Publications by authors named "Roger D Cox"

81 Publications

Palmitoylated small GTPase ARL15 is translocated within Golgi network during adipogenesis.

Biol Open 2021 12 13;10(12). Epub 2021 Dec 13.

Mammalian Genetics Unit, MRC Harwell Institute, Harwell Oxford, Oxfordshire, OX11 0RD, UK.

The small GTPase ARF family member ARL15 gene locus is associated in population studies with increased risk of type 2 diabetes, lower adiponectin and higher fasting insulin levels. Previously, loss of ARL15 was shown to reduce insulin secretion in a human β-cell line and loss-of-function mutations are found in some lipodystrophy patients. We set out to understand the role of ARL15 in adipogenesis and showed that endogenous ARL15 palmitoylated and localised in the Golgi of mouse liver. Adipocyte overexpression of palmitoylation-deficient ARL15 resulted in redistribution to the cytoplasm and a mild reduction in expression of some adipogenesis-related genes. Further investigation of the localisation of ARL15 during differentiation of a human white adipocyte cell line showed that ARL15 was predominantly co-localised with a marker of the cis face of Golgi at the preadipocyte stage and then translocated to other Golgi compartments after differentiation was induced. Finally, co-immunoprecipitation and mass spectrometry identified potential interacting partners of ARL15, including the ER-localised protein ARL6IP5. Together, these results suggest a palmitoylation dependent trafficking-related role of ARL15 as a regulator of adipocyte differentiation via ARL6IP5 interaction. This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/bio.058420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8689486PMC
December 2021

Linking the obesity rs1421085 variant circuitry to cellular, metabolic, and organismal phenotypes in vivo.

Sci Adv 2021 Jul 21;7(30). Epub 2021 Jul 21.

Mammalian Genetics Unit, MRC Harwell Institute, Oxfordshire OX11 0RD, UK.

Variants in FTO have the strongest association with obesity; however, it is still unclear how those noncoding variants mechanistically affect whole-body physiology. We engineered a deletion of the rs1421085 conserved cis-regulatory module (CRM) in mice and confirmed in vivo that the CRM modulates and gene expression and mitochondrial function in adipocytes. The CRM affects molecular and cellular phenotypes in an adipose depot-dependent manner and affects organismal phenotypes that are relevant for obesity, including decreased high-fat diet-induced weight gain, decreased whole-body fat mass, and decreased skin fat thickness. Last, we connected the CRM to a genetically determined effect on steroid patterns in males that was dependent on nutritional challenge and conserved across mice and humans. Together, our data establish cross-species conservation of the rs1421085 regulatory circuitry at the molecular, cellular, metabolic, and organismal level, revealing previously unknown contextual dependence of the variant's action.
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http://dx.doi.org/10.1126/sciadv.abg0108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8294759PMC
July 2021

The Gene Variant Hastens Diabetes Progression by Impairing Glucose-Induced Insulin Secretion.

Diabetes 2021 05 10;70(5):1145-1156. Epub 2021 Feb 10.

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, U.K.

The ATP-sensitive K (K) channel controls blood glucose levels by coupling glucose metabolism to insulin secretion in pancreatic β-cells. E23K, a common polymorphism in the pore-forming K channel subunit () gene, has been linked to increased risk of type 2 diabetes. Understanding the risk-allele-specific pathogenesis has the potential to improve personalized diabetes treatment, but the underlying mechanism has remained elusive. Using a genetically engineered mouse model, we now show that the K23 variant impairs glucose-induced insulin secretion and increases diabetes risk when combined with a high-fat diet (HFD) and obesity. K-channels in β-cells with two K23 risk alleles (KK) showed decreased ATP inhibition, and the threshold for glucose-stimulated insulin secretion from KK islets was increased. Consequently, the insulin response to glucose and glycemic control was impaired in KK mice fed a standard diet. On an HFD, the effects of the KK genotype were exacerbated, accelerating diet-induced diabetes progression and causing β-cell failure. We conclude that the K23 variant increases diabetes risk by impairing insulin secretion at threshold glucose levels, thus accelerating loss of β-cell function in the early stages of diabetes progression.
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http://dx.doi.org/10.2337/db20-0691DOI Listing
May 2021

A regulatory variant at 3q21.1 confers an increased pleiotropic risk for hyperglycemia and altered bone mineral density.

Cell Metab 2021 03 28;33(3):615-628.e13. Epub 2021 Jan 28.

Metabolism Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Cell Circuits and Epigenomics, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02131, USA; University of Hohenheim, Institute of Nutritional Science, Stuttgart 70599, Germany. Electronic address:

Skeletal and glycemic traits have shared etiology, but the underlying genetic factors remain largely unknown. To identify genetic loci that may have pleiotropic effects, we studied Genome-wide association studies (GWASs) for bone mineral density and glycemic traits and identified a bivariate risk locus at 3q21. Using sequence and epigenetic modeling, we prioritized an adenylate cyclase 5 (ADCY5) intronic causal variant, rs56371916. This SNP changes the binding affinity of SREBP1 and leads to differential ADCY5 gene expression, altering the chromatin landscape from poised to repressed. These alterations result in bone- and type 2 diabetes-relevant cell-autonomous changes in lipid metabolism in osteoblasts and adipocytes. We validated our findings by directly manipulating the regulator SREBP1, the target gene ADCY5, and the variant rs56371916, which together imply a novel link between fatty acid oxidation and osteoblast differentiation. Our work, by systematic functional dissection of pleiotropic GWAS loci, represents a framework to uncover biological mechanisms affecting pleiotropic traits.
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http://dx.doi.org/10.1016/j.cmet.2021.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928941PMC
March 2021

Maternal and offspring high-fat diet leads to platelet hyperactivation in male mice offspring.

Sci Rep 2021 01 14;11(1):1473. Epub 2021 Jan 14.

Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading - Harborne Building, Reading, RG6 6AS, UK.

Maternal over-nutrition increases the risk of diabetes and cardiovascular events in offspring. While prominent effects on cardiovascular health are observed, the impact on platelet physiology has not been studied. Here, we examined whether maternal high-fat diet (HF) ingestion affects the platelet function in lean and obese offspring. C57BL6/N mice dams were given a HF or control (C) diet for 8 weeks before and during pregnancy. Male and female offspring received C or HF diets for 26 weeks. Experimental groups were: C/C, dam and offspring fed standard laboratory diet; C/HF dam fed standard laboratory diet and offspring fed HF diet; HF/C and HF/HF. Phenotypic and metabolic tests were performed and blood collected for platelet studies. Compared to C/C, offspring HF groups were obese, with fat accumulation, hyperglycaemia and insulin resistance. Female offspring did not present platelet hyperactivity, hence we focused on male offspring. Platelets from HF/HF mice were larger, hyperactive and presented oxidative stress when compared to