Apoptotic cell identity induces distinct functional responses to IL-4 in efferocytic macrophages.

Macrophages are functionally heterogeneous cells essential for apoptotic cell clearance. Apoptotic cells are defined by homogeneous characteristics, ignoring their original cell lineage identity. We found that in an interleukin-4 (IL-4)-enriched environment, the sensing of apoptotic neutrophils by macrophages triggered their tissue remodeling signature. Engulfment of apoptotic hepatocytes promoted a tolerogenic phenotype, whereas phagocytosis of T cells had little effect on IL-4-induced gene expression. In a mouse model of parasite-induced pathology, the transfer of macrophages conditioned with IL-4 and apoptotic neutrophils promoted parasitic egg clearance. Knockout of phagocytic receptors required for the uptake of apoptotic neutrophils and partially T cells, but not hepatocytes, exacerbated helminth infection. These findings suggest that the identity of apoptotic cells may contribute to the development of distinct IL-4-driven immune programs in macrophages.

The TRPM2 ion channel regulates metabolic and thermogenic adaptations in adipose tissue of cold-exposed mice.

Introduction: During thermogenesis, adipose tissue (AT) becomes more active and enhances oxidative metabolism. The promotion of this process in white AT (WAT) is called "browning" and, together with the brown AT (BAT) activation, is considered as a promising approach to counteract obesity and metabolic diseases. Transient receptor potential cation channel, subfamily M, member 2 (TRPM2), is an ion channel that allows extracellular Ca2+ influx into the cytosol, and is gated by adenosine diphosphate ribose (ADPR), produced from NAD+ degradation. The aim of this study was to investigate the relevance of TRPM2 in the regulation of energy metabolism in BAT, WAT, and liver during thermogenesis. Methods: Wild type (WT) and Trpm2-/- mice were exposed to 6°C and BAT, WAT and liver were collected to evaluate mRNA, protein levels and ADPR content. Furthermore, O2 consumption, CO2 production and energy expenditure were measured in these mice upon thermogenic stimulation. Finally, the effect of the pharmacological inhibition of TRPM2 was assessed in primary adipocytes, evaluating the response upon stimulation with the ß-adrenergic receptor agonist CL316,243. Results: Trpm2-/- mice displayed lower expression of browning markers in AT and lower energy expenditure in response to thermogenic stimulus, compared to WT animals. Trpm2 gene overexpression was observed in WAT, BAT and liver upon cold exposure. In addition, ADPR levels and mono/poly-ADPR hydrolases expression were higher in mice exposed to cold, compared to control mice, likely mediating ADPR generation. Discussion: Our data indicate TRPM2 as a fundamental player in BAT activation and WAT browning. TRPM2 agonists may represent new pharmacological strategies to fight obesity.

Fatty acid synthesis suppresses dietary polyunsaturated fatty acid use.

Dietary polyunsaturated fatty acids (PUFA) are increasingly recognized for their health benefits, whereas a high production of endogenous fatty acids - a process called de novo lipogenesis (DNL) - is closely linked to metabolic diseases. Determinants of PUFA incorporation into complex lipids are insufficiently understood and may influence the onset and progression of metabolic diseases. Here we show that fatty acid synthase (FASN), the key enzyme of DNL, critically determines the use of dietary PUFA in mice and humans. Moreover, the combination of FASN inhibition and PUFA-supplementation decreases liver triacylglycerols (TAG) in mice fed with high-fat diet. Mechanistically, FASN inhibition causes higher PUFA uptake via the lysophosphatidylcholine transporter MFSD2A, and a diacylglycerol O-acyltransferase 2 (DGAT2)-dependent incorporation of PUFA into TAG. Overall, the outcome of PUFA supplementation may depend on the degree of endogenous DNL and combining PUFA supplementation and FASN inhibition might be a promising approach to target metabolic disease.

A liquid chromatography-tandem mass spectrometry based method for the quantification of adenosine nucleotides and NAD precursors and products in various biological samples.

