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Agpat5 (1-acylglycerol-3-phosphate O-acyltransferase 5) is a broadly expressed lipid regulatory enzyme involved in glycerophospholipid metabolism. Multiple genetic studies in mice and humans have identified that Agpat5 is associated with plasma insulin, cholesterol, and alanine aminotransferase levels. Despite the strong genetic evidence on Agpat5, no study has investigated its liver-specific role in physiology. Here, we conducted a series of metabolic studies under four distinct dietary conditions to assess the impact of liver-specific Agpat5 deletion on plasma insulin levels, glucose tolerance, plasma cholesterol levels, and hepatic steatosis. Liver-specific deletion of Agpat5 did not affect plasma insulin levels, glucose tolerance, plasma cholesterol levels, or hepatic steatosis in mice fed a chow diet, high-fat diet, or Western diet. However, when mice consumed a chow diet combined with liquid sucrose, liver-specific deletion of Agpat5 resulted in significantly decreased plasma insulin levels and improved glucose tolerance without alterations in body weight or fat mass. Using global lipidomics, we identified that Agpat5 specifically modulated levels of phosphatidylglycerol and cardiolipin within the livers of mice consuming liquid sucrose. Overall, our findings indicate a liver-specific role of Agpat5 in contributing to hyperinsulinemia and glucose tolerance in the absence of body weight changes when consuming liquid sucrose., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Brian W. Parks reports financial support was provided by National Heart Lung and Blood Institute. Judith A. Simcox reports financial support was provided by Howard Hughes Medical Institute. Judith A. Simcox reports financial support was provided by Juvenile Diabetes Research Foundation Limited. Judith A. Simcox reports financial support was provided by National Institute of Diabetes and Digestive and Kidney Diseases. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The availability of genome-wide transcriptomic and proteomic datasets is ever-increasing and often not used beyond initial publication. Here, we applied module-based coexpression network analysis to a comprehensive catalog of 35 mouse genome-wide liver expression datasets (encompassing more than 3800 mice) with the goal of identifying and validating unknown genes involved in cholesterol metabolism. From these 35 datasets, we identified a conserved module of genes enriched with cholesterol biosynthetic genes. Using a systematic approach across the 35 datasets, we identified three genes (Rdh11, Echdc1, and Aldoc) with no known role in cholesterol metabolism. We then performed functional validation studies and show that each gene is capable of regulating cholesterol metabolism. For the glycolytic gene, Aldoc, we demonstrate that it contributes to de novo cholesterol biosynthesis and regulates cholesterol and triglyceride levels in mice. As Aldoc is located within a genome-wide significant genome-wide association studies locus for human plasma cholesterol levels, our studies establish Aldoc as a causal gene within this locus. Through our work, we develop a framework for leveraging mouse genome-wide liver datasets for identifying and validating genes involved in cholesterol metabolism., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

We and others have previously shown that genetic association can be used to make causal connections between gene loci and small molecules measured by mass spectrometry in the bloodstream and in tissues. We identified a locus on mouse chromosome 7 where several phospholipids in liver showed strong genetic association to distinct gene loci. In this study, we integrated gene expression data with genetic association data to identify a single gene at the chromosome 7 locus as the driver of the phospholipid phenotypes. The gene encodes α/β-hydrolase domain 2 (Abhd2), one of 23 members of the ABHD gene family. We validated this observation by measuring lipids in a mouse with a whole-body deletion of Abhd2. The Abhd2KO mice had a significant increase in liver levels of phosphatidylcholine and phosphatidylethanolamine. Unexpectedly, we also found a decrease in two key mitochondrial lipids, cardiolipin and phosphatidylglycerol, in male Abhd2KO mice. These data suggest that Abhd2 plays a role in the synthesis, turnover, or remodeling of liver phospholipids., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Price et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Thermoneutral housing has been shown to promote more accurate and robust development of several pathologies in mice. Raising animal housing temperatures a few degrees may create a relatively straightforward opportunity to improve translatability of mouse models. In this commentary, we discuss the changes of physiology induced in mice housed at thermoneutrality, and review techniques for measuring systemic thermogenesis, specifically those affecting storage and mobilization of lipids in adipose depots. Environmental cues are a component of the information integrated by the brain to calculate food consumption and calorie deposition. We show that relative humidity is one of those cues, inducing a rapid sensory response that is converted to a more chronic susceptibility to obesity. Given high inter-institutional variability in the regulation of relative humidity, study reproducibility may be improved by consideration of this factor. We evaluate a "humanized" environmental cycling protocol, where mice sleep in warm temperature housing, and are cool during the wake cycle. We show that this protocol suppresses adaptation to cool exposure, with consequence for adipose-associated lipid storage. To evaluate systemic cues in mice housed at thermoneutral temperatures, we characterized the circulating lipidome, and show that sera are highly depleted in some HDL-associated phospholipids, specifically phospholipids containing the essential fatty acid, 18:2 linoleic acid, and its derivative, arachidonic acid (20:4) and related ether-phospholipids. Given the role of these fatty acids in inflammatory responses, we propose they may underlie the differences in disease progression observed at thermoneutrality., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Objective: To investigate the role of adipocyte Pcpe2 (procollagen C-endopeptidase enhancer 2) in SR-BI (scavenger receptor class BI)-mediated HDL-C (high-density lipoprotein cholesterol) uptake and contributions to adipose lipid storage., Approach and Results: Pcpe2, a glycoprotein devoid of intrinsic proteolytic activity, is believed to participate in extracellular protein-protein interactions, supporting SR-BI- mediated HDL-C uptake. In published studies, Pcpe2 deficiency increased the development of atherosclerosis by reducing SR-BI-mediated HDL-C catabolism, but the biological impact of this deficiency on adipocyte SR-BI-mediated HDL-C uptake is unknown. Differentiated cells from Ldlr-/-/Pcpe2-/- (Pcpe2-/-) mouse adipose tissue showed elevated SR-BI protein levels, but significantly reduced HDL-C uptake compared to Ldlr-/- (control) adipose tissue. SR-BI-mediated HDL-C uptake was restored by preincubation of cells with exogenous Pcpe2. In diet-fed mice lacking Pcpe2, significant reductions in visceral, subcutaneous, and brown adipose tissue mass were observed, despite elevations in plasma triglyceride and cholesterol concentrations. Significant positive correlations exist between adipose mass and Pcpe2 expression in both mice and humans., Conclusions: Overall, these findings reveal a novel and unexpected function for Pcpe2 in modulating SR-BI expression and function as it relates to adipose tissue expansion and cholesterol balance in both mice and humans.

