Alkylglycerol Monooxygenase Research Articles

Peripheralized sepiapterin reductase inhibition as a safe analgesic therapy.

The development of novel analgesics for chronic pain in the last 2decades has proven virtually intractable, typically failing due to lack of efficacy and dose-limiting side effects. Identified through unbiased gene expression profiling experiments in rats and confirmed by human genome-wide association studies, the role of excessive tetrahydrobiopterin (BH4) in chronic pain has been validated by numerous clinical and preclinical studies. BH4 is an essential cofactor for aromatic amino acid hydroxylases, nitric oxide synthases, and alkylglycerol monooxygenase so a lack of BH4 leads to a range of symptoms in the periphery and central nervous system (CNS). An ideal therapeutic goal therefore would be to block excessive BH4 production, while preventing potential BH4 rundown. In this review, we make the case that sepiapterin reductase (SPR) inhibition restricted to the periphery (i.e., excluded from the spinal cord and brain), is an efficacious and safe target to alleviate chronic pain. First, we describe how different cell types that engage in BH4 overproduction and contribute to pain hypersensitivity, are themselves restricted to peripheral tissues and show their blockade is sufficient to alleviate pain. We discuss the likely safety profile of peripherally restricted SPR inhibition based on human genetic data, the biochemical alternate routes of BH4 production in various tissues and species, and the potential pitfalls to predictive translation when using rodents. Finally, we propose and discuss possible formulation and molecular strategies to achieve peripherally restricted, potent SPR inhibition to treat not only chronic pain but other conditions where excessive BH4 has been demonstrated to be pathological.

Alterations in ether lipid metabolism and the consequences for the mouse lipidome

Alkylglycerol monooxygenase (AGMO) and plasmanylethanolamine desaturase (PEDS1) are enzymes involved in ether lipid metabolism. While AGMO degrades plasmanyl lipids by oxidative cleavage of the ether bond, PEDS1 exclusively synthesizes a specific subclass of ether lipids, the plasmalogens, by introducing a vinyl ether double bond into plasmanylethanolamine phospholipids. Ether lipids are characterized by an ether linkage at the sn-1 position of the glycerol backbone and they are found in membranes of different cell types. Decreased plasmalogen levels have been associated with neurological diseases like Alzheimer's disease. Agmo-deficient mice do not present an obvious phenotype under unchallenged conditions. In contrast, Peds1 knockout mice display a growth phenotype. To investigate the molecular consequences of Agmo and Peds1 deficiency on the mouse lipidome, five tissues from each mouse model were isolated and subjected to high resolution mass spectrometry allowing the characterization of up to 2013 lipid species from 42 lipid subclasses. Agmo knockout mice moderately accumulated plasmanyl and plasmenyl lipid species. Peds1-deficient mice manifested striking changes characterized by a strong reduction of plasmenyl lipids and a concomitant massive accumulation of plasmanyl lipids resulting in increased total ether lipid levels in the analyzed tissues except for the class of phosphatidylethanolamines where total levels remained remarkably constant also in Peds1 knockout mice. The rate-limiting enzyme in ether lipid metabolism, FAR1, was not upregulated in Peds1-deficient mice, indicating that the selective loss of plasmalogens is not sufficient to activate the feedback mechanism observed in total ether lipid deficiency.

Whole genome SNPs among 8 chicken breeds enable identification of genetic signatures that underlie breed features

