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The identification of pathways necessary for photoreceptor and retinal pigment epithelium (RPE) function is critical to uncover therapies for blindness. Here we report the discovery of adiponectin receptor 1 (AdipoR1) as a regulator of these cells’ functions. Docosahexaenoic acid (DHA) is avidly retained in photoreceptors, while mechanisms controlling DHA uptake and retention are unknown. Thus, we demonstrate that AdipoR1 ablation results in DHA reduction. In situ hybridization reveals photoreceptor and RPE cell AdipoR1 expression, blunted in AdipoR1 −/− mice. We also find decreased photoreceptor-specific phosphatidylcholine containing very long-chain polyunsaturated fatty acids and severely attenuated electroretinograms. These changes precede progressive photoreceptor degeneration in AdipoR1 −/− mice. RPE-rich eyecup cultures from AdipoR1 −/− reveal impaired DHA uptake. AdipoR1 overexpression in RPE cells enhances DHA uptake, whereas AdipoR1 silencing has the opposite effect. These results establish AdipoR1 as a regulatory switch of DHA uptake, retention, conservation and elongation in photoreceptors and RPE, thus preserving photoreceptor cell integrity. Docosahexaenoic acid is a major and important retinal fatty acid that is recruited and retained in the photoreceptor membrane via an unknown mechanism. Here, Rice et al . show that adiponectin receptor 1 is a key molecular switch for docosahexaenoic acid membrane homeostasis and photoreceptor cell function.

Tang et al . present the first large-scale, gene-specific library of knockout mice. They disrupt 472 genes encoding secreted or transmembrane proteins and report the results of a comprehensive phenotypic analysis. Large collections of knockout organisms facilitate the elucidation of gene functions. Here we used retroviral insertion or homologous recombination to disrupt 472 genes encoding secreted and membrane proteins in mice, providing a resource for studying a large fraction of this important class of drug target. The knockout mice were subjected to a systematic phenotypic screen designed to uncover alterations in embryonic development, metabolism, the immune system, the nervous system and the cardiovascular system. The majority of knockout lines exhibited altered phenotypes in at least one of these therapeutic areas. To our knowledge, a comprehensive phenotypic assessment of a large number of mouse mutants generated by a gene-specific approach has not been described previously.

Humans with haploinsufficiency of , an orphan GPCR, are thin. knockout (KO) mice are also thin with improved glucose homeostasis. We wanted to confirm these findings in KO mice and determine whether decreased energy intake and/or increased energy expenditure contributed to the thin phenotype. KO mice were generated by homologous recombination. All studies compared female and male KO mice to their wild type (WT) littermates. Body composition was measured by DXA and QMR technologies. Glucose homeostasis was evaluated by measuring glucose and insulin levels during oral glucose tolerance tests (OGTTs). Food intake was measured in group-housed mice. In singly housed mice, energy expenditure was measured in Oxymax indirect calorimetry chambers, and locomotor activity was measured in Oxymax and Photobeam Activity System chambers. In all 12 cohorts of adult female or male mice, KO mice had less body fat; pooled data showed that, compared to WT littermates (n = 103), KO mice (n = 118) had 49% less body fat and 4% less LBM ( < 0.001 for each). KO mice also had 8% less body fat at weaning (P < 0.05), and during the month after weaning as the thin phenotype became more exaggerated, KO mice ate significantly less than, but had energy expenditure and activity levels comparable to, their WT littermates. During OGTTs, KO mice showed improved glucose tolerance (glucose AUC 23% lower in females, < 0.05, and 26% lower in males, P < 0.001), accompanied by significantly decreased insulin levels and significantly increased insulin sensitivity indices. KO mice are thin at weaning, are hypophagic as the thin phenotype becomes more exaggerated, and exhibit improved glucose tolerance and insulin sensitivity as healthy-appearing adults. These results suggest that inhibiting GPR75 in obese humans may safely decrease energy intake and body fat while improving glucose tolerance and insulin sensitivity.