Adenine nucleotides (AN) are ubiquitous metabolites that regulate cellular energy metabolism and modulate cell communication and inflammation. To understand how disturbances in AN balance arise and affect cellular function, robust quantification techniques for these metabolites are crucial. However, due to their hydrophilicity, simultaneous quantification of AN across various biological samples has been challenging. Here we present a hydrophilic interaction high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based method for the quantification of 26 adenosine nucleotides and precursors as well as metabolic products of nicotinamide adenine dinucleotide (NAD) in plasma, liver, and adipose tissue samples as well as cell culture supernatants and cells. Method validation was performed with regard to linearity, accuracy, precision, matrix effects, and carryover. Finally, analysis of cell culture supernatants derived from intestinal organoids and RAW 264.7 cells illustrates that the here described method is a reliable and easy-to-use tool to quantify AN and opens up new avenues to understand the role of AN generation and breakdown for cellular functions.

Short-term dietary changes can result in mucosal and systemic immune depression.

Omnivorous animals, including mice and humans, tend to prefer energy-dense nutrients rich in fat over plant-based diets, especially for short periods of time, but the health consequences of this short-term consumption of energy-dense nutrients are unclear. Here, we show that short-term reiterative switching to 'feast diets', mimicking our social eating behavior, breaches the potential buffering effect of the intestinal microbiota and reorganizes the immunological architecture of mucosa-associated lymphoid tissues. The first dietary switch was sufficient to induce transient mucosal immune depression and suppress systemic immunity, leading to higher susceptibility to Salmonella enterica serovar Typhimurium and Listeria monocytogenes infections. The ability to respond to antigenic challenges with a model antigen was also impaired. These observations could be explained by a reduction of CD4+ T cell metabolic fitness and cytokine production due to impaired mTOR activity in response to reduced microbial provision of fiber metabolites. Reintroducing dietary fiber rewired T cell metabolism and restored mucosal and systemic CD4+ T cell functions and immunity. Finally, dietary intervention with human volunteers confirmed the effect of short-term dietary switches on human CD4+ T cell functionality. Therefore, short-term nutritional changes cause a transient depression of mucosal and systemic immunity, creating a window of opportunity for pathogenic infection.

TREM2 Regulates the Removal of Apoptotic Cells and Inflammatory Processes during the Progression of NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver pathology worldwide. In mice and humans, NAFLD progression is characterized by the appearance of TREM2-expressing macrophages in the liver. However, their mechanistic contributions to disease progression have not been completely elucidated. Here, we show that TREM2+ macrophages prevent the generation of a pro-inflammatory response elicited by LPS-laden lipoproteins in vitro. Further, Trem2 expression regulates bone-marrow-derived macrophages (BMDMs) and Kupffer cell capacity to phagocyte apoptotic cells in vitro, which is dependent on CD14 activation. In line with this, loss of Trem2 resulted in an increased pro-inflammatory response, which ultimately aggravated liver fibrosis in murine models of NAFLD. Similarly, in a human NAFLD cohort, plasma levels of TREM2 were increased and hepatic TREM2 expression was correlated with higher levels of liver triglycerides and the acquisition of a fibrotic gene signature. Altogether, our results suggest that TREM2+ macrophages have a protective function during the progression of NAFLD, as they are involved in the processing of pro-inflammatory lipoproteins and phagocytosis of apoptotic cells and, thereby, are critical contributors for the re-establishment of liver homeostasis.

Cold-Induced Lipoprotein Clearance in Cyp7b1-Deficient Mice.

Brown adipose tissue (BAT) has emerged as an appealing therapeutic target for cardio metabolic diseases. BAT is a heat-producing organ and upon activation substantially lowers hyperlipidemia. In response to cold exposure, not only the uptake of lipids into BAT is increased but also the Cyp7b1-mediated synthesis of bile acids (BA) from cholesterol in the liver is triggered. In addition to their role for intestinal lipid digestion, BA act as endocrine signals that can activate thermogenesis in BAT. When exposed to cold temperatures, Cyp7b1 -/- mice have compromised BAT function along with reduced fecal bile acid levels. Here, we aim to evaluate the role of Cyp7b1 for BAT-dependent lipid clearance. Using metabolic studies with radioactive tracers, we show that in response to a cold stimulus, BAT-mediated clearance of fatty acids derived from triglyceride-rich lipoproteins (TRL), and their remnants are reduced in Cyp7b1 -/- mice. The impaired lipid uptake can be explained by reduced BAT lipoprotein lipase (LPL) levels and compromised organ activity in Cyp7b1 -/- mice, which may be linked to impaired insulin signaling. Overall, our findings reveal that alterations of systemic lipoprotein metabolism mediated by cold-activated BAT are dependent, at least in part, on CYP7Β1.

