypes in Inflammatory Settings.

In addition to their essential role in hemostasis and thrombosis, platelets also modulate inflammatory reactions and immune responses. This is achieved by specialized surface receptors as well as secretory products including inflammatory mediators and cytokines. Platelets can support and facilitate the recruitment of leukocytes into inflamed tissue. The various properties of platelet function make it less surprising that circulating platelets are different within one individual. Platelets have different physical properties leading to distinct subtypes of platelets based either on their function (procoagulant, aggregatory, secretory) or their age (reticulated/immature, non-reticulated/mature). To understand the significance of platelet phenotypic variation, qualitatively distinguishable platelet phenotypes should be studied in a variety of physiological and pathological circumstances. The advancement in proteomics instrumentation and tools (such as mass spectrometry-driven approaches) improved the ability to perform studies beyond that of foundational work. Despite the wealth of knowledge around molecular processes in platelets, knowledge gaps in understanding platelet phenotypes in health and disease exist. In this review, we report an overview of the role of platelet subpopulations in inflammation and a selection of tools for investigating the role of platelet subpopulations in inflammation.

Peripheral serotonin lacks effects on endothelial adhesion molecule expression in acute inflammation.

BACKGROUND: Peripheral, non-neuronal serotonin promotes the recruitment of neutrophils to sites of acute inflammation and tissue damage. Direct effects of serotonin on neutrophil function were shown to be involved. However, the influence of serotonin on the endothelial counterpart is unknown. OBJECTIVES: To investigate whether serotonin alters the function of endothelial cells in leukocyte recruitment during acute inflammation. METHODS: We used two murine models of acute inflammation: intraperitoneal lipopolysaccharide (LPS) injection and mesenteric ischemia/reperfusion injury (I/R). To study effects of peripheral serotonin, leukocyte recruitment and endothelial adhesion molecule expression were compared in wild type (WT) and tryptophan hydroxylase 1 deficient (Tph1-/- ) mice, which are unable to synthesize peripheral serotonin. RESULTS: As expected, neutrophil transmigration into the peritoneal cavity following LPS injection was impaired in Tph1-/- mice. Abdominal blood vessels, however, showed no difference in adhesion molecule expression. In the early reperfusion phase after mesenteric I/R, the number of rolling leukocytes was significantly lower in Tph1-/- compared to WT. In line with the LPS model, endothelial adhesion molecule expression was independent of serotonin. In vitro experiments using human umbilical vein endothelial cells (HUVECs) confirmed that serotonin does not affect endothelial adhesion molecules. CONCLUSIONS: The inflammatory release of peripheral serotonin is dispensable for the regulation of endothelial adhesion molecules.

Reticulated Platelets-Which Functions Have Been Established by In Vivo and In Vitro Data?

Reticulated platelets (RP) are the youngest platelet fraction released into the circulation. These immature platelets have increased RNA content, a larger cell volume, more dense granules, higher levels of surface activation markers and are thought to be more reactive compared to their mature counterparts. RP have been associated with cardiovascular disease, diabetes and increased mortality. Yet only a few animal studies investigating RP have been conducted so far and further investigations are warranted. Established methods to count RP are flow cytometry (staining with thiazole orange or SYTO13) or fully automated hematology analyzers (immature platelet fraction, IPF). IPF has been established as a diagnostic parameter in thrombocytopenia, cardiovascular disease and, in particular, the response to antiplatelet therapy. This review seeks to provide an overview of the key features of RP as well as preanalytical and analytical aspects that need to be considered when working with this platelet population.

Endocrine, Metabolic and Pharmacological Effects of Thyronamines (TAM), Thyroacetic Acids (TA) and Thyroid Hormone Metabolites (THM) - Evidence from in vitro, Cellular, Experimental Animal and Human Studies.