Purpose of Review: More than one hundred loci have been identified from human genome-wide association studies (GWAS) for blood lipids. Despite the success of GWAS in identifying loci, subsequent prioritization of causal genes related to these loci remains a challenge. To address this challenge, recent work suggests that candidate causal genes within loci can be prioritized through cross-species integration using genome-wide data from the mouse., Recent Findings: Mouse model systems provide unparalleled access to primary tissues, like the liver, that are not readily available for human studies. Given the key role the liver plays in controlling blood lipid levels and the wealth of liver genome-wide transcript and protein data available in the mouse, these data can be leveraged. Using coexpression network analysis approaches with mouse genome-wide data, coupled with cross-species analysis of human lipid GWAS, causal genes within lipid loci can be prioritized. Prioritization through both mouse and human along with biochemical validation provide a systematic and valuable method to discover lipid metabolism genes., Summary: The prioritization of causal lipid genes within GWAS loci is a challenging process requiring a multidisciplinary approach. Integration of data types across species, such as the mouse, can aid in causal gene prioritization., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

To elucidate the contributions of specific lipid species to metabolic traits, we integrated global hepatic lipid data with other omics measures and genetic data from a cohort of about 100 diverse inbred strains of mice fed a high-fat/high-sucrose diet for 8 weeks. Association mapping, correlation, structure analyses, and network modeling revealed pathways and genes underlying these interactions. In particular, our studies lead to the identification of Ifi203 and Map2k6 as regulators of hepatic phosphatidylcholine homeostasis and triacylglycerol accumulation, respectively. Our analyses highlight mechanisms for how genetic variation in hepatic lipidome can be linked to physiological and molecular phenotypes, such as microbiota composition., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Background & Aims: Liver fibrosis is a multifactorial trait that develops in response to chronic liver injury. Our aim was to characterize the genetic architecture of carbon tetrachloride (CCl 4 )-induced liver fibrosis using the Hybrid Mouse Diversity Panel, a panel of more than 100 genetically distinct mouse strains optimized for genome-wide association studies and systems genetics., Methods: Chronic liver injury was induced by CCl 4 injections twice weekly for 6 weeks. Four hundred thirty-seven mice received CCl 4 and 256 received vehicle, after which animals were euthanized for liver histology and gene expression. Using automated digital image analysis, we quantified fibrosis as the collagen proportionate area of the whole section, excluding normal collagen., Results: We discovered broad variation in fibrosis among the Hybrid Mouse Diversity Panel strains, demonstrating a significant genetic influence. Genome-wide association analyses revealed significant and suggestive loci underlying susceptibility to fibrosis, some of which overlapped with loci identified in mouse crosses and human population studies. Liver global gene expression was assessed by RNA sequencing across the strains, and candidate genes were identified using differential expression and expression quantitative trait locus analyses. Gene set enrichment analyses identified the underlying pathways, of which stellate cell involvement was prominent, and coexpression network modeling identified modules associated with fibrosis., Conclusions: Our results provide a rich resource for the design of experiments to understand mechanisms underlying fibrosis and for rational strain selection when testing antifibrotic drugs., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Identifying the causal gene(s) that connects genetic variation to a phenotype is a challenging problem in genome-wide association studies (GWASs). Here, we develop a systematic approach that integrates mouse liver co-expression networks with human lipid GWAS data to identify regulators of cholesterol and lipid metabolism. Through our approach, we identified 48 genes showing replication in mice and associated with plasma lipid traits in humans and six genes on the X chromosome. Among these 54 genes, 25 have no previously identified role in lipid metabolism. Based on functional studies and integration with additional human lipid GWAS datasets, we pinpoint Sestrin1 as a causal gene associated with plasma cholesterol levels in humans. Our validation studies demonstrate that Sestrin1 influences plasma cholesterol in multiple mouse models and regulates cholesterol biosynthesis. Our results highlight the power of combining mouse and human datasets for prioritization of human lipid GWAS loci and discovery of lipid genes., Competing Interests: Declaration of Interests D.W.L. has received funding from, and is a scientific advisory board member of, Aeonian Pharmaceuticals, which seeks to develop novel, selective mTOR inhibitors for the treatment of various diseases., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Responses to a high fat, high sucrose (HFHS) diet vary greatly among inbred strains of mice. We sought to examine the epigenetic (DNA methylation) changes underlying these differences as well as variation in weight loss when switched to a low-fat chow diet. We surveyed DNA methylation from livers of 45 inbred mouse strains fed a HFHS diet for 8 weeks using reduced-representation bisulfite sequencing (RRBS). We observed a total of 1,045,665 CpGs of which 83 candidate sites were significantly associated with HFHS diet. Many of these CpGs correlated strongly with gene expression or clinical traits such as body fat percentage and plasma glucose. Five inbred strains were then studied in the context of weight loss to test for evidence of epigenetic "memory." The mice were first fed a HFHS diet for 6 weeks followed by a low-fat chow diet for 4 weeks. Four of the five strains returned to initial levels of body fat while one strain, A/J, retained almost 50% of the fat gained. A total of 36 of the HFHS diet responsive CpGs exhibited evidence of persistent epigenetic modifications following weight normalization, including CpGs near the genes Scd1 and Cdk1 . Our study identifies DNA methylation changes in response to a HFHS diet challenge that revert more slowly than overall body fat percentage in weight loss and provides evidence for epigenetic mediated "memory.", (Copyright © 2019 Edillor, Parks, Mehrabian, Lusis and Pellegrini.)