Many different chicken breeds are found around the world, their features vary among them, and they are valuable resources. Currently, there is a huge lack of knowledge of the genetic determinants responsible for phenotypic and biochemical properties of these breeds of chickens. Understanding the underlying genetic mechanisms that explain across-breed variation can help breeders develop improved chicken breeds. The whole-genomes of 140 chickens from 7 Shandong native breeds and 20 introduced recessive white chickens from China were re-sequenced. Comparative population genomics based on autosomal single nucleotide polymorphisms (SNPs) revealed geographically based clusters among the chickens. Through genome-wide scans for selective sweeps, we identified thyroid stimulating hormone receptor (TSHR, reproductive traits, circadian rhythm), erythrocyte membrane protein band 4.1 like 1 (EPB41L1, body size), and alkylglycerol monooxygenase (AGMO, aggressive behavior), as major candidate breed-specific determining genes in chickens. In addition, we used a machine learning classification model to predict chicken breeds based on the SNPs significantly associated with recourse characteristics, and the prediction accuracy was 92%, which can effectively achieve the breed identification of Laiwu Black chickens. We provide the first comprehensive genomic data of the Shandong indigenous chickens. Our analyses revealed phylogeographic patterns among the Shandong indigenous chickens and candidate genes that potentially contribute to breed-specific traits of the chickens. In addition, we developed a machine learning-based prediction model using SNP data to identify chicken breeds. The genetic basis of indigenous chicken breeds revealed in this study is useful to better understand the mechanisms underlying the resource characteristics of chicken.

Adaptations of the 3T3-L1 adipocyte lipidome to defective ether lipid catabolism upon Agmo knockdown

Little is known about the physiological role of alkylglycerol monooxygenase (AGMO), the only enzyme capable of cleaving the 1-O-alkyl ether bond of ether lipids. Expression and enzymatic activity of this enzyme can be detected in a variety of tissues including adipose tissue. This labile lipolytic membrane-bound protein uses tetrahydrobiopterin as a cofactor, and mice with reduced tetrahydrobiopterin levels have alterations in body fat distribution and blood lipid concentrations. In addition, manipulation of AGMO in macrophages led to significant changes in the cellular lipidome, and alkylglycerolipids, the preferred substrates of AGMO, were shown to accumulate in mature adipocytes. Here, we investigated the roles of AGMO in lipid metabolism by studying 3T3-L1 adipogenesis. AGMO activity was induced over 11 days using an adipocyte differentiation protocol. We show that RNA interference-mediated knockdown of AGMO did not interfere with adipocyte differentiation or affect lipid droplet formation. Furthermore, lipidomics revealed that plasmalogen phospholipids were preferentially accumulated upon Agmo knockdown, and a significant shift toward longer and more polyunsaturated acyl side chains of diacylglycerols and triacylglycerols could be detected by mass spectrometry. Our results indicate that alkylglycerol catabolism has an influence not only on ether-linked species but also on the degree of unsaturation in the massive amounts of triacylglycerols formed during in vitro 3T3-L1 adipocyte differentiation.

Priapism caused by partial deficiency of tetrahydrobiopterin through hypofunction of the sympathetic neurons in sepiapterin reductase gene‐disrupted mice

6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor for aromatic L-amino acid hydroxylases, including tyrosine hydroxylase (TH), alkylglycerol monooxygenase, and three types of nitric oxide (NO) synthases (NOS). Sepiapterin reductase (SPR) catalyzes the third step of BH4 biosynthesis. SPR gene-disrupted (Spr-/- ) mice exhibit a dystonic posture, low body weight, hyperphenylalaninemia, and unstable hypertension with endothelial dysfunction. In this study, we found that Spr-/- mice suffered from a high incidence of severe priapism. Their erections persisted for months. The biopterin, BH4, and norepinephrine contents, and TH protein levels in the penile tissue of Spr-/- mice without and with priapism were significantly reduced compared to those of Spr+/+ mice. In contrast, their neural NOS (nNOS) protein levels were increased, and the cyclic guanosine monophosphate (cGMP) levels were remarkably elevated in the penises of Spr-/- mice with priapism. The symptoms were relieved by repeated administration of BH4. The biopterin, BH4, and norepinephrine contents were increased in penile homogenates from BH4-supplemented Spr-/- mice, and the TH protein levels tended to increase, and their nitrite plus nitrate levels were significantly lower than those of vehicle-treated Spr-/- mice and were approximately the same as vehicle- and BH4-supplemented Spr+/+ mice. Thus, we deduced that the priapism of Spr-/- mice is primarily caused by hypofunction of the sympathetic neurons due to cofactor depletion and the loss of TH protein and, further, dysregulation of the NO/cGMP signaling pathway, which would be caused by disinhibition of nNOS-containing neurons and/or abnormal catabolism of cyclic nucleotides is suggested.