Thyroid hormone metabolites (THM) with few or no iodine substituents such as 3,5-T2, the thyronamines 3-T1AM and T0AM, and their oxidation products, the thyroacetic acids (TA) formed by monoamine oxidases, have recently attracted major interest due to their metabolic actions which are in part distinct from those of the classical thyromimetic hormone T3, the major ligand of T3 receptors. This review compiles and discusses in vitro effects of 3,5-T2, TAM and TA reported for thyrocytes, pancreatic islets and hepatocytes as well as findings from in vivo studies in mouse models after single or repeated administration of pharmacological doses of these agents. Comparison of the 3,5-T2 effects on the transcriptome with not yet published proteome data in livers of obese mice on high fat diet indicate a distinct anti-steatotic effect of this THM. Furthermore, uptake, metabolism, and cellular actions via various receptors such as trace amine-associated receptors (TAAR), alpha-adrenergic, GPCR and T3 receptors are discussed. Studies on postulated pathways of biosynthesis of 3-T1AM, its effects on the HPT-axis and thyroid gland as well as insulin secretion are reviewed. 3-T1AM also acts on hepatocytes and interferes with TRPM8-dependent signaling in human cell lines related to the eye compartment. Human studies are presented which address potential biosynthesis routes of 3,5-T2 and 3-T1AM from THM precursors, especially T3. The current state of diagnostic analytics of these minor THM in human blood is portrayed comparing and critically discussing the still divergent findings based on classical immunoassay and recently developed liquid-chromatography/mass- spectrometry methods, which allow quantification of the thyronome spectrum from one single small volume serum sample. The clinical perspectives of use and potential abuse of these biologically active THM is addressed.

Extracellular HtrA2 Induces Apoptosis in Human Umbilical Vein Endothelial Cells.

The serine protease high-temperature-required protein A2 (HtrA2) has been identified as a key intracellular molecule promoting apoptosis in cells during ischemia reperfusion (IR) injury. IR injury in ST-segment elevation myocardial infarction (STEMI) contributes to overall myocardial damage. HtrA2 has further been shown to be significantly increased in the serum of patients with STEMI. In the present pilot study, we use human umbilical vein endothelial cells (HUVECs) to investigate whether extracellular HtrA2 induces apoptosis using Annexin V staining. Furthermore, we examine whether HtrA2 is released extracellularly after staurosporine-induced apoptosis using ELISA. We find that HtrA2 is released upon induction of apoptosis by staurosporine into the cell culture medium. Furthermore, treatment of HUVECs with extracellular HtrA2-induces apoptosis, while the addition of anti-HtrA2 antibodies reduces both HtrA2- and staurosporine-induced endothelial cell apoptosis. In conclusion, we show here that extracellular HtrA2 induces apoptosis in human endothelial cells, although the exact molecular mechanisms have to be investigated in future.

Platelet Contributions to Myocardial Ischemia/Reperfusion Injury.

Obstruction of a coronary artery causes ischemia of heart tissue leading to myocardial infarction. Prolonged oxygen deficiency provokes tissue necrosis, which can result in heart failure and death of the patient. Therefore, restoration of coronary blood flow (reperfusion of the ischemic area) by re-canalizing the affected vessel is essential for a better patient outcome. Paradoxically, sudden reperfusion also causes tissue injury, thereby increasing the initial ischemic damage despite restoration of blood flow (=ischemia/reperfusion injury, IRI). Myocardial IRI is a complex event that involves various harmful mechanisms (e.g., production of reactive oxygen species and local increase in calcium ions) as well as inflammatory cells and signals like chemokines and cytokines. An involvement of platelets in the inflammatory reaction associated with IRI was discovered several years ago, but the underlying mechanisms are not yet fully understood. This mini review focusses on platelet contributions to the intricate picture of myocardial IRI. We summarize how upregulation of platelet surface receptors and release of immunomodulatory mediators lead to aggravation of myocardial IRI and subsequent cardiac damage by different mechanisms such as recruitment and activation of immune cells or modification of the cardiac vascular endothelium. In addition, evidence for cardioprotective roles of distinct platelet factors during IRI will be discussed.

In vivo Effects of Repeated Thyronamine Administration in Male C57BL/6J Mice.