Deleterious changes in energy metabolism have been linked to aging and disease vulnerability, while activation of mitochondrial pathways has been linked to delayed aging by caloric restriction (CR). The basis for these associations is poorly understood, and the scope of impact of mitochondrial activation on cellular function has yet to be defined. Here, we show that mitochondrial regulator PGC-1a is induced by CR in multiple tissues, and at the cellular level, CR-like activation of PGC-1a impacts a network that integrates mitochondrial status with metabolism and growth parameters. Transcriptional profiling reveals that diverse functions, including immune pathways, growth, structure, and macromolecule homeostasis, are responsive to PGC-1a. Mechanistically, these changes in gene expression were linked to chromatin remodeling and RNA processing. Metabolic changes implicated in the transcriptional data were confirmed functionally including shifts in NAD metabolism, lipid metabolism, and membrane lipid composition. Delayed cellular proliferation, altered cytoskeleton, and attenuated growth signaling through post-transcriptional and post-translational mechanisms were also identified as outcomes of PGC-1a-directed mitochondrial activation. Furthermore, in vivo in tissues from a genetically heterogeneous mouse population, endogenous PGC-1a expression was correlated with this same metabolism and growth network. These data show that small changes in metabolism have broad consequences that arguably would profoundly alter cell function. We suggest that this PGC-1a sensitive network may be the basis for the association between mitochondrial function and aging where small deficiencies precipitate loss of function across a spectrum of cellular activities., (© 2019 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

We studied sex differences in over 50 cardio-metabolic traits in a panel of 100 diverse inbred strains of mice. The results clearly showed that the effects of sex on both clinical phenotypes and gene expression depend on the genetic background. In support of this, genetic loci associated with the traits frequently showed sex specificity. For example, Lyplal1, a gene implicated in human obesity, was shown to underlie a sex-specific locus for diet-induced obesity. Global gene expression analyses of tissues across the panel implicated adipose tissue "beiging" and mitochondrial functions in the sex differences. Isolated mitochondria showed gene-by-sex interactions in oxidative functions, such that some strains (C57BL/6J) showed similar function between sexes, whereas others (DBA/2J and A/J) showed increased function in females. Reduced adipose mitochondrial function in males as compared to females was associated with increased susceptibility to obesity and insulin resistance. Gonadectomy studies indicated that gonadal hormones acting in a tissue-specific manner were responsible in part for the sex differences., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Increasing non-shivering thermogenesis (NST), which expends calories as heat rather than storing them as fat, is championed as an effective way to combat obesity and metabolic disease. Innate mechanisms constraining the capacity for NST present a fundamental limitation to this approach, yet are not well understood. Here, we provide evidence that Regulator of Calcineurin 1 ( RCAN1 ), a feedback inhibitor of the calcium-activated protein phosphatase calcineurin (CN), acts to suppress two distinctly different mechanisms of non-shivering thermogenesis (NST): one involving the activation of UCP1 expression in white adipose tissue, the other mediated by sarcolipin (SLN) in skeletal muscle. UCP1 generates heat at the expense of reducing ATP production, whereas SLN increases ATP consumption to generate heat. Gene expression profiles demonstrate a high correlation between Rcan1 expression and metabolic syndrome. On an evolutionary timescale, in the context of limited food resources, systemic suppression of prolonged NST by RCAN1 might have been beneficial; however, in the face of caloric abundance, RCAN1-mediated suppression of these adaptive avenues of energy expenditure may now contribute to the growing epidemic of obesity., (© 2018 The Authors.)

Elevated hepatic ceramide levels have been implicated in both insulin resistance (IR) and hepatic steatosis. To understand the factors contributing to hepatic ceramide levels in mice of both sexes, we have quantitated ceramides in a reference population of mice, the Hybrid Mouse Diversity Panel that has been previously characterized for a variety of metabolic syndrome traits. We observed significant positive correlations between Cer(d18:1/16:0) and IR/hepatic steatosis, consistent with previous findings, although the relationship broke down between sexes, as females were less insulin resistant, but had higher Cer(d18:1/16:0) levels than males. The sex difference was due in part to testosterone-mediated repression of ceramide synthase 6. One ceramide species, Cer(d18:1/20:0), was present at higher levels in males and was associated with IR only in males. Clear evidence of gene-by-sex and gene-by-diet interactions was observed, including sex-specific genome-wide association study results. Thus, our studies show clear differences in how hepatic ceramides are regulated between the sexes, which again suggests that the physiological roles of certain hepatic ceramides differ between the sexes., (Copyright © 2018 by the American Society for Biochemistry and Molecular Biology, Inc.)

Inter-tissue communication via secreted proteins has been established as a vital mechanism for proper physiologic homeostasis. Here, we report a bioinformatics framework using a mouse reference population, the Hybrid Mouse Diversity Panel (HMDP), which integrates global multi-tissue expression data and publicly available resources to identify and functionally annotate novel circuits of tissue-tissue communication. We validate this method by showing that we can identify known as well as novel endocrine factors responsible for communication between tissues. We further show the utility of this approach by identification and mechanistic characterization of two new endocrine factors. Adipose-derived Lipocalin-5 is shown to enhance skeletal muscle mitochondrial function, and liver-secreted Notum promotes browning of white adipose tissue, also known as "beiging." We demonstrate the general applicability of the method by providing in vivo evidence for three additional novel molecules mediating tissue-tissue interactions., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Subcutaneous adipose tissue stores excess lipids and maintains energy balance. We performed expression quantitative trait locus (eQTL) analyses by using abdominal subcutaneous adipose tissue of 770 extensively phenotyped participants of the METSIM study. We identified cis-eQTLs for 12,400 genes at a 1% false-discovery rate. Among an approximately 680 known genome-wide association study (GWAS) loci for cardio-metabolic traits, we identified 140 coincident cis-eQTLs at 109 GWAS loci, including 93 eQTLs not previously described. At 49 of these 140 eQTLs, gene expression was nominally associated (p < 0.05) with levels of the GWAS trait. The size of our dataset enabled identification of five loci associated (p < 5 × 10 -8 ) with at least five genes located >5 Mb away. These trans-eQTL signals confirmed and extended the previously reported KLF14-mediated network to 55 target genes, validated the CIITA regulation of class II MHC genes, and identified ZNF800 as a candidate master regulator. Finally, we observed similar expression-clinical trait correlations of genes associated with GWAS loci in both humans and a panel of genetically diverse mice. These results provide candidate genes for further investigation of their potential roles in adipose biology and in regulating cardio-metabolic traits., (Copyright © 2017. Published by Elsevier Inc.)

