Differential cell type-specific function of the aryl hydrocarbon receptor and its repressor in diet-induced obesity and fibrosis.

OBJECTIVE: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor regulating xenobiotic responses as well as physiological metabolism. Dietary AhR ligands activate the AhR signaling axis, whereas AhR activation is negatively regulated by the AhR repressor (AhRR). While AhR-deficient mice are known to be resistant to diet-induced obesity (DIO), the influence of the AhRR on DIO has not been assessed so far. METHODS: In this study, we analyzed AhRR-/- mice and mice with a conditional deletion of either AhRR or AhR in myeloid cells under conditions of DIO and after supplementation of dietary AhR ligands. Moreover, macrophage metabolism was assessed using Seahorse Mito Stress Test and ROS assays as well as transcriptomic analysis. RESULTS: We demonstrate that global AhRR deficiency leads to a robust, but not as profound protection from DIO and hepatosteatosis as AhR deficiency. Under conditions of DIO, AhRR-/- mice did not accumulate TCA cycle intermediates in the circulation in contrast to wild-type (WT) mice, indicating protection from metabolic dysfunction. This effect could be mimicked by dietary supplementation of AhR ligands in WT mice. Because of the predominant expression of the AhRR in myeloid cells, AhRR-deficient macrophages were analyzed for changes in metabolism and showed major metabolic alterations regarding oxidative phosphorylation and mitochondrial activity. Unbiased transcriptomic analysis revealed increased expression of genes involved in de novo lipogenesis and mitochondrial biogenesis. Mice with a genetic deficiency of the AhRR in myeloid cells did not show alterations in weight gain after high fat diet (HFD) but demonstrated ameliorated liver damage compared to control mice. Further, deficiency of the AhR in myeloid cells also did not affect weight gain but led to enhanced liver damage and adipose tissue fibrosis compared to controls. CONCLUSIONS: AhRR-deficient mice are resistant to diet-induced metabolic syndrome. Although conditional ablation of either the AhR or AhRR in myeloid cells did not recapitulate the phenotype of the global knockout, our findings suggest that enhanced AhR signaling in myeloid cells deficient for AhRR protects from diet-induced liver damage and fibrosis, whereas myeloid cell-specific AhR deficiency is detrimental.

Triglyceride cycling enables modification of stored fatty acids.

Triglyceride cycling is the process of continuous degradation and re-synthesis of triglyceride in cellular stores. We show in 3T3-L1 adipocytes that triglycerides are subject to rapid turnover and re-arrangement of fatty acids with an estimated half-life of 2-4 h. We develop a tracing technology that can simultaneously and quantitatively follow the metabolism of multiple fatty acids to study the triglyceride futile substrate cycle directly and with molecular species resolution. Our approach is based on alkyne fatty acid tracers and mass spectrometry. The triglyceride cycling is connected to modification of released fatty acids by elongation and desaturation. Through cycling and modification, saturated fatty acids are slowly converted to monounsaturated fatty acids, and linoleic acid to arachidonic acid. We conclude that triglyceride cycling renders stored fatty acids accessible for metabolic alteration. The overall process facilitates cellular adjustments to the stored fatty acid pool to meet changing needs of the cell.

Creld2 function during unfolded protein response is essential for liver metabolism homeostasis.

The unfolded protein response (UPR) is associated with hepatic metabolic function, yet it is not well understood how endoplasmic reticulum (ER) disturbance might influence metabolic homeostasis. Here, we describe the physiological function of Cysteine-rich with EGF-like domains 2 (Creld2), previously characterized as a downstream target of the ER-stress signal transducer Atf6. To this end, we generated Creld2-deficient mice and induced UPR by injection of tunicamycin. Creld2 augments protein folding and creates an interlink between the UPR axes through its interaction with proteins involved in the cellular stress response. Thereby, Creld2 promotes tolerance to ER stress and recovery from acute stress. Creld2-deficiency leads to a dysregulated UPR and causes the development of hepatic steatosis during ER stress conditions. Moreover, Creld2-dependent enhancement of the UPR assists in the regulation of energy expenditure. Furthermore, we observed a sex dimorphism in human and mouse livers with only male patients showing an accumulation of CRELD2 protein during the progression from non-alcoholic fatty liver disease to non-alcoholic steatohepatitis and only male Creld2-deficient mice developing hepatic steatosis upon aging. These results reveal a Creld2 function at the intersection between UPR and metabolic homeostasis and suggest a mechanism in which chronic ER stress underlies fatty liver disease in males.

Hepatic synthesis of triacylglycerols containing medium-chain fatty acids is dominated by diacylglycerol acyltransferase 1 and efficiently inhibited by etomoxir.