A Gas Chromatography Mass Spectrometry-Based Method for the Quantification of Short Chain Fatty Acids.

Short Chain Fatty Acids (SCFAs) are produced by the gut microbiota and are present in varying concentrations in the intestinal lumen, in feces but also in the circulatory system. By interacting with different cell types in the body, they have a great impact on host metabolism and their exact quantification is indispensable. Here, we present a derivatization-free method for the gas chromatography mass spectrometry (GC-MS) based quantification of SCFAs in plasma, feces, cecum, liver and adipose tissue. SCFAs were extracted using ethanol and concentrated by alkaline vacuum centrifugation. To allow volatility for separation by GC, samples were acidified with succinic acid. Analytes were detected in selected ion monitoring (SIM) mode and quantified using deuterated internal standards and external calibration curves. Method validation rendered excellent linearity (R2 > 0.99 for most analytes), good recovery rates (95-117%), and good reproducibility (RSD: 1-4.5%). Matrix effects were ruled out in plasma, feces, cecum, liver and fat tissues where most abundant SCFAs were detected and accurately quantified. Finally, applicability of the method was assessed using samples derived from conventionally raised versus germ-free mice or mice treated with antibiotics. Altogether, a reliable, fast, derivatization-free GC-MS method for the quantification of SCFAs in different biological matrices was developed allowing for the study of the (patho)physiological role of SCFAs in metabolic health.

Susceptibility to diet-induced obesity at thermoneutral conditions is independent of UCP1.

Activation of uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) upon cold stimulation leads to substantial increase in energy expenditure to defend body temperature. Increases in energy expenditure after a high-caloric food intake, termed diet-induced thermogenesis, are also attributed to BAT. These properties render BAT a potential target to combat diet-induced obesity. However, studies investigating the role of UCP1 to protect against diet-induced obesity are controversial and rely on the phenotyping of a single constitutive UCP1-knockout model. To address this issue, we generated a novel UCP1-knockout model by Cre-mediated deletion of exon 2 in the UCP1 gene. We studied the effect of constitutive UCP1 knockout on metabolism and the development of diet-induced obesity. UCP1 knockout and wild-type mice were housed at 30°C and fed a control diet for 4 wk followed by 8 wk of high-fat diet. Body weight and food intake were monitored continuously over the course of the study, and indirect calorimetry was used to determine energy expenditure during both feeding periods. Based on Western blot analysis, thermal imaging and noradrenaline test, we confirmed the lack of functional UCP1 in knockout mice. However, body weight gain, food intake, and energy expenditure were not affected by loss of UCP1 function during both feeding periods. We introduce a novel UCP1-KO mouse enabling the generation of conditional UCP1-knockout mice to scrutinize the contribution of UCP1 to energy metabolism in different cell types or life stages. Our results demonstrate that UCP1 does not protect against diet-induced obesity at thermoneutrality.NEW & NOTEWORTHY We provide evidence that the abundance of UCP1 does not influence energy metabolism at thermoneutrality studying a novel Cre-mediated UCP1-KO mouse model. This model will be a foundation for a better understanding of the contribution of UCP1 in different cell types or life stages to energy metabolism.

Oxysterol 7-α Hydroxylase (CYP7B1) Attenuates Metabolic-Associated Fatty Liver Disease in Mice at Thermoneutrality.