The etiology of nonalcoholic fatty liver disease is complex and influenced by factors such as obesity, insulin resistance, hyperlipidemia, and sex. We now report a study on sex difference in hepatic steatosis in the context of genetic variation using a population of inbred strains of mice. While male mice generally exhibited higher concentration of hepatic TG levels on a high-fat high-sucrose diet, sex differences showed extensive interaction with genetic variation. Differences in percentage body fat were the best predictor of hepatic steatosis among the strains and explained about 30% of the variation in both sexes. The difference in percent gonadal fat and HDL explained 9.6% and 6.7% of the difference in hepatic TGs between the sexes, respectively. Genome-wide association mapping of hepatic TG revealed some striking differences in genetic control of hepatic steatosis between females and males. Gonadectomy increased the hepatic TG to body fat percentage ratio among male, but not female, mice. Our data suggest that the difference between the sexes in hepatic TG can be partly explained by differences in body fat distribution, plasma HDL, and genetic regulation. Future studies are required to understand the molecular interactions between sex, genetics, and the environment., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Previous studies had shown that the integration of genome wide expression profiles, in metabolic tissues, with genetic and phenotypic variance, provided valuable insight into the underlying molecular mechanisms. We used RNA-Seq to characterize hypothalamic transcriptome in 99 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP), a reference resource population for cardiovascular and metabolic traits. We report numerous novel transcripts supported by proteomic analyses, as well as novel non coding RNAs. High resolution genetic mapping of transcript levels in HMDP, reveals both local and trans expression Quantitative Trait Loci (eQTLs) demonstrating 2 trans eQTL 'hotspots' associated with expression of hundreds of genes. We also report thousands of alternative splicing events regulated by genetic variants. Finally, comparison with about 150 metabolic and cardiovascular traits revealed many highly significant associations. Our data provide a rich resource for understanding the many physiologic functions mediated by the hypothalamus and their genetic regulation., Competing Interests: The authors declare that no competing interests exist.

Background: Functional regulatory regions in eukaryotic genomes are characterized by the disruption of nucleosomes leading to accessible chromatin. The modulation of chromatin accessibility is one of the key mediators of transcriptional regulation, and variation in chromatin accessibility across individuals has been linked to complex traits and disease susceptibility. While mechanisms responsible for chromatin variation across individuals have been investigated, the overwhelming majority of chromatin variation remains unexplained. Furthermore, the processes through which the variation of chromatin accessibility contributes to phenotypic diversity remain poorly understood., Results: We profiled chromatin accessibility in liver from seven strains of mice with phenotypic diversity in response to a high-fat/high-sucrose (HF/HS) diet and identified reproducible chromatin variation across the individuals. We found that sites of variable chromatin accessibility were more likely to coincide with particular classes of transposable elements (TEs) than sites with common chromatin signatures. Evolutionarily younger long interspersed nuclear elements (LINEs) are particularly likely to harbor variable chromatin sites. These younger LINEs are enriched for binding sites of immune-associated transcription factors, whereas older LINEs are enriched for liver-specific transcription factors. Genomic region enrichment analysis indicates that variable chromatin sites at TEs may function to regulate liver metabolic pathways. CRISPR-Cas9 deletion of a number of variable chromatin sites at TEs altered expression of nearby metabolic genes. Finally, we show that polymorphism of TEs and differential DNA methylation at TEs can both influence chromatin variation., Conclusions: Our results demonstrate that specific classes of TEs show variable chromatin accessibility across strains of mice that display phenotypic diversity in response to a HF/HS diet. These results indicate that chromatin variation at TEs is an important contributor to phenotypic variation among populations.

We previously reported quantitation of gut microbiota in a panel of 89 different inbred strains of mice, and we now examine the question of sex differences in microbiota composition. When the total population of 689 mice was examined together, several taxa exhibited significant differences in abundance between sexes but a larger number of differences were observed at the single strain level, suggesting that sex differences can be obscured by host genetics and environmental factors. We also examined a subset of mice on chow and high fat diets and observed sex-by-diet interactions. We further investigated the sex differences using gonadectomized and hormone treated mice from 3 different inbred strains. Principal coordinate analysis with unweighted UniFrac distances revealed very clear effects of gonadectomy and hormone replacement on microbiota composition in all 3 strains. Moreover, bile acid analyses showed gender-specific differences as well as effects of gonodectomy, providing one possible mechanism mediating sex differences in microbiota composition.

Impaired estrogen receptor α (ERα) action promotes obesity and metabolic dysfunction in humans and mice; however, the mechanisms underlying these phenotypes remain unknown. Considering that skeletal muscle is a primary tissue responsible for glucose disposal and oxidative metabolism, we established that reduced ERα expression in muscle is associated with glucose intolerance and adiposity in women and female mice. To test this relationship, we generated muscle-specific ERα knockout (MERKO) mice. Impaired glucose homeostasis and increased adiposity were paralleled by diminished muscle oxidative metabolism and bioactive lipid accumulation in MERKO mice. Aberrant mitochondrial morphology, overproduction of reactive oxygen species, and impairment in basal and stress-induced mitochondrial fission dynamics, driven by imbalanced protein kinase A-regulator of calcineurin 1-calcineurin signaling through dynamin-related protein 1, tracked with reduced oxidative metabolism in MERKO muscle. Although muscle mitochondrial DNA (mtDNA) abundance was similar between the genotypes, ERα deficiency diminished mtDNA turnover by a balanced reduction in mtDNA replication and degradation. Our findings indicate the retention of dysfunctional mitochondria in MERKO muscle and implicate ERα in the preservation of mitochondrial health and insulin sensitivity as a defense against metabolic disease in women., (Copyright © 2016, American Association for the Advancement of Science.)