OBJECTIVES: Thyronamines are decarboxylated and deiodinated metabolites of thyroid hormones (THs). Of all possible thyronamine variants, only 3-iodothyronamine (3-T1AM) and iodine-free thyronamine (T0AM) have been detected in vivo. While intensive research has been done on the (patho-)physiological action of 3-T1AM, the role of T0AM has been studied less intensively. STUDY DESIGN: We determined whether a single pharmacological dose (50 mg/kg, i.p.) or repeated administration (5 mg/kg/day, i.p., for 7 days) of T0AM affects metabolism, cardiovascular function, or thermoregulation in male C57BL/6J mice. Since selenium (Se) is important for proper TH function and Se metabolism is affected by TH, we additionally analyzed Se concentrations in liver, serum, and kidney using total reflection X-ray analysis. RESULTS: A single injection of T0AM had no effect on heart rate, temperature, or activity as assessed by radio telemetry. Likewise, daily administration of T0AM did not alter body weight, food or water intake, heart rate, blood pressure, brown adipose tissue thermogenesis, or body temperature, and no significant differences in hepatic glycogen content or mRNA expression of genes involved in cardiovascular function or metabolic control were determined. Also, the X-ray analysis of Se concentrations revealed no significant changes. However, hepatic T0AM was significantly increased in the treated animals. CONCLUSIONS: In summary, our data demonstrate that T0AM elicits no obvious metabolic, cardiovascular, or thermoregulatory activities in mice. As T0AM does also not interfere with TH or Se metabolism, we conclude that the deiodination of 3-T1AM to T0AM constitutes an efficient inactivation mechanism, terminating the actions of the more powerful precursor.

BMPs as new insulin sensitizers: enhanced glucose uptake in mature 3T3-L1 adipocytes via PPARγ and GLUT4 upregulation.

Insulin-resistance is the main cause of type 2 diabetes. Here we describe the identification and characterization of BMP2 and BMP6 as new insulin-sensitizing growth factors in mature adipocytes. We show that BMP2 and BMP6 lead to enhanced insulin-mediated glucose uptake in both insulin-sensitive and -insensitive adipocytes. We exclude a direct effect of BMP2 or BMP6 on translocation of GLUT4 to the plasma membrane and demonstrate that these BMPs increase GLUT4 protein levels equipotent to Rosiglitazone. BMPs induce expression of PPARγ as the crucial mediator for the insulin-sensitizing effect. A comprehensive RNA-Seq analysis in mature adipocytes revealed regulation of both BMP/Smad and PPARγ target genes. The effects of BMP2 and BMP6 are not completely redundant and include regulation of genes involved in glucose and fatty acid metabolism and adipokine expression. Collectively, these findings suggest the BMP2 and BMP6 pathway(s) as promising new drug targets to treat insulin resistance.

A validated LC-MS/MS method for cellular thyroid hormone metabolism: Uptake and turnover of mono-iodinated thyroid hormone metabolites by PCCL3 thyrocytes.

Tyrosine and phenolic ring de-iodination of thyroid hormones (TH) is crucial for regulating their physiological activity. Furthermore, reactions such as de-carboxylation to thyronamines (TAM) and de-amination to thyroacetic acids (TAc) produce TH metabolites (THM) with distinct biological properties. This needs to be considered when studying effects of TH and THM. The accurate and precise quantitative analysis of TH and THM in cell culture supernatants and cell lysates are key procedures required for studying the in vitro metabolism of TH. We report here the development of a liquid-liquid extraction/isotope dilution-liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the quantification of 9 thyronines (TN) and 6 TAM in human hepatocellular carcinoma Hep G2 cell lysate extracts. In addition, we adapted the method to quantify TH, TAM and TAc, in cell lysates of FBS-depleted rat thyroid epithelium PCCL3 cells. The methods for both cell lines were validated by rigorous assessment of linearity, limits of quantification and detection (LLOQ and LLOD respectively), intra- and inter-day accuracy, precision, process efficiency (PE), matrix effect (ME) and relative recovery (RE). Calibration curves covering 11 concentrations (based on 400 µl of lysate) were linear in the range 0.016-50 nM and 0.010-50 nM for Hep G2 and PCCL3 cells respectively. The lower limits of quantification were in the range 0.031 to 1 nM. We applied the PCCL3 version of the LC-MS/MS method to the analysis of lysed cell extracts from PCCL3 cells that had been incubated with 3-iodo-L-thyronine (T1), 3-iodothyronamine (3-T1AM) and 3-iodothyroacetic acid (3-T1Ac). Over the course of 30 minutes incubation 3-T1AM was de-iodinated to 4-[4-(2-aminoethylphenoxy)]phenol (thyronamine, T0AM) and de-aminated to 3-T1Ac respectively, whilst T1 underwent de-iodination to T0. This data indicates avid metabolism of these mono-iodinated compounds and the utility of LC-MS/MS to quantify such cellular metabolism.