AGMO Inhibitor Reduces 3T3-L1 Adipogenesis.

Alkylglycerol monooxygenase (AGMO) is a tetrahydrobiopterin (BH4)-dependent enzyme with major expression in the liver and white adipose tissue that cleaves alkyl ether glycerolipids. The present study describes the disclosure and biological characterization of a candidate compound (Cp6), which inhibits AGMO with an IC50 of 30–100 µM and 5–20-fold preference of AGMO relative to other BH4-dependent enzymes, i.e., phenylalanine-hydroxylase and nitric oxide synthase. The viability and metabolic activity of mouse 3T3-L1 fibroblasts, HepG2 human hepatocytes and mouse RAW264.7 macrophages were not affected up to 10-fold of the IC50. However, Cp6 reversibly inhibited the differentiation of 3T3-L1 cells towards adipocytes, in which AGMO expression was upregulated upon differentiation. Cp6 reduced the accumulation of lipid droplets in adipocytes upon differentiation and in HepG2 cells exposed to free fatty acids. Cp6 also inhibited IL-4-driven differentiation of RAW264.7 macrophages towards M2-like macrophages, which serve as adipocyte progenitors in adipose tissue. Collectively, the data suggest that pharmacologic AGMO inhibition may affect lipid storage.

When the genome bluffs: a tandem duplication event during generation of a novel Agmo knockout mouse model fools routine genotyping

BackgroundGenome editing in mice using either classical approaches like homologous recombination or CRISPR/Cas9 has been reported to harbor off target effects (insertion/deletion, frame shifts or gene segment duplications) that lead to mutations not only in close proximity to the target site but also outside. Only the genomes of few engineered mouse strains have been sequenced. Since the role of the ether-lipid cleaving enzyme alkylglycerol monooxygenase (AGMO) in physiology and pathophysiology remains enigmatic, we created a knockout mouse model for AGMO using EUCOMM stem cells but unforeseen genotyping issues that did not agree with Mendelian distribution and enzyme activity data prompted an in-depth genomic validation of the mouse model.ResultsWe report a gene segment tandem duplication event that occurred during the generation of an Agmo knockout-first allele by homologous recombination. Only low homology was seen between the breakpoints. While a single copy of the recombinant 18 kb cassette was integrated correctly around exon 2 of the Agmo gene, whole genome nanopore sequencing revealed a 94 kb duplication in the Agmo locus that contains Agmo wild-type exons 1–3. The duplication fooled genotyping by routine PCR, but could be resolved using qPCR-based genotyping, targeted locus amplification sequencing and nanopore sequencing. Despite this event, this Agmo knockout mouse model lacks AGMO enzyme activity and can therefore be used to study its physiological role.ConclusionsA duplication event occurred at the exact locus of the homologous recombination and was not detected by conventional quality control filters such as FISH or long-range PCR over the recombination sites. Nanopore sequencing provides a cost convenient method to detect such underrated off-target effects, suggesting its use for additional quality assessment of gene editing in mice and also other model organisms.

The Emerging Physiological Role of AGMO 10 Years after Its Gene Identification.

The gene encoding alkylglycerol monooxygenase (AGMO) was assigned 10 years ago. So far, AGMO is the only known enzyme capable of catalysing the breakdown of alkylglycerols and lyso-alkylglycerophospholipids. With the knowledge of the genetic information, it was possible to relate a potential contribution for mutations in the AGMO locus to human diseases by genome-wide association studies. A possible role for AGMO was implicated by genetic analyses in a variety of human pathologies such as type 2 diabetes, neurodevelopmental disorders, cancer, and immune defence. Deficient catabolism of stored lipids carrying an alkyl bond by an absence of AGMO was shown to impact on the overall lipid composition also outside the ether lipid pool. This review focuses on the current evidence of AGMO in human diseases and summarises experimental evidence for its role in immunity, energy homeostasis, and development in humans and several model organisms. With the progress in lipidomics platform and genetic identification of enzymes involved in ether lipid metabolism such as AGMO, it is now possible to study the consequence of gene ablation on the global lipid pool and further on certain signalling cascades in a variety of model organisms in more detail.