Genetics provides a potentially powerful approach to dissect host-gut microbiota interactions. Toward this end, we profiled gut microbiota using 16s rRNA gene sequencing in a panel of 110 diverse inbred strains of mice. This panel has previously been studied for a wide range of metabolic traits and can be used for high-resolution association mapping. Using a SNP-based approach with a linear mixed model, we estimated the heritability of microbiota composition. We conclude that, in a controlled environment, the genetic background accounts for a substantial fraction of abundance of most common microbiota. The mice were previously studied for response to a high-fat, high-sucrose diet, and we hypothesized that the dietary response was determined in part by gut microbiota composition. We tested this using a cross-fostering strategy in which a strain showing a modest response, SWR, was seeded with microbiota from a strain showing a strong response, A×B19. Consistent with a role of microbiota in dietary response, the cross-fostered SWR pups exhibited a significantly increased response in weight gain. To examine specific microbiota contributing to the response, we identified various genera whose abundance correlated with dietary response. Among these, we chose Akkermansia muciniphila, a common anaerobe previously associated with metabolic effects. When administered to strain A×B19 by gavage, the dietary response was significantly blunted for obesity, plasma lipids, and insulin resistance. In an effort to further understand host-microbiota interactions, we mapped loci controlling microbiota composition and prioritized candidate genes. Our publicly available data provide a resource for future studies., (© 2015 Org et al.; Published by Cold Spring Harbor Laboratory Press.)

Prior efforts to identify regulators of hematopoietic stem cell physiology have relied mainly on candidate gene approaches with genetically modified mice. Here we used a genome-wide association study (GWAS) strategy with the hybrid mouse diversity panel to identify the genetic determinants of hematopoietic stem/progenitor cell (HSPC) frequency. Among 108 strains, we observed ∼120- to 300-fold variation in three HSPC populations. A GWAS analysis identified several loci that were significantly associated with HSPC frequency, including a locus on chromosome 5 harboring the homeodomain-only protein gene (Hopx). Hopx previously had been implicated in cardiac development but was not known to influence HSPC biology. Analysis of the HSPC pool in Hopx-/- mice demonstrated significantly reduced cell frequencies and impaired engraftment in competitive repopulation assays, thus providing functional validation of this positional candidate gene. These results demonstrate the power of GWAS in mice to identify genetic determinants of the hematopoietic system., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

To identify genetic and environmental factors contributing to the pathogenesis of non-alcoholic fatty liver disease, we examined liver steatosis and related clinical and molecular traits in more than 100 unique inbred mouse strains, which were fed a diet rich in fat and carbohydrates. A &gt;30-fold variation in hepatic TG accumulation was observed among the strains. Genome-wide association studies revealed three loci associated with hepatic TG accumulation. Utilizing transcriptomic data from the liver and adipose tissue, we identified several high-confidence candidate genes for hepatic steatosis, including Gde1, a glycerophosphodiester phosphodiesterase not previously implicated in triglyceride metabolism. We confirmed the role of Gde1 by in vivo hepatic over-expression and shRNA knockdown studies. We hypothesize that Gde1 expression increases TG production by contributing to the production of glycerol-3-phosphate. Our multi-level data, including transcript levels, metabolite levels, and gut microbiota composition, provide a framework for understanding genetic and environmental interactions underlying hepatic steatosis.

Background: Bariatric surgery is associated to improvements in obesity-associated comorbidities thought to be mediated by a decrease of adipose inflammation. However, the molecular mechanisms behind these beneficial effects are poorly understood., Methodology/principal Findings: We analyzed RNA-seq expression profiles in adipose tissue from 22 obese women before and 3 months after surgery. Of 15,972 detected genes, 1214 were differentially expressed after surgery at a 5% false discovery rate. Upregulated genes were mostly involved in the basal cellular machinery. Downregulated genes were enriched in metabolic functions of adipose tissue. At baseline, 26 modules of coexpressed genes were identified. The four most stable modules reflected the innate and adaptive immune responses of adipose tissue. A first module reflecting a non-specific signature of innate immune cells, mainly macrophages, was highly conserved after surgery with the exception of DUSP2 and CD300C. A second module reflected the adaptive immune response elicited by T lymphocytes; after surgery, a disconnection was observed between genes involved in T-cell signaling and mediators of the signal transduction such as CXCR1, CXCR2, GPR97, CCR7 and IL7R. A third module reflected neutrophil-mediated inflammation; after surgery, several genes were dissociated from the module, including S100A8, S100A12, CD300E, VNN2, TUBB1 and FAM65B. We also identified a dense network of 19 genes involved in the interferon-signaling pathway which was strongly preserved after surgery, with the exception of DDX60, an antiviral factor involved in RIG-I-mediated interferon signaling. A similar loss of connection was observed in lean mice compared to their obese counterparts., Conclusions/significance: These results suggest that improvements of the inflammatory state following surgery might be explained by a disruption of immuno-inflammatory cascades involving a few crucial molecules which could serve as potential therapeutic targets.