3-Iodothyronamine Decreases Expression of Genes Involved in Iodide Metabolism in Mouse Thyroids and Inhibits Iodide Uptake in PCCL3 Thyrocytes.

BACKGROUND: 3-Iodothyronamine (3-T1AM) is an endogenous decarboxylated thyroid hormone (TH) metabolite. Pharmacological doses of 3-T1AM decrease heart rate, body temperature, and metabolic rate in rodents-effects that are contrary to classic TH excess. Furthermore, a single dose of 3-T1AM was shown to suppress the hypothalamic-pituitary-thyroid (HPT) axis in rats. It was hypothesized that 3-T1AM might play a role in the fine-tuning of TH action and might have a direct regulatory effect on the thyroid gland. METHODS: This study tested whether repeated 3-T1AM treatment interfered with thyroid function and the HPT axis in mice. Therefore, male C57BL/6 mice were intraperitoneally injected with 5 mg/kg of 3-T1AM or vehicle daily for seven days. Additionally, the effects of 3-T1AM on the differentiated rat thyrocyte cell line PCCL3 were analyzed. RESULTS: Repeated administration of 3-T1AM decreased thyroidal mRNA content of the sodium iodide symporter (Nis), thyroglobulin, and pendrin in mice. No interference with the HPT axis was observed, as determined by unaltered pituitary mRNA levels of triiodothyronine-responsive genes, including thyrotropin subunit ß. Furthermore, 3-T1AM treatment did not change transcript levels of hepatic triiodothyronine-responsive genes, such as deiodinase 1. In line with this, serum TH concentrations were not changed after the treatment period of seven days. In concordance with the in vivo findings, 3-T1AM decreased the thyrotropin-dependent expression of Nis and functional iodide uptake in PCCL3 cells in vitro. Additionally, uptake and metabolism of 3-T1AM by PCCL3 cells was observed, as well as 3-T1AM-dependent changes in intracellular Ca2+ concentration that might be involved in mediating the reported effects. CONCLUSIONS: In conclusion, 3-T1AM application decreased expression of selected TH synthesis genes by acting directly on the thyroid gland, and it might therefore affect TH synthesis without involvement of the HPT axis.

3-Iodothyroacetic acid lacks thermoregulatory and cardiovascular effects in vivo.

BACKGROUND AND PURPOSE: 3-Iodothyronamine (3-T1 AM) is an endogenous thyroid hormone derivative reported to induce strong hypothermia and bradycardia within minutes upon injection in rodents. Although 3-T1 AM is rapidly converted to several other metabolites in vivo, these strong pharmacological responses were solely attributed to 3-T1 AM, leaving potential contributions of downstream products untested. We therefore examined the cardiometabolic effects of 3-iodothyroacetic acid (TA1 ), the main degradation product of 3-T1 AM. EXPERIMENTAL APPROACH: We used a sensitive implantable radiotelemetry system in C57/Bl6J mice to study the effects of TA1 on body temperature and heart rate, as well as other metabolic parameters. KEY RESULTS: Interestingly, despite using pharmacological TA1 doses, we observed no effects on heart rate or body temperature after a single TA1 injection (50 mg·kg(-1) , i.p.) compared to sham-injected controls. Repeated administration of TA1 (5 mg·kg(-1) , i.p. for 7 days) likewise did not alter body weight, food and water intake, heart rate, blood pressure, brown adipose tissue (BAT) thermogenesis or body temperature. Moreover, mRNA expression of tissue specific genes in heart, kidney, liver, BAT and lung was also not altered by TA1 compared to sham-injected controls. CONCLUSIONS AND IMPLICATIONS: Our data therefore conclusively demonstrate that TA1 does not contribute to the cardiovascular or thermoregulatory effects observed after 3-T1 AM administration in mice, suggesting that the oxidative deamination constitutes an important deactivation mechanism for 3-T1 AM with possible implications for cardiovascular and thermoregulatory functions.