Genome diversity of Chinese indigenous chicken and the selective signatures in Chinese gamecock chicken

Gamecock chickens are one of the earliest recorded birds in China, and have accumulated some unique morphological and behavioral signatures such as large body size, muscularity and aggressive behavior, whereby being excellent breeding materials and a good model for studying bird muscular development and behavior. In this study, we sequenced 126 chicken genomes from 19 populations, including four commercial chicken breeds that are commonly farmed in China, 13 nationwide Chinese typical indigenous chicken breeds (including two Chinese gamecock breeds), one red jungle fowl from Guangxi Province of China and three gamecock chickens from Laos. Combined with 31 published chicken genomes from three populations, a comparative genomics analysis was performed across 157 chickens. We found a severe confounding effect on potential cold adaptation exerted by introgression from commercial chickens into Chinese indigenous chickens, and argued that the genetic introgression from commercial chickens into indigenous chickens should be seriously considered for identifying selection footprint in indigenous chickens. LX gamecock chickens might have played a core role in recent breeding and conservation of other Chinese gamecock chickens. Importantly, AGMO (Alkylglycerol monooxygenase) and CPZ (Carboxypeptidase Z) might be crucial for determining the behavioral pattern of gamecock chickens, while ISPD (Isoprenoid synthase domain containing) might be essential for the muscularity of gamecock chickens. Our results can further the understanding of the evolution of Chinese gamecock chickens, especially the genetic basis of gamecock chickens revealed here was valuable for us to better understand the mechanisms underlying the behavioral pattern and the muscular development in chicken.

Alkylglycerol monooxygenase, a heterotaxy candidate gene, regulates left-right patterning via Wnt signaling

Congenital heart disease (CHD) is a major cause of morbidity in the pediatric population yet its genetic and molecular causes remain poorly defined. Previously, we identified AGMO as a candidate heterotaxy disease gene, a disorder of left-right (LR) patterning that can have a profound effect on cardiac function. AGMO is the only known alkylglycerol monooxygenase, an orphan tetrahydrobiopterin dependent enzyme that cleaves the ether linkage in alkylglycerols. However, whether AGMO plays a role in LR patterning was unexplored. Here we reveal that Agmo is required for correct development of the embryonic LR axis in Xenopus embryos recapitulating the patient’s heterotaxy phenotype. Mechanistically, we demonstrate that Agmo is a regulator of canonical Wnt signaling, required during gastrulation for normal formation of the left – right organizer. Mutational analysis demonstrates that this function is dependent on Agmo’s alkylglycerol monooxygenase activity. Together, our findings identify Agmo as a regulator of canonical Wnt signaling, demonstrate a role for Agmo in embryonic axis formation, and provide insight into the poorly understood developmental requirements for ether lipid cleavage.

Alternative splicing of the Wnt trafficking protein, Wntless and its effects on protein-protein interactions