Obesity is associated with increased breast cancer (BrCA) incidence. Considering that inactivation of estrogen receptor (ER)α promotes obesity and metabolic dysfunction in women and female mice, understanding the mechanisms and tissue-specific sites of ERα action to combat metabolic-related disease, including BrCA, is of clinical importance. To study the role of ERα in adipose tissue we generated fat-specific ERα knock-out (FERKO) mice. Herein we show that ERα deletion increased adipocyte size, fat pad weight, and tissue expression and circulating levels of the secreted glycoprotein, lipocalin 2 (Lcn2), an adipokine previously associated with BrCA development. Chromatin immunoprecipitation and luciferase reporter studies showed that ERα binds the Lcn2 promoter to repress its expression. Because adipocytes constitute an important cell type of the breast microenvironment, we examined the impact of adipocyte ERα deletion on cancer cell behavior. Conditioned medium from ERα-null adipocytes and medium containing pure Lcn2 increased proliferation and migration of a subset of BrCA cells in culture. The proliferative and promigratory effects of ERα-deficient adipocyte-conditioned medium on BrCA cells was reversed by Lcn2 deletion. BrCA cell responsiveness to exogenous Lcn2 was heightened in cell types where endogenous Lcn2 expression was minimal, but components of the Lcn2 signaling pathway were enriched, i.e. SLC22A17 and 3-hydroxybutyrate dehydrogenase (BDH2). In breast tumor biopsies from women diagnosed with BrCA we found that BDH2 expression was positively associated with adiposity and circulating Lcn2 levels. Collectively these data suggest that reduction of ERα expression in adipose tissue promotes adiposity and is linked with the progression and severity of BrCA via increased adipocyte-specific Lcn2 production and enhanced tumor cell Lcn2 sensitivity., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Insulin resistance (IR) is a complex trait with multiple genetic and environmental components. Confounded by large differences between the sexes, environment, and disease pathology, the genetic basis of IR has been difficult to dissect. Here we examine IR and related traits in a diverse population of more than 100 unique male and female inbred mouse strains after feeding a diet rich in fat and refined carbohydrates. Our results show dramatic variation in IR among strains of mice and widespread differences between sexes that are dependent on genotype. We uncover more than 15 genome-wide significant loci and validate a gene, Agpat5, associated with IR. We also integrate plasma metabolite levels and global gene expression from liver and adipose tissue to identify metabolite quantitative trait loci (mQTL) and expression QTL (eQTL), respectively. Our results provide a resource for analysis of interactions between diet, sex, and genetic background in IR., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Metabolic diseases result from multiple genetic and environmental factors. We report here that one manner in which environmental factors can contribute to metabolic disease progression is through modification to chromatin. We demonstrate that high fat diet leads to chromatin remodeling in the livers of C57BL/6J mice, as compared with mice fed a control diet, and that these chromatin changes are associated with changes in gene expression. We further show that the regions of greatest variation in chromatin accessibility are targeted by liver transcription factors, including HNF4α, CCAAT/enhancer-binding protein α (CEBP/α), and FOXA1. Repeating the chromatin and gene expression profiling in another mouse strain, DBA/2J, revealed that the regions of greatest chromatin change are largely strain-specific and that integration of chromatin, gene expression, and genetic data can be used to characterize regulatory regions. Our data indicate dramatic changes in the epigenome due to diet and demonstrate strain-specific dynamics in chromatin remodeling., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Macrophage G2A and CD36 lipid receptors are thought to mediate efferocytosis following tissue injury and thereby prevent excessive inflammation that could compromise tissue repair. To test this, we subjected mice lacking G2A or CD36 receptor to bleomycin-induced lung injury and measured efferocytosis, inflammation, and fibrosis. Loss of CD36 (but not G2A) delayed clearance of apoptotic alveolar cells (mean 78% increase in apoptotic cells 7 days postinjury), potentiated inflammation (mean 56% increase in lung neutrophils and 75% increase in lung KC levels 7 days postinjury, 51% increase in lung macrophages 14 days postinjury), and reduced lung fibrosis (mean 41% and 29% reduction 14 and 21 days postinjury, respectively). Reduced fibrosis in CD36(-/-) mice was associated with lower levels of profibrotic TH2 cytokines (IL-9, IL-13, IL-4), decreased expression of the M2 macrophage marker Arginase-1, and reduced interstitial myofibroblasts. G2A, on the other hand, was required for optimal clearance of apoptotic neutrophils during zymosan-induced peritoneal inflammation (50.3% increase in apoptotic neutrophils and 30.6% increase in total neutrophils 24 h following zymosan administration in G2A(-/-) mice). Thus, CD36 is required for timely removal of apoptotic cells in the context of lung injury and modulates subsequent inflammatory and fibrotic processes relevant to fibrotic lung disease.

Obesity is a highly heritable disease driven by complex interactions between genetic and environmental factors. Human genome-wide association studies (GWAS) have identified a number of loci contributing to obesity; however, a major limitation of these studies is the inability to assess environmental interactions common to obesity. Using a systems genetics approach, we measured obesity traits, global gene expression, and gut microbiota composition in response to a high-fat/high-sucrose (HF/HS) diet of more than 100 inbred strains of mice. Here we show that HF/HS feeding promotes robust, strain-specific changes in obesity that are not accounted for by food intake and provide evidence for a genetically determined set point for obesity. GWAS analysis identified 11 genome-wide significant loci associated with obesity traits, several of which overlap with loci identified in human studies. We also show strong relationships between genotype and gut microbiota plasticity during HF/HS feeding and identify gut microbial phylotypes associated with obesity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We have developed an association-based approach using classical inbred strains of mice in which we correct for population structure, which is very extensive in mice, using an efficient mixed-model algorithm. Our approach includes inbred parental strains as well as recombinant inbred strains in order to capture loci with effect sizes typical of complex traits in mice (in the range of 5% of total trait variance). Over the last few years, we have typed the hybrid mouse diversity panel (HMDP) strains for a variety of clinical traits as well as intermediate phenotypes and have shown that the HMDP has sufficient power to map genes for highly complex traits with resolution that is in most cases less than a megabase. In this essay, we review our experience with the HMDP, describe various ongoing projects, and discuss how the HMDP may fit into the larger picture of common diseases and different approaches.

Objective: Atherosclerosis is a chronic inflammatory disease initiated by monocyte recruitment and retention in the vessel wall. An important mediator of monocyte endothelial interaction is the chemokine interleukin (IL)-8. The oxidation products of phospholipids, including oxidized 1-palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine (Ox-PAPC), accumulate in atherosclerotic lesions and strongly induce IL-8 in human aortic endothelial cells (HAECs). The goal of this study was to identify the proximal events leading to induction of IL-8 by Ox-PAPC in vascular endothelial cells., Methods and Results: In a systems genetics analysis of HAECs isolated from 96 different human donors, we showed that heparin-binding EGF-like growth factor (HBEGF) transcript levels are strongly correlated to IL-8 induction by Ox-PAPC. The silencing and overexpression of HBEGF in HAECs confirmed the role of HBEGF in regulating IL-8 expression. HBEGF has been shown to be stored in an inactive form and activation is dependent on processing by a dysintegrin and metalloproteinases (ADAM) to a form that can activate the epidermal growth factor (EGF) receptor. Ox-PAPC was shown to rapidly induce HBEGF processing and EGF receptor activation in HAECs. Using siRNA we identified 3 ADAMs that regulate IL-8 induction and directly demonstrated that Ox-PAPC increases ADAM activity in the cells using a substrate cleavage assay. We provide evidence for one mechanism of Ox-PAPC activation of ADAM involving covalent binding of Ox-PAPC to cysteine on ADAM. Free thiol cysteine analogs showed inhibition of IL-8 induction by Ox-PAPC, and both a cysteine analog and a cell surface thiol blocker strongly inhibited ADAM activity induction by Ox-PAPC. Using microarray analyses, we determined that this ADAM pathway may regulate at least 30% of genes induced by Ox-PAPC in HAECs., Conclusions: This study is the first report demonstrating a role for the ADAM-HBEGF-EGF receptor axis in Ox-PAPC induction of IL-8 in HAECs. These studies highlight a role for specific ADAMs as initiators of Ox-PAPC action and provide evidence for a role of covalent interaction of Ox-PAPC in activation of ADAMs.