Stimulation of fat accumulation in hepatocytes by PGE2-dependent repression of hepatic lipolysis, ß-oxidation and VLDL-synthesis.

Hepatic steatosis is recognized as hepatic presentation of the metabolic syndrome. Hyperinsulinaemia, which shifts fatty acid oxidation to de novo lipogenesis and lipid storage in the liver, appears to be a principal elicitor particularly in the early stages of disease development. The impact of PGE2, which has previously been shown to attenuate insulin signaling and hence might reduce insulin-dependent lipid accumulation, on insulin-induced steatosis of hepatocytes was studied. The PGE2-generating capacity was enhanced in various obese mouse models by the induction of cyclooxygenase 2 and microsomal prostaglandin E-synthases (mPGES1, mPGES2). PGE2 attenuated the insulin-dependent induction of SREBP-1c and its target genes glucokinase and fatty acid synthase. Nevertheless, PGE2 enhanced incorporation of glucose into hepatic triglycerides synergistically with insulin. This was most likely due to a combination of a PGE2-dependent repression of (1) the key lipolytic enzyme adipose triglyceride lipase, (2) carnitine-palmitoyltransferase 1, a key regulator of mitochondrial ß-oxidation, and (3) microsomal transfer protein, as well as (4) apolipoprotein B, key components of the VLDL synthesis. Repression of PGC1α, a common upstream regulator of these genes, was identified as a possible cause. In support of this hypothesis, overexpression of PGC1α completely blunted the PGE2-dependent fat accumulation. PGE2 enhanced lipid accumulation synergistically with insulin, despite attenuating insulin signaling and might thus contribute to the development of hepatic steatosis. Induction of enzymes involved in PGE2 synthesis in in vivo models of obesity imply a potential role of prostanoids in the development of NAFLD and NASH.

Oncostatin M produced in Kupffer cells in response to PGE2: possible contributor to hepatic insulin resistance and steatosis.

Hepatic insulin resistance is a major contributor to hyperglycemia in metabolic syndrome and type II diabetes. It is caused in part by the low-grade inflammation that accompanies both diseases, leading to elevated local and circulating levels of cytokines and cyclooxygenase (COX) products such as prostaglandin E(2) (PGE(2)). In a recent study, PGE(2) produced in Kupffer cells attenuated insulin-dependent glucose utilization by interrupting the intracellular signal chain downstream of the insulin receptor in hepatocytes. In addition to directly affecting insulin signaling in hepatocytes, PGE(2) in the liver might affect insulin resistance by modulating cytokine production in non-parenchymal cells. In accordance with this hypothesis, PGE(2) stimulated oncostatin M (OSM) production by Kupffer cells. OSM in turn attenuated insulin-dependent Akt activation and, as a downstream target, glucokinase induction in hepatocytes, most likely by inducing suppressor of cytokine signaling 3 (SOCS3). In addition, it inhibited the expression of key enzymes of hepatic lipid metabolism. COX-2 and OSM mRNA were induced early in the course of the development of non-alcoholic steatohepatitis (NASH) in mice. Thus, induction of OSM production in Kupffer cells by an autocrine PGE(2)-dependent feed-forward loop may be an additional, thus far unrecognized, mechanism contributing to hepatic insulin resistance and the development of NASH.