BackgroundWntless (Wls) is a protein that regulates secretion of Wnt signaling molecules from Wnt-producing cells. Wnt signaling is known to be critical for several developmental and homeostatic processes. However, Wnt-independent functions of Wls are now being elucidated. Primates express an alternative splice variant of Wls (here termed WlsX). WlsX contains an alternatively spliced COOH-terminus, and does not appear to be able to sustain significant levels of WNT secretion because of its inability to undergo retrograde trafficking to the endoplasmic reticulum. The functional significance for this alternatively spliced form of Wls has not yet been elucidated. We previously identified a cohort of Wls interacting proteins using a combination of yeast 2-hybrid and candidate gene approaches.ResultsIn the present study, we analyzed the interaction of WlsX with previously identified Wls interactors, and additionally screened for novel protein interactors of WlsX utilizing a membrane yeast two hybrid screen. Three novel Wls interactors, Glycoprotein M6A (GPM6A), Alkylglycerol Monooxygenase (AGMO), and ORAI1 were identified. Each of these novel WlsX interactors, as well as all other Wls interacting proteins identified previously, with the exception of the mu-opioid receptor, were found to interact with both Wls and WlsX splice forms. We show that WlsX can form homodimers, but that WlsX may not interact with Wls.ConclusionsWlsX has the ability to form homodimers and to interact with most known Wls interacting proteins. Taken together, our results suggest that Wls and WlsX may have overlapping, but distinct functions, including sensitivity to opioid drugs. While studies have focused on the ability of Wls interacting proteins to affect Wnt secretion, future efforts will explore the reciprocal regulation of these proteins by Wls, possibly via Wnt-independent mechanisms.

Lipodisqs for eukaryote lipidomics with retention of viability: Sensitivity and resistance to Leucobacter infection linked to C.elegans cuticle composition.

Lipodisq™ nanoparticles have been used to extract surface lipids from the cuticle of two strains (wild type, N2 and the bacteria-resistant strain, agmo-1) of the C. elegans nematode without loss of viability. The extracted lipids were characterized by thin layer chromatography and MALDI-TOF-MS. The lipid profiles differed between the two strains. The extracted lipids from the bacteria-resistant strain, agmo-1, contained ether-linked (O-alkyl chain) lipids, in contrast to the wild-type strain which contained exclusively ester- linked (O-acyl) lipids. This observation is consistent with the loss of a functional alkylglycerol monooxygenase (AGMO) in the bacterial resistant strain agmo-1. The presence and abundance of other lipid species also differs between the wild-type N2 and agmo-1 nematodes, suggesting that the agmo-1 mutant strain attempts to compensate for the increase in ether-linked lipids by modulating other lipid-synthesis pathways. Together these differences not only affect the fragility of the cuticle and the buoyancy of the worm in aqueous buffer, but also interactions with surface-adhering bacteria. The much greater chemical stability of O-alkyl, non-hydrolysable linked lipids compared with hydrolysable O-acyl linked lipids, may be the origin of the resistance of the agmo-1 strain to bacterial infection, providing a more resilient cuticle for the nematode. Additionally, we show that lipid extraction with a polymer of styrene and maleic acid (SMA) provides a viable route to lipidomics studies with minimal perturbation of the organism.

Influence of Genetic Polymorphism Towards Pulmonary Tuberculosis Susceptibility.

Tuberculosis (TB) is still remains the major threat for human health worldwide. Several case-control, candidate-gene, family studies and genome-wide association studies (GWAS) suggested the association of host genetic factors to TB susceptibility or resistance in various ethnic populations. Moreover, these factors modulate the host immune responses to tuberculosis. Studies have reported genetic markers to predict TB development in human leukocyte antigen (HLA) and non-HLA genes like killer immunoglobulin-like receptor (KIR), toll-like receptors (TLRs), cytokine/chemokines and their receptors, vitamin D receptor (VDR) and SLC11A1 etc. Highly polymorphic HLA loci may influence antigen presentation specificities by modifying peptide binding motifs. The recent meta-analysis studies revealed the association of several HLA alleles in particular class II HLA-DRB1 with TB susceptibility and valuable marker for disease development especially in Asian populations. Case-control studies have found the association of HLA-DR2 in some populations, but not in other populations, this could be due to an ethnic specific association of gene variants. Recently, GWAS conducted in case-control and family based studies in Russia, Chinese Han, Morocco, Uganda and Tanzania revealed the association of genes such as ASAP1, Alkylglycerol monooxygenase (AGMO), Forkhead BoxP1 (FOXP1), C-terminal domain phosphatase 1 (UBLCP1) and intergenic SNP rs932347C/T with TB. Whereas, SNP rs10956514A/G were not associated with TB in western Chinese Han and Tibetan population. In this review, we summarize the recent findings of genetic variants with susceptibility/resistance to TB.