Ambient temperature is an important determinant of both the alternative bile acid synthesis pathway controlled by oxysterol 7-α hydroxylase (CYP7B1) and the progression of metabolic-associated fatty liver disease (MAFLD). Here, we investigated whether CYP7B1 is involved in the etiology of MAFLD under conditions of low and high energy expenditure. For this, Cyp7b1-/- and wild type (WT) mice were fed a choline-deficient high-fat diet and housed either at 30 °C (thermoneutrality) or at 22 °C (mild cold). To study disease phenotype and underlying mechanisms, plasma and organ samples were analyzed to determine metabolic parameters, immune cell infiltration by immunohistology and flow cytometry, lipid species including hydroxycholesterols, bile acids and structural lipids. In WT and Cyp7b1-/- mice, thermoneutral housing promoted MAFLD, an effect that was more pronounced in CYP7B1-deficient mice. In these mice, we found higher plasma alanine aminotransferase activity, hyperlipidemia, hepatic accumulation of potentially harmful lipid species, aggravated liver fibrosis, increased inflammation and immune cell infiltration. Bile acids and hydroxycholesterols did not correlate with aggravated MAFLD in Cyp7b1-/- mice housed at thermoneutrality. Notably, an up-regulation of lipoprotein receptors was detected at 22 °C but not at 30 °C in livers of Cyp7b1-/- mice, suggesting that accelerated metabolism of lipoproteins carrying lipotoxic molecules counteracts MAFLD progression.

Endothelial Lipase Is Involved in Cold-Induced High-Density Lipoprotein Turnover and Reverse Cholesterol Transport in Mice.

The physiologic activation of thermogenic brown and white adipose tissues (BAT/WAT) by cold exposure triggers heat production by adaptive thermogenesis, a process known to ameliorate hyperlipidemia and protect from atherosclerosis. Mechanistically, it has been shown that thermogenic activation increases lipoprotein lipase (LPL)-dependent hydrolysis of triglyceride-rich lipoproteins (TRL) and accelerates the generation of cholesterol-enriched remnants and high-density lipoprotein (HDL), which promotes cholesterol flux from the periphery to the liver. HDL is also subjected to hydrolysis by endothelial lipase (EL) (encoded by LIPG). Genome-wide association studies have identified various variants of EL that are associated with altered HDL cholesterol levels. However, a potential role of EL in BAT-mediated HDL metabolism has not been investigated so far. In the present study, we show that in mice, cold-stimulated activation of thermogenic adipocytes induced expression of Lipg in BAT and inguinal WAT but that loss of Lipg did not affect gene expression of thermogenic markers. Furthermore, in both wild type (WT) and Lipg-deficient mice, activation of thermogenesis resulted in a decline of HDL cholesterol levels. However, cold-induced remodeling of the HDL lipid composition was different between WT and Lipg-deficient mice. Notably, radioactive tracer studies with double-labeled HDL indicated that cold-induced hepatic HDL cholesterol clearance was lower in Lipg-deficient mice. Moreover, this reduced clearance was associated with impaired macrophage-to-feces cholesterol transport. Overall, these data indicate that EL is a determinant of HDL lipid composition, cholesterol flux, and HDL turnover in conditions of high thermogenic activity.

Endogenous Fatty Acid Synthesis Drives Brown Adipose Tissue Involution.

Thermoneutral conditions typical for standard human living environments result in brown adipose tissue (BAT) involution, characterized by decreased mitochondrial mass and increased lipid deposition. Low BAT activity is associated with poor metabolic health, and BAT reactivation may confer therapeutic potential. However, the molecular drivers of this BAT adaptive process in response to thermoneutrality remain enigmatic. Using metabolic and lipidomic approaches, we show that endogenous fatty acid synthesis, regulated by carbohydrate-response element-binding protein (ChREBP), is the central regulator of BAT involution. By transcriptional control of lipogenesis-related enzymes, ChREBP determines the abundance and composition of both storage and membrane lipids known to regulate organelle turnover and function. Notably, ChREBP deficiency and pharmacological inhibition of lipogenesis during thermoneutral adaptation preserved mitochondrial mass and thermogenic capacity of BAT independently of mitochondrial biogenesis. In conclusion, we establish lipogenesis as a potential therapeutic target to prevent loss of BAT thermogenic capacity as seen in adult humans.

Lysosomal lipoprotein processing in endothelial cells stimulates adipose tissue thermogenic adaptation.