Radiation exposure from a number of terrestrial sources is associated with an increased risk for atherosclerosis. Recently, concern over whether exposure to cosmic radiation might pose a similar risk for astronauts has increased. To address this question, we examined the effect of 2 to 5 Gy iron ions ((56)Fe), a particularly damaging component of cosmic radiation, targeted to specific arterial sites in male apolipoprotein E-deficient (apoE(-/-)) mice. Radiation accelerated the development of atherosclerosis in irradiated portions of the aorta independent of any systemic effects on plasma lipid profiles or circulating leukocytes. Further, radiation exposure resulted in a more rapid progression of advanced aortic root lesions, characterized by larger necrotic cores associated with greater numbers of apoptotic macrophages and reduced lesional collagen compared to sham-treated mice. Intima media thickening of the carotid arteries was also exacerbated. Exposure to (56)Fe ions can therefore accelerate the development of atherosclerotic lesions and promote their progression to an advanced stage characterized by compositional changes indicative of increased thrombogenicity and instability. We conclude that the potential consequences of radiation exposure for astronauts on prolonged deep-space missions are a major concern. Knowledge gained from further studies with animal models should lead to a better understanding of the pathophysiological effects of accelerated ion radiation to better estimate atherogenic risk and develop appropriate countermeasures to mitigate its damaging effects.

Objective: To characterize modifications of high-density lipoprotein (HDL) in autoimmune gld mice that may be relevant to premature atherosclerosis in systemic lupus erythematosus, and to assess their relationship to specific aspects of autoimmune disease., Methods: HDL cholesterol (HDL-C), apolipoprotein A-I (Apo A-I), paraoxonase 1 (PON1) activity, hepatic gene expression, and HDL biogenesis were measured in aging female gld and wild-type congenic mice. Autoantibodies, lymphoid organs, and cytokines were analyzed by enzyme-linked immunosorbent assay, flow cytometry, and multiplex assay, respectively., Results: Plasma HDL-C, HDL Apo A-I, and HDL-associated PON1 activity were reduced in aging gld mice in association with the development of autoimmunity, independent of changes in hepatic Apo A-I and PON1 expression or HDL biogenesis. Hepatic induction of the acute-phase reactant serum amyloid A1 resulted in its incorporation into HDL in gld mice. Deletion of the lipid-sensitive receptor G2A in gld mice (G2A-/- gld) attenuated reductions in HDL-C and PON1 activity without altering hepatic Apo A-I and PON1 expression, HDL biogenesis, or levels of acute-phase proinflammatory cytokines. Plasma anti-Apo A-I autoantibodies were elevated in aging gld mice commensurate with detectable increases in Apo A-I immune complexes. Autoantibody levels were lower in aging G2A-/- gld mice compared with gld mice, and anti-Apo A-I autoantibody levels were significantly related to HDL-C concentrations (r=-0.645, P<0.00004) and PON1 activity (r=-0.555, P<0.0007) among autoimmune gld and G2A-/- gld mice., Conclusion: Autoantibodies against Apo A-I contribute to reducing HDL-C and PON1 activity in autoimmune gld mice independently of hepatic HDL biogenesis, suggesting that functional impairment and premature clearance of HDL immune complexes may be principal mechanisms involved., (Copyright © 2011 by the American College of Rheumatology.)

Objective: Deletion of the lysophospholipid-sensitive receptor, G2A, in low-density lipoprotein receptor knockout (LDLR(-/-)) mice elevates plasma high-density lipoprotein (HDL) cholesterol and suppresses atherosclerosis. However, chemotactic action of G2A in monocytes/macrophages, in addition to its modulatory effect on HDL, may contribute to the proatherogenic action of G2A., Methods and Results: We determined that deletion of G2A in LDLR(-/-) mice increases the ApoA1, ApoE, and cholesterol content of plasma HDL fractions. Hepatocytes were shown to express G2A and hepatocytes from G2A-deficient LDLR(-/-) mice secreted more ApoA1 and ApoE in HDL fractions compared to their G2A-sufficient counterparts. The atheroprotective and HDL modulatory effects of G2A deficiency were dependent on the presence of ApoE, as deletion of G2A in ApoE(-/-) and ApoE(-/-)LDLR(-/-) mice failed to raise HDL and did not suppress atherosclerosis. G2A deficiency in bone marrow-derived cells of LDLR(-/-) mice had no effect on atherosclerosis or HDL, whereas G2A deficiency in resident tissues was sufficient to raise HDL and suppress atherosclerosis., Conclusions: These data demonstrate that the chemotactic function of G2A in bone marrow-derived monocytes does not modulate atherosclerosis in LDLR(-/-) mice and suggest an ApoE-dependent function for G2A in the control of hepatic HDL metabolism that might contribute to its proatherogenic action.

Lysophosphatidylcholine (LPC) is a chemotactic lysolipid produced during inflammation by the hydrolytic action of phospholipase A(2) enzymes. LPC stimulates chemotaxis of T cells in vitro through activation of the G protein-coupled receptor, G2A. This has led to the proposition that G2A contributes to the recruitment of T cells to sites of inflammation and thus promotes chronic inflammatory autoimmune diseases associated with the generation and subsequent tissue infiltration of auto-antigen-specific effector T cells. However, one study suggests that G2A may negatively regulate T cell proliferative responses to antigen receptor engagement and thereby attenuates autoimmunity by reducing the generation of autoreactive T cells. To address the relative contribution of these G2A-mediated effects to the pathophysiology of T cell-mediated autoimmune disease, we examined the impact of G2A inactivation on the onset and severity of murine experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). Wild type (G2A(+/+)) and G2A-deficient (G2A(-/-)) C57BL/6J mice exhibited a similar incidence and onset of disease following immunization with MOG(35-55) peptide. Disease severity was only moderately reduced in G2A(-/-) mice. Similar numbers of MOG(35-55) specific T cells were generated in secondary lymphoid organs of MOG(35-55)-immunized G2A(+/+) and G2A(-/-) mice. Comparable numbers of T cells were detected in spinal cords of G2A(+/+) and G2A(-/-) mice. We conclude that the proposed anti-proliferative and chemotactic functions of G2A are not manifested in vivo and therefore therapeutic targeting of G2A is unlikely to be beneficial in the treatment of MS.