We used gene knockout mice to explore the role of Angiopoietin-like-4 (Angptl4) in lipid metabolism as well as to generate anti-Angptl4 mAbs with pharmacological activity. Angptl4 -/- mice had lower triglyceride (TG) levels resulting both from increased very low-density lipoprotein (VLDL) clearance and decreased VLDL production and had modestly lower cholesterol levels. Also, both Angptl4 -/- suckling mice and adult mice fed a high-fat diet showed reduced viability associated with lipogranulomatous lesions of the intestines and their draining lymphatics and mesenteric lymph nodes. Treating C57BL/6J, ApoE -/-, LDLr -/-, and db/db mice with the anti-Angptl4 mAb 14D12 recapitulated the lipid and histopathologic phenotypes noted in Angptl4 -/- mice. This demonstrates that the knockout phenotype reflects not only the physiologic function of the Angptl4 gene but also predicts the pharmacologic consequences of Angptl4 protein inhibition with a neutralizing antibody in relevant models of human disease.

Obesity is a major public health problem. Understanding which genes contribute to obesity may better predict individual risk and allow development of new therapies. Because obesity of a mouse gene knockout (KO) line predicts an association of the orthologous human gene with obesity, we reviewed data from the Lexicon Genome5000 high throughput phenotypic screen (HTS) of mouse gene KOs to identify KO lines with high body fat. KO lines were generated using homologous recombination or gene trapping technologies. HTS body composition analyses were performed on adult wild-type and homozygous KO littermate mice from 3758 druggable mouse genes having a human ortholog. Body composition was measured by either DXA or QMR on chow-fed cohorts from all 3758 KO lines and was measured by QMR on independent high fat diet-fed cohorts from 2488 of these KO lines. Where possible, comparisons were made to HTS data from the International Mouse Phenotyping Consortium (IMPC). Body fat data are presented for 75 KO lines. Of 46 KO lines where independent external published and/or IMPC KO lines are reported as obese, 43 had increased body fat. For the remaining 29 novel high body fat KO lines, and are supported by data from additional independent KO cohorts, 6 ( and ) are supported by data from additional internal cohorts, and the remaining 21 including and were studied with HTS cohorts only. These data support the finding of high body fat in 43 independent external published and/or IMPC KO lines. A novel obese phenotype was identified in 29 additional KO lines, with 27 still lacking the external confirmation now provided for and KO mice. Undoubtedly, many mammalian obesity genes remain to be identified and characterized.

In humans, single nucleotide polymorphisms (SNPs) near the adjacent protein kinase D1 ( ) and G2/M-phase-specific E3 ubiquitin protein ligase ( ) genes on chromosome 14 are associated with obesity. To date, no published evidence links inactivation of either gene to changes in body fat. These two genes are also adjacent on mouse chromosome 12. Because obesity genes are highly conserved between humans and mice, we analyzed body fat in adult and knockout (KO) mice to determine whether inactivating either gene leads to obesity in mice and, by inference, probably in humans. The and KO lines were generated by gene trapping and by homologous recombination methodologies, respectively. Body fat was measured by DEXA in adult mice fed chow from weaning and by QMR in a separate cohort of mice fed high-fat diet (HFD) from weaning. Glucose homeostasis was evaluated with oral glucose tolerance tests (OGTTs) performed on adult mice fed HFD from weaning. Body fat was increased in multiple cohorts of KO mice relative to their wild-type (WT) littermates. When data from all KO (n=32) and WT (n=31) mice were compared, KO mice showed increases of 11% in body weight ( <0.01), 65% in body fat ( <0.001), 48% in % body fat ( <0.001), and an insignificant 3% decrease in lean body mass. KO mice were also glucose intolerant during an OGTT ( <0.05). In contrast, KO and WT mice had comparable body fat levels and glucose tolerance. Significant obesity and glucose intolerance were observed in , but not , KO mice. The conservation of obesity genes between mice and humans strongly suggests that the obesity-associated SNPs located near the human and genes are linked to variants that decrease the amount of functional human G2E3.