Regulation of Tetrahydrobiopterin (BH4) Synthesis in the Nematode C. elegans

5,6,7,8‐tetrahydrobiopterin (BH4) is a cofactor required by the aromatic amino acid hydroxylases (AAAHs), nitric oxide synthases, and alkylglycerol monooxygenase (Werner et al., 2011, Biochem. J. 438: 397). Among the AAAHs are the rate‐limiting synthetic enzymes for serotonin and dopamine synthesis, and for conversion of phenylalanine (Phe) to tyrosine. In humans, the activity of phenylalanine hydroxylase (PAH) is essential for Phe catabolism; dysfunction of PAH, either from mutations in the PAH gene or in genes required for BH4 synthesis, results in phenylketonuria. Regulation of BH4 levels is important for many other physiological functions. The first and rate‐limiting step in BH4 de novo synthesis is performed by GTP Cyclohydrolase I (GTPCH1). Like in many biosynthesis pathways, this first enzyme is regulated by ‘end product feedback inhibition’ whereby BH4 inhibits GTPCH1 activity. In mammals, BH4 does not bind to GTPCH1 alone, but requires an additional protein called GTPCH1 feedback regulatory protein (GFRP) to inhibit GTPCH1. Crystal structures of rat GTPCH1 and GFRP with the BH4 analog BH2 show that the biopterin binds at the interface between the proteins (Maita et al., 2004, J Biol Chem 279: 51534). Phe increases BH4 production via GTPCH1 stimulation, also in a GFRP‐dependent fashion, similarly binding at the interface between GTPCH1 and GFRP. We have recently described the function and expression of genes required for BH4 synthesis and regeneration in the nematode C. elegans (Loer et al., 2015, Genetics 200: 237). Here we present work on the function of the gene gfrp‐1, which encodes a ortholog of mammalian GFRP, in the regulation of BH4 synthesis in C. elegans. Sequence analysis and structural predictions suggest the worm GFRP will function like the mammalian protein, binding both BH4 and Phe. In mammals, GTPCH1 and GFRP can also bind the selective inhibitor 2,4‐Diamino‐6‐hydroxypyrimidine (DAHP), which is structurally similar to both GTP (substrate) and BH4 (pathway end product). At low concentrations, DAHP apparently inhibits GTPCH1 like BH4 by binding with GFRP; this inhibition is GFRP‐dependent. At higher concentrations, DAHP acts as a competitive inhibitor, binding at the GTPCH1 active site like GTP; this inhibition is GFRP‐independent (Xie et al., 1998, J Biol Chem 273: 21091). Using a reduction‐of‐function mutant in the worm GTPCH1 gene [cat‐4(e3015) – S148N], we find that DAHP reduces BH4 production, as assessed by a significant reduction in serotonin immunoreactivity in whole worms. Treatment with Phe significantly increases serotonin levels in cat‐4(e3015) worms. We are currently assessing the effects of mutations in gfrp‐1 on serotonin levels (hence BH4 levels) in single mutants and cat‐4; gfrp‐1 double mutants, and whether the effects of DAHP and Phe on BH4 synthesis are GFRP‐dependent.Support or Funding InformationSupported by a Univ of San Diego Faculty Research Grant and an endowment from the Fletcher Jones Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oxidative cyclization of prodigiosin by an alkylglycerol monooxygenase-like enzyme.

Prodiginines, tripyrrole alkaloids displaying a wide array of bioactivities, occur as linear and cyclic congeners. Identification of an unclustered biosynthetic gene led to the discovery of the enzyme responsible for catalyzing the regiospecific C–H activation and cyclization of prodigiosin to form cycloprodigiosin in Pseudoalteromonas rubra. This enzyme is closely related to alkylglycerol monooxygenase, and unrelated to RedG, the Rieske oxygenase that produces cyclized prodiginines in Streptomyces, implying convergent evolution.