OBJECTIVE: Medium-chain fatty acids (MCFAs) play an increasing role in human nutrition. In the liver, one fraction is used for synthesis of MCFA-containing triacylglycerol (MCFA-TG), and the rest is used for oxidative energy production or ketogenesis. We investigated which enzymes catalyse the synthesis of MCFA-TG and how inhibition of MCFA-TG synthesis or fatty acid (FA) oxidation influences the metabolic fate of the MCFAs. METHODS: FA metabolism was followed by time-resolved tracing of alkyne-labelled FAs in freshly isolated mouse hepatocytes. Quantitative data were obtained by mass spectrometry of several hundred labelled lipid species. Wild-type hepatocytes and cells from diacylglycerol acyltransferase (DGAT)1-/- mice were treated with inhibitors against DGAT1, DGAT2, or FA ß-oxidation. RESULTS: Inhibition or deletion of DGAT1 resulted in a reduction of MCFA-TG synthesis by 70%, while long-chain (LC)FA-TG synthesis was reduced by 20%. In contrast, DGAT2 inhibition increased MCFA-TG formation by 50%, while LCFA-TG synthesis was reduced by 5-25%. Inhibition of ß-oxidation by the specific inhibitor teglicar strongly increased MCFA-TG synthesis. In contrast, the widely used ß-oxidation inhibitor etomoxir blocked MCFA-TG synthesis, phenocopying DGAT1 inhibition. CONCLUSIONS: DGAT1 is the major enzyme for hepatic MCFA-TG synthesis. Its loss can only partially be compensated by DGAT2. Specific inhibition of ß-oxidation leads to a compensatory increase in MCFA-TG synthesis, whereas etomoxir blocks both ß-oxidation and MCFA-TG synthesis, indicating a strong off-target effect on DGAT1.

Lipid-Droplet Formation Drives Pathogenic Group 2 Innate Lymphoid Cells in Airway Inflammation.

Innate lymphoid cells (ILCs) play an important role in the control and maintenance of barrier immunity. However, chronic activation of ILCs results in immune-mediated pathology. Here, we show that tissue-resident type 2 ILCs (ILC2s) display a distinct metabolic signature upon chronic activation. In the context of allergen-driven airway inflammation, ILC2s increase their uptake of both external lipids and glucose. Externally acquired fatty acids are transiently stored in lipid droplets and converted into phospholipids to promote the proliferation of ILC2s. This metabolic program is imprinted by interleukin-33 (IL-33) and regulated by the genes Pparg and Dgat1, which are both controlled by glucose availability and mTOR signaling. Restricting dietary glucose by feeding mice a ketogenic diet largely ablated ILC2-mediated airway inflammation by impairing fatty acid metabolism and the formation of lipid droplets. Together, these results reveal that pathogenic ILC2 responses require lipid metabolism and identify ketogenic diet as a potent intervention strategy to treat airway inflammation.

Multiplexed and single cell tracing of lipid metabolism.

Cellular lipid metabolism is a complex network process comprising dozens of enzymes, multiple organelles and more than a thousand lipid species. Tracing metabolic reactions in this network is a major technological and scientific challenge. Using a click-chemistry mass spectrometry reporter strategy, we have developed a specific, highly sensitive and robust tracing procedure for alkyne-labeled lipids. The method enables sample multiplexing, which improves sample comparison. We demonstrate this by a time-resolved analysis of hepatocyte glycerolipid metabolism with parallel quantitative monitoring of 120 labeled lipid species. The subfemtomole sensitivity enabled a single cell analysis of fatty acid incorporation into neutral and membrane lipids. The results demonstrate the robustness of lipid homeostasis at the single cell level.

Localization of 1-deoxysphingolipids to mitochondria induces mitochondrial dysfunction.

1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids that are elevated in the plasma of patients with type 2 diabetes and hereditary sensory and autonomic neuropathy type 1 (HSAN1). Clinically, diabetic neuropathy and HSAN1 are very similar, suggesting the involvement of deoxySLs in the pathology of both diseases. However, very little is known about the biology of these lipids and the underlying pathomechanism. We synthesized an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, to trace the metabolism and localization of deoxySLs. Our results indicate that the metabolism of these lipids is restricted to only some lipid species and that they are not converted to canonical sphingolipids or fatty acids. Furthermore, exogenously added alkyne-doxSA [(2S,3R)-2-aminooctadec-17-yn-3-ol] localized to mitochondria, causing mitochondrial fragmentation and dysfunction. The induced mitochondrial toxicity was also shown for natural doxSA, but not for sphinganine, and was rescued by inhibition of ceramide synthase activity. Our findings therefore indicate that mitochondrial enrichment of an N-acylated doxSA metabolite may contribute to the neurotoxicity seen in diabetic neuropathy and HSAN1. Hence, we provide a potential explanation for the characteristic vulnerability of peripheral nerves to elevated levels of deoxySLs.