In response to cold exposure, thermogenic adipocytes internalize large amounts of fatty acids after lipoprotein lipase-mediated hydrolysis of triglyceride-rich lipoproteins (TRL) in the capillary lumen of brown adipose tissue (BAT) and white adipose tissue (WAT). Here, we show that in cold-exposed mice, vascular endothelial cells in adipose tissues endocytose substantial amounts of entire TRL particles. These lipoproteins subsequently follow the endosomal-lysosomal pathway, where they undergo lysosomal acid lipase (LAL)-mediated processing. Endothelial cell-specific LAL deficiency results in impaired thermogenic capacity as a consequence of reduced recruitment of brown and brite/beige adipocytes. Mechanistically, TRL processing by LAL induces proliferation of endothelial cells and adipocyte precursors via beta-oxidation-dependent production of reactive oxygen species, which in turn stimulates hypoxia-inducible factor-1α-dependent proliferative responses. In conclusion, this study demonstrates a physiological role for TRL particle uptake into BAT and WAT and establishes endothelial lipoprotein processing as an important determinant of adipose tissue remodeling during thermogenic adaptation.

Inulin Supplementation Disturbs Hepatic Cholesterol and Bile Acid Metabolism Independent from Housing Temperature.

Dietary fibers are fermented by gut bacteria into the major short chain fatty acids (SCFAs) acetate, propionate, and butyrate. Generally, fiber-rich diets are believed to improve metabolic health. However, recent studies suggest that long-term supplementation with fibers causes changes in hepatic bile acid metabolism, hepatocyte damage, and hepatocellular cancer in dysbiotic mice. Alterations in hepatic bile acid metabolism have also been reported after cold-induced activation of brown adipose tissue. Here, we aim to investigate the effects of short-term dietary inulin supplementation on liver cholesterol and bile acid metabolism in control and cold housed specific pathogen free wild type (WT) mice. We found that short-term inulin feeding lowered plasma cholesterol levels and provoked cholestasis and mild liver damage in WT mice. Of note, inulin feeding caused marked perturbations in bile acid metabolism, which were aggravated by cold treatment. Our studies indicate that even relatively short periods of inulin consumption in mice with an intact gut microbiome have detrimental effects on liver metabolism and function.

Effects of Pharmacological Thermogenic Adipocyte Activation on Metabolism and Atherosclerotic Plaque Regression.

Thermogenic adipocytes burn nutrients in order to produce heat. Upon activation, brown adipose tissue (BAT) clears vast amounts of lipids and glucose from the circulation and thus substantially lowers plasma lipid levels. As a consequence, BAT activation protects from the development of atherosclerosis. However, it is unclear if pharmacologic activation of BAT can be exploited therapeutically to reduce plaque burden in established atherosclerotic disease. Here we study the impact of thermogenic adipose tissues on plaque regression in a mouse model of atherosclerosis. Thermogenic adipocytes in atherosclerotic low-density lipoprotein (LDL) receptor (LDLR)-deficient mice were pharmacologically activated by dietary CL316,243 (CL) treatment for 4 weeks and the outcomes on metabolically active tissues, plasma lipids and atherosclerosis were analyzed. While the chronic activation of thermogenic adipocytes reduced adiposity, increased browning of white adipose tissue (WAT), altered liver gene expression, and reduced plasma triglyceride levels, atherosclerotic plaque burden remained unchanged. Our findings suggest that despite improving adiposity and plasma triglycerides, pharmacologic activation of thermogenic adipocytes is not able to reverse atherosclerosis in LDLR-deficient mice.

Assessment of Uptake and Biodistribution of Radiolabeled Cholesterol in Mice Using Gavaged Recombinant Triglyceride-rich Lipoprotein Particles (rTRL).

The here described method can be used to estimate the uptake of orally provided cholesterol in mice. Briefly, mice are gavaged with radiolabeled cholesterol and 4 h later, organ distribution of the radiolabel is determined by liquid scintillation counting. The method has been applied successfully to determine dietary cholesterol handling of mice housed at different ambient temperatures.

The TREM2-APOE Pathway Drives the Transcriptional Phenotype of Dysfunctional Microglia in Neurodegenerative Diseases.