Exome Sequencing Identifies Two Variants of the Alkylglycerol Monooxygenase Gene as a Cause of Relapses in Visceral Leishmaniasis in Children, in Sudan.

Visceral leishmaniasis (kala-azar, KA) is the most severe form of leishmaniasis, characterized by fever, weight loss, hepatosplenomegaly, and lymphadenopathy. During an outbreak of KA in Babar El Fugara (Sudan), 5.7% of cured patients displayed relapses, with familial clustering in half the cases. We performed whole-exome sequencing on 10 relapsing individuals and 11 controls from 5 nuclear families. Rare homozygous and compound-heterozygous nonsense (c.1213C > T, rs139309795, p.Arg405*) and missense (c.701A > G, rs143439626, p.Lys234Arg) mutations of the alkylglycerol monooxygenase (AGMO) gene were associated with KA relapse in 3 families. Sequencing in additional family members confirmed the segregation of these mutations with relapse and revealed an autosomal dominant mode of transmission. These mutations were detected heterozygous in 2 subjects among 100 unrelated individuals with KA who never relapsed after cure, suggesting incomplete penetrance of AGMO deficiency. AGMO is expressed in hematopoietic cells, and is strongly expressed in the liver. AGMO modulates PAF production by mouse macrophages, suggesting that it may act through the PAF/PAF receptor pathway previously shown to have anti-Leishmania activity. This is the first demonstration that relapses after a first episode of KA are due to differences in human genetic susceptibility and not to modifications of parasite pathogenicity.

Neuropeptide FF increases M2 activation and self-renewal of adipose tissue macrophages.

The quantity and activation state of adipose tissue macrophages (ATMs) impact the development of obesity-induced metabolic diseases. Appetite-controlling hormones play key roles in obesity; however, our understanding of their effects on ATMs is limited. Here, we have shown that human and mouse ATMs express NPFFR2, a receptor for the appetite-reducing neuropeptide FF (NPFF), and that NPFFR2 expression is upregulated by IL-4, an M2-polarizing cytokine. Plasma levels of NPFF decreased in obese patients and high-fat diet-fed mice and increased following caloric restriction. NPFF promoted M2 activation and increased the proliferation of murine and human ATMs. Both M2 activation and increased ATM proliferation were abolished in NPFFR2-deficient ATMs. Mechanistically, the effects of NPFF involved the suppression of E3 ubiquitin ligase RNF128 expression, resulting in enhanced stability of phosphorylated STAT6 and increased transcription of the M2 macrophage-associated genes IL-4 receptor α (Il4ra), arginase 1 (Arg1), IL-10 (Il10), and alkylglycerol monooxygenase (Agmo). NPFF induced ATM proliferation concomitantly with the increase in N-Myc downstream-regulated gene 2 (Ndrg2) expression and suppressed the transcription of Ifi200 cell-cycle inhibitor family members and MAF bZIP transcription factor B (Mafb), a negative regulator of macrophage proliferation. NPFF thus plays an important role in supporting healthy adipose tissue via the maintenance of metabolically beneficial ATMs.

Neuropeptide FF increases M2 activation and self-renewal of adipose tissue macrophages.