Receptor tyrosine phosphatase gamma (PTPRG, or RPTPγ) is a mammalian receptor-like tyrosine phosphatase which is highly expressed in the nervous system as well as other tissues. Its function and biochemical characteristics remain largely unknown. We created a knockdown (KD) line of this gene in mouse by retroviral insertion that led to 98-99% reduction of RPTPγ gene expression. The knockdown mice displayed antidepressive-like behaviors in the tail-suspension test, confirming observations by Lamprianou et al. 2006. We investigated this phenotype in detail using multiple behavioral assays. To see if the antidepressive-like phenotype was due to the loss of phosphatase activity, we made a knock-in (KI) mouse in which a mutant, RPTPγ C1060S, replaced the wild type. We showed that human wild type RPTPγ protein, expressed and purified, demonstrated tyrosine phosphatase activity, and that the RPTPγ C1060S mutant was completely inactive. Phenotypic analysis showed that the KI mice also displayed some antidepressive-like phenotype. These results lead to a hypothesis that an RPTPγ inhibitor could be a potential treatment for human depressive disorders. In an effort to identify a natural substrate of RPTPγ for use in an assay for identifying inhibitors, \"substrate trapping\" mutants (C1060S, or D1028A) were studied in binding assays. Expressed in HEK293 cells, these mutant RPTPγs retained a phosphorylated tyrosine residue, whereas similarly expressed wild type RPTPγ did not. This suggested that wild type RPTPγ might auto-dephosphorylate which was confirmed by an in vitro dephosphorylation experiment. Using truncation and mutagenesis studies, we mapped the auto-dephosphorylation to the Y1307 residue in the D2 domain. This novel discovery provides a potential natural substrate peptide for drug screening assays, and also reveals a potential functional regulatory site for RPTPγ. Additional investigation of RPTPγ activity and regulation may lead to a better understanding of the biochemical underpinnings of human depression.

Testicular orphan nuclear receptor 4 (TR4) is a member of the nuclear receptor superfamily for which a ligand has not yet been found. In vitro data obtained from various cell lines suggest that TR4 functions as a master regulator to modulate many signaling pathways, yet the in vivo physiological roles of TR4 remain unclear. Here, we report the generation of mice lacking TR4 by means of targeted gene disruption ( TR4-/-). The number of TR4-/- pups generated by the mating of TR4+/- mice is well under that predicted by the normal Mendelian ratio, and TR4-/- mice demonstrate high rates of early postnatal mortality, as well as significant growth retardation. Additionally, TR4-/- females show defects in reproduction and maternal behavior, with pups of TR4-/- dams dying soon after birth with no indication of milk intake. These results provide in vivo evidence that TR4 plays important roles in growth, embryonic and early postnatal pup survival, female reproductive function, and maternal behavior.

Abstract Genes regulating body fat are shared by mice and humans, and mouse knockout phenotypes for known drug targets correlate well with drug efficacy, suggesting that mouse knockout phenotyping can identify anti-obesity drug targets. Mice with an intestine-specific Acsl5 knockout are protected from high-fat diet (HFD)-induced obesity, insulin resistance, glucose intolerance and hepatic steatosis, and show increased GLP-1 levels, delayed gastric emptying (GE), and decreased food consumption (FC). Here we provide data on these and further outcomes in mice with a global Acsl5 knockout and in mice receiving ACSL5 inhibitors (ACSL5i). We generated Acsl5 knockout mice by homologous recombination and identified potent ACSL5i by compound library screening, iterative medicinal chemistry optimization, and by testing whether compounds inhibit oral triglyceride absorption. We found that both genetic and pharmacologic ACSL5 inhibition reproduce the intestine-specific knockout metabolic phenotype. Importantly, the ACSL5i LP-856866 lowered FC in wild-type but not Acsl5 knockout mice, indicating targeted ACSL5 inhibition. Acsl5 knockout mice had increased fecal free fatty acids but not triglycerides, and adding the lipase inhibitor orlistat to an oral triglyceride load reversed the delayed GE associated with genetic and pharmacologic ACSL5 inhibition; these findings, and the marked GLP-1 release after mice with genetic and pharmacologic ACSL5 inhibition received an oral triglyceride load, suggest ileal brake activation. We conclude that HFD-fed Acsl5 knockout mice exhibit a favorable metabolic phenotype, driven by ileal brake activation, which is phenocopied by orally available small molecule ACSL5i.

Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1-/- B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1-/- CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.