FXR Isoforms Control Different Metabolic Functions in Liver Cells via Binding to Specific DNA Motifs.

BACKGROUND & AIMS: The nuclear receptor subfamily 1 group H member 4 (NR1H4, also called FXR) is a ligand-activated transcription factor that, upon binding of bile acids, regulates the expression of genes involved in bile acid, fat, sugar, and amino acid metabolism. Transcript variants encode the FXR isoforms alpha 1, alpha 2, alpha 3, and alpha 4, which activate different genes that regulate metabolism. Little is known about the mechanisms by which the different isoforms regulate specific genes or how the expression of these genes affects the outcomes of patients given drugs that target FXR. METHODS: We determined genome-wide binding of FXR isoforms in mouse liver organoids that express individual FXR isoforms using chromatin immunoprecipitation, followed by sequencing analysis and DNA motif discovery. We validated regulatory DNA sequences by mobility shift assays and with luciferase reporters using mouse and human FXR isoforms. We analyzed mouse liver organoids and HepG2 cells that expressed the FXR isoforms using chromatin immunoprecipitation, quantitative polymerase chain reaction, and immunoblot assays. Organoids were analyzed for mitochondrial respiration, lipid droplet content, and triglyceride excretion. We used the FXR ligand obeticholic acid to induce FXR activity in organoids, cell lines, and mice. We collected data on the binding of FXR in mouse liver and the expression levels of FXR isoforms and gene targets in human liver tissue and primary human hepatocytes from the Gene Expression Omnibus. RESULTS: In mouse liver cells, 89% of sites that bound FXR were bound by only FXRα2 or FXRα4, via direct interactions with the DNA sequence motif ER-2. Via DNA binding, these isoforms regulated metabolic functions in liver cells, including carbon metabolism and lipogenesis. Incubation with obeticholic acid increased mitochondrial pyruvate transport and reduced insulin-induced lipogenesis in organoids that expressed FXRα2 but not FXRα1. In human liver tissues, levels of FXRα2 varied significantly and correlated with expression of genes predicted to be regulated via an ER-2 motif. CONCLUSIONS: Most metabolic effects regulated by FXR in mouse and human liver cells are regulated by the FXRα2 isoform via specific binding to ER-2 motifs. The expression level of FXRα2 in liver might be used to predict responses of patients to treatment with FXR agonists.

Advanced omics techniques shed light on CD1d-mediated lipid antigen presentation to iNKT cells.

Invariant natural killer T cells (iNKT cells) can be activated through binding antigenic lipid/CD1d complexes to their TCR. Antigenic lipids are processed, loaded, and displayed in complex with CD1d by lipid antigen presenting cells (LAPCs). The mechanism of lipid antigen presentation via CD1d is highly conserved with recent work showing adipocytes are LAPCs that, besides having a role in lipid storage, can activate iNKT cells and play an important role in systemic metabolic disease. Recent studies shed light on parameters potentially dictating cytokine output and how obesity-associated metabolic disease may affect such parameters. By following a lipid antigen's journey, we identify five key areas which may dictate cytokine skew: co-stimulation, structural properties of the lipid antigen, stability of lipid antigen/CD1d complexes, intracellular and extracellular pH, and intracellular and extracellular lipid environment. Recent publications indicate that the combination of advanced omics-type approaches and machine learning may be a fruitful way to interconnect these 5 areas, with the ultimate goal to provide new insights for therapeutic exploration.

The pseudokinase TRIB3 controls adipocyte lipid homeostasis and proliferation in vitro and in vivo.

OBJECTIVE: In vivo studies in humans and mice have implicated the pseudokinase Tribbles 3 (TRIB3) in various aspects of energy metabolism. Whilst cell-based studies indicate a role for TRIB3 in adipocyte differentiation and function, it is unclear if and how these cellular functions may contribute to overall metabolic health. METHODS: We investigated the metabolic phenotype of whole-body Trib3 knockout (Trib3KO) mice, focusing on adipocyte and adipose tissue functions. In addition, we combined lipidomics, transcriptomics, interactomics and phosphoproteomics analyses to elucidate cell-intrinsic functions of TRIB3 in pre- and mature adipocytes. RESULTS: Trib3KO mice display increased adiposity, but their insulin sensitivity remains unaltered. Trib3KO adipocytes are smaller and display higher Proliferating Cell Nuclear Antigen (PCNA) levels, indicating potential alterations in either i) proliferation-differentiation balance, ii) impaired expansion after cell division, or iii) an altered balance between lipid storage and release, or a combination thereof. Lipidome analyses suggest TRIB3 involvement in the latter two processes, as triglyceride storage is reduced and membrane composition, which can restrain cellular expansion, is altered. Integrated interactome, phosphoproteome and transcriptome analyses support a role for TRIB3 in all three cellular processes through multiple cellular pathways, including Mitogen Activated Protein Kinase- (MAPK/ERK), Protein Kinase A (PKA)-mediated signaling and Transcription Factor 7 like 2 (TCF7L2) and Beta Catenin-mediated gene expression. CONCLUSIONS: Our findings support TRIB3 playing multiple distinct regulatory roles in the cytoplasm, nucleus and mitochondria, ultimately controlling adipose tissue homeostasis, rather than affecting a single cellular pathway.