A general synthetic approach was developed for the preparation of a series of 6,6-bicyclic malonamides, a class of ligands that provide a preorganized binding site for f-block ions (particularly trivalent lanthanides). The approach described is convenient to introduce a variety of functional groups at the amide nitrogens to tune the properties of the ligand without altering the preorganized binding. Each of the ten derivatives (that represent a range of functionality, including R = alkyl, hydroxy, phenyl, ester, perfluorocarbon) reported here derives from a single, readily prepared dialdehyde intermediate. This intermediate is converted to the final products via reductive amination with an appropriately functionalized benzylamine, followed by hydrogenolysis and lactam formation. Because derivatization occurs late in the synthesis, the approach is general, requiring only modification of the purification procedures for each new derivative. To aid in the purification of the bicyclic malonamides, we report a novel complexation-based purification method that takes advantage of the high affinity of the ligand for f-block metals.

Objective: Lysophosphatidylcholine is a major product of low-density lipoprotein (LDL) oxidation and secretory phospholipase A2-mediated lipid hydrolysis within atherosclerotic lesions. The G2A receptor mediates chemotaxis of cultured macrophages and T cells to lysophosphatidylcholine, supporting a pro-atherogenic role for this receptor in vivo. We investigated the ability of G2A to modulate atherosclerosis in mice., Methods and Results: We measured atherosclerosis in G2A+/+ and G2A-/- LDL receptor knockout (LDLR-/-) mice. Consistent with a previous study, early lesion size at the aortic sinus was unaffected by G2A deficiency. However, G2A deficiency attenuated lesion progression at this site (42% to 44% reduction in average lesion area) and led to robust suppression of atherosclerosis throughout the aorta after short and extended periods of diet intervention (reduction in aortic lesion coverage: 62% to 73% at 9 weeks, 75% to 84% at 20 weeks). In G2A-/- LDLR-/- mice, intimal macrophage accumulation at lesion-prone sites of the aorta was significantly reduced in the absence of any detectable effect on T cell recruitment. Examination of lipoprotein profiles revealed elevated levels of circulating high-density lipoprotein (HDL) cholesterol in G2A-/- LDLR-/- mice compared with their G2A+/+ LDLR-/- counterparts after extended periods of diet intervention (54% increase in mean HDL cholesterol concentration)., Conclusions: G2A provides a pro-atherogenic stimulus in vivo consistent with its chemotactic action but to which a pleiotropy of effects, including modulation of lipoprotein metabolism, may also contribute.

This report describes an investigation into the coordination chemistry of trivalent lanthanides in solution and the solid state with acyclic and preorganized bicyclic malonamide ligands. Two experimental investigations were performed: solution binding affinities were determined through single-phase spectrophotometric titrations and the extent of conformational change upon binding was investigated with single-crystal X-ray crystallography. Both experimental methods compare the bicyclic malonamide (BMA), which is designed to be preorganized for binding trivalent lanthanides, to an analogous acyclic malonamide. Results from the spectrophotometric titrations indicate that BMA exhibits a 10-100x increase in binding affinity to Ln(III) over acyclic malonamide. In addition, BMA forms compounds with high ligand-metal ratios, even when competing with water and nitrate ligands for binding sites. The crystal structures exhibit no significant differences in the nature of the binding between Ln(III) and the BMA or acyclic malonamide. These results support the conclusion that rational ligand design can lead to compounds that enhance the binding affinities within a ligand class.

Lysophosphatidylcholine (LPC) is considered a major proatherogenic component of oxidized low density lipoprotein based on its proinflammatory actions in vitro. LPC stimulates macrophage and T-cell chemotaxis via the G protein-coupled receptor G2A and may thus promote inflammatory cell infiltration during atherosclerotic lesion development. However, G2A also mediates proapoptotic effects of LPC and may therefore promote the death of inflammatory cells within developing lesions. To determine how these effects of LPC modify atherogenesis, we examined atherosclerotic lesion development in G2A-sufficient and G2A-deficient low density lipoprotein receptor knockout mice. Although LPC species capable of activating G2A-dependent responses were increased during lesion development, G2A-deficient mice developed lesions similar in size to those in their G2A-sufficient counterparts. Loss of G2A during atherosclerotic lesion development did not reduce macrophage and T-cell infiltration but instead resulted in increased lesional macrophage content associated with reduced numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeled cells and decreased collagen deposition. These data indicate that the ability of LPC to stimulate macrophage and T-cell chemotaxis via G2A is not manifested in vivo and that G2A-mediated proapoptotic rather than chemotactic action is most penetrant during atherogenesis and may modify the stability of atherosclerotic lesions by promoting macrophage death.

This report describes affinity measurements for two, water-soluble, methyl-alkylated diamides incorporating the malonamide functionality, N,N,N',N' tetramethylmalonamide (TMMA) and a bicyclic diamide (1a), toward actinide metal cations (An) in acidic nitrate solutions. Ligand complexation to actinides possessing oxidation states ranging from +3 to +6 was monitored through optical absorbance spectroscopy, and formation constants were obtained from the refinement of the spectrophotometric titration data sets. Species analysis gives evidence for the formation of 1, 4, 1, and 2 spectrophotometrically observable complexes by TMMA to An(III, IV, V, and VI), respectively, while for 1a, the respective numbers are 3, 4, 2, and 2. Consistent with the preorganization of 1a toward actinide binding, a significant difference is found in the magnitudes of their respective formation constants at each complexation step. It has been found that the binding affinity for TMMA follows the well-established order An(V) < An(III) < An(VI) < An(IV). However, with 1a, Np(V) forms stronger complexes than Am(III). The complexation of 1a with Np(V) and Pu(VI) at an acidity of 1.0 M is followed by reduction to Np(IV) and Pu(IV), whereas TMMA does not perturb the initial oxidation state for these dioxocations. These measurements of diamide binding affinity mark the first time single-component optical absorbance spectra have been reported for a span of actinide-diamide complexes covering all common oxidation states in aqueous solution.