The quantity and activation state of adipose tissue macrophages (ATMs) impact the development of obesity-induced metabolic diseases. Appetite-controlling hormones play key roles in obesity; however, our understanding of their effects on ATMs is limited. Here, we have shown that human and mouse ATMs express NPFFR2, a receptor for the appetite-reducing neuropeptide FF (NPFF), and that NPFFR2 expression is upregulated by IL-4, an M2-polarizing cytokine. Plasma levels of NPFF decreased in obese patients and high-fat diet-fed mice and increased following caloric restriction. NPFF promoted M2 activation and increased the proliferation of murine and human ATMs. Both M2 activation and increased ATM proliferation were abolished in NPFFR2-deficient ATMs. Mechanistically, the effects of NPFF involved the suppression of E3 ubiquitin ligase RNF128 expression, resulting in enhanced stability of phosphorylated STAT6 and increased transcription of the M2 macrophage-associated genes IL-4 receptor α (Il4ra), arginase 1 (Arg1), IL-10 (Il10), and alkylglycerol monooxygenase (Agmo). NPFF induced ATM proliferation concomitantly with the increase in N-Myc downstream-regulated gene 2 (Ndrg2) expression and suppressed the transcription of Ifi200 cell-cycle inhibitor family members and MAF bZIP transcription factor B (Mafb), a negative regulator of macrophage proliferation. NPFF thus plays an important role in supporting healthy adipose tissue via the maintenance of metabolically beneficial ATMs.

Organic anion transporters, OAT1 and OAT3, are crucial biopterin transporters involved in bodily distribution of tetrahydrobiopterin and exclusion of its excess

Tetrahydrobiopterin (BH4) is a common coenzyme of phenylalanine-, tyrosine-, and tryptophan hydroxylases, alkylglycerol monooxygenase, and NO synthases (NOS). Synthetic BH4 is used medicinally for BH4-responsive phenylketonuria and inherited BH4 deficiency. BH4 supplementation has also drawn attention as a therapy for various NOS-related cardio-vascular diseases, but its use has met with limited success in decreasing BH2, the oxidized form of BH4. An increase in the BH2/BH4 ratio leads to NOS dysfunction. Previous studies revealed that BH4 supplementation caused a rapid urinary loss of BH4 accompanied by an increase in the blood BH2/BH4 ratio and an involvement of probenecid-sensitive but unknown transporters was strongly suggested in these processes. Here we show that OAT1 and OAT3 enabled cells to take up BP (BH4 and/or BH2) in a probenecid-sensitive manner using rat kidney slices and transporter-expressing cell systems, LLC-PK1 cells and Xenopus oocytes. Both OAT1 and OAT3 preferred BH2 and sepiapterin as their substrate roughly 5- to 10-fold more than BH4. Administration of probenecid acutely reduced the urinary exclusion of endogenous BP accompanied by a rise in blood BP in vivo. These results indicated that OAT1 and OAT3 played crucial roles: (1) in determining baseline levels of blood BP by excluding endogenous BP through the urine, (2) in the rapid distribution to organs of exogenous BH4 and the exclusion to urine of a BH4 excess, particularly when BH4 was administered, and (3) in scavenging blood BH2 by cellular uptake as the gateway to the salvage pathway of BH4, which reduces BH2 back to BH4.

The alkylglycerol monooxygenase (AGMO) gene previously involved in autism also causes a novel syndromic form of primary microcephaly in a consanguineous Saudi family

Autosomal recessive primary microcephaly (MCPH) refers to a genetically heterogeneous group of neurodevelopmental disorders in which patients exhibit a marked decrease in occipitofrontal head circumference at birth and a variable degree of intellectual disability. To date, 18 genes have been reported for MCPH worldwide. We enrolled a consanguineous family from Saudi Arabia presenting with primary microcephaly, developmental delay, short stature and intellectual disability. Whole exome sequencing (WES) with 100× coverage was performed on two affected siblings after defining common regions of homozygosity through genome-wide single nucleotide polymorphism (SNP) microarray genotyping. WES data analysis, confirmed by subsequent Sanger sequence validation, identified a novel homozygous deletion mutation (c.967delA; p.Glu324Lysfs12*) in exon 10 of the alkylglycerol monooxygenase (AGMO) gene on chromosome 7p21.2. Population screening of 178 ethnically matched control chromosomes and consultation of the Exome Aggregation Consortium database, containing 60,706 individuals' exomes worldwide, confirmed that this mutation was not present outside the family. To the best of our knowledge, this is the first evidence of an AGMO mutation underlying primary microcephaly and intellectual disability in humans. Our findings further expand the genetic heterogeneity of MCPH in familial cases.