Cytokine Output of Adipocyte-iNKT Cell Interplay Is Skewed by a Lipid-Rich Microenvironment.

The complex direct and indirect interplay between adipocytes and various adipose tissue (AT)-resident immune cells plays an important role in maintaining local and whole-body insulin sensitivity. Adipocytes can directly interact with and activate AT-resident invariant natural killer T (iNKT) cells through CD1d-dependent presentation of lipid antigens, which is associated with anti-inflammatory cytokine production in lean AT (IL-4, IL-10). Whether alterations in the microenvironment, i.e., increased free fatty acids concentrations or altered cytokine/adipokine profiles as observed in obesity, directly affect adipocyte-iNKT cell communication and subsequent cytokine output is currently unknown. Here we show that the cytokine output of adipocyte-iNKT cell interplay is skewed by a lipid-rich microenvironment. Incubation of mature 3T3-L1 adipocytes with a mixture of saturated and unsaturated fatty acids specifically reduced insulin sensitivity and increased lipolysis. Reduced activation of the CD1d-invariant T-Cell Receptor (TCR) signaling axis was observed in Jurkat reporter cells expressing the invariant NKT TCR, while co-culture assays with a iNKT hybridoma cell line (DN32.D3) skewed the cytokine output toward reduced IL-4 secretion and increased IFNγ secretion. Importantly, co-culture assays of mature 3T3-L1 adipocytes with primary iNKT cells isolated from visceral AT showed a similar shift in cytokine output. Collectively, these data indicate that iNKT cells display considerable plasticity with respect to their cytokine output, which can be skewed toward a more pro-inflammatory profile in vitro by microenvironmental factors like fatty acids.

Brain-Sparing Sympathofacilitators Mitigate Obesity without Adverse Cardiovascular Effects.

Anti-obesity drugs in the amphetamine (AMPH) class act in the brain to reduce appetite and increase locomotion. They are also characterized by adverse cardiovascular effects with origin that, despite absence of any in vivo evidence, is attributed to a direct sympathomimetic action in the heart. Here, we show that the cardiac side effects of AMPH originate from the brain and can be circumvented by PEGylation (PEGyAMPH) to exclude its central action. PEGyAMPH does not enter the brain and facilitates SNS activity via theß2-adrenoceptor, protecting mice against obesity by increasing lipolysis and thermogenesis, coupled to higher heat dissipation, which acts as an energy sink to increase energy expenditure without altering food intake or locomotor activity. Thus, we provide proof-of-principle for a novel class of exclusively peripheral anti-obesity sympathofacilitators that are devoid of any cardiovascular and brain-related side effects.

Sympathetic neuron-associated macrophages contribute to obesity by importing and metabolizing norepinephrine.

The cellular mechanism(s) linking macrophages to norepinephrine (NE)-mediated regulation of thermogenesis have been a topic of debate. Here we identify sympathetic neuron-associated macrophages (SAMs) as a population of cells that mediate clearance of NE via expression of solute carrier family 6 member 2 (SLC6A2), an NE transporter, and monoamine oxidase A (MAOA), a degradation enzyme. Optogenetic activation of the sympathetic nervous system (SNS) upregulates NE uptake by SAMs and shifts the SAM profile to a more proinflammatory state. NE uptake by SAMs is prevented by genetic deletion of Slc6a2 or inhibition of the encoded transporter. We also observed an increased proportion of SAMs in the SNS of two mouse models of obesity. Genetic ablation of Slc6a2 in SAMs increases brown adipose tissue (BAT) content, causes browning of white fat, increases thermogenesis, and leads to substantial and sustained weight loss in obese mice. We further show that this pathway is conserved, as human sympathetic ganglia also contain SAMs expressing the analogous molecular machinery for NE clearance, which thus constitutes a potential target for obesity treatment.