Microglia play a pivotal role in the maintenance of brain homeostasis but lose homeostatic function during neurodegenerative disorders. We identified a specific apolipoprotein E (APOE)-dependent molecular signature in microglia from models of amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and Alzheimer's disease (AD) and in microglia surrounding neuritic ß-amyloid (Aß)-plaques in the brains of people with AD. The APOE pathway mediated a switch from a homeostatic to a neurodegenerative microglia phenotype after phagocytosis of apoptotic neurons. TREM2 (triggering receptor expressed on myeloid cells 2) induced APOE signaling, and targeting the TREM2-APOE pathway restored the homeostatic signature of microglia in ALS and AD mouse models and prevented neuronal loss in an acute model of neurodegeneration. APOE-mediated neurodegenerative microglia had lost their tolerogenic function. Our work identifies the TREM2-APOE pathway as a major regulator of microglial functional phenotype in neurodegenerative diseases and serves as a novel target that could aid in the restoration of homeostatic microglia.

Metabolic Circuit Involving Free Fatty Acids, microRNA 122, and Triglyceride Synthesis in Liver and Muscle Tissues.

BACKGROUND & AIMS: Effective treatments are needed for hepatic steatosis characterized by accumulation of triglycerides in hepatocytes, which leads to hepatocellular carcinoma. MicroRNA 122 (MIR122) is expressed only in the liver, where it regulates lipid metabolism. We investigated the mechanism by which free fatty acids (FFAs) regulate MIR122 expression and the effect of MIR122 on triglyceride synthesis. METHODS: We analyzed MIR122 promoter activity and validated its target mRNAs by transfection of Luciferase reporter plasmids into Huh7, BNL-1ME, and HEK293 cultured cell lines. We measured levels of microRNAs and mRNAs by quantitative real-time PCR analysis of RNA extracted from plasma, liver, muscle, and adipose tissues of C57BL/6 mice given the FFA-inducer CL316243. MIR122 was inhibited using an inhibitor of MIR122. Metabolic profiles of mice were determined using metabolic chambers and by histologic analyses of liver tissues. We performed RNA sequence analyses to identify metabolic pathways involving MIR122. RESULTS: We validated human Agpat1 and Dgat1 mRNAs, involved in triglyceride synthesis, as targets of MIR122. FFAs increased MIR122 expression in livers of mice by activating the retinoic acid-related orphan receptor alpha, and induced secretion of MIR122 from liver to blood. Circulating MIR122 entered muscle and adipose tissues of mice, reducing mRNA levels of genes involved in triglyceride synthesis. Mice injected with an inhibitor of MIR122 and then given CL316243, accumulated triglycerides in liver and muscle tissues, and had reduced rates of ß-oxidation. There was a positive correlation between level of FFAs and level of MIR122 in plasma samples from 6 healthy individuals, collected before and during fasting. CONCLUSIONS: In biochemical and histologic studies of plasma, liver, muscle, and adipose tissues from mice, we found that FFAs increase hepatic expression and secretion of MIR122, which regulates energy storage vs expenditure in liver and peripheral tissues. Strategies to reduce triglyceride levels, by increasing MIR122, might be developed for treatment of metabolic syndrome.

Cold-induced conversion of cholesterol to bile acids in mice shapes the gut microbiome and promotes adaptive thermogenesis.

Adaptive thermogenesis is an energy-demanding process that is mediated by cold-activated beige and brown adipocytes, and it entails increased uptake of carbohydrates, as well as lipoprotein-derived triglycerides and cholesterol, into these thermogenic cells. Here we report that cold exposure in mice triggers a metabolic program that orchestrates lipoprotein processing in brown adipose tissue (BAT) and hepatic conversion of cholesterol to bile acids via the alternative synthesis pathway. This process is dependent on hepatic induction of cytochrome P450, family 7, subfamily b, polypeptide 1 (CYP7B1) and results in increased plasma levels, as well as fecal excretion, of bile acids that is accompanied by distinct changes in gut microbiota and increased heat production. Genetic and pharmacological interventions that targeted the synthesis and biliary excretion of bile acids prevented the rise in fecal bile acid excretion, changed the bacterial composition of the gut and modulated thermogenic responses. These results identify bile acids as important metabolic effectors under conditions of sustained BAT activation and highlight the relevance of cholesterol metabolism by the host for diet-induced changes of the gut microbiota and energy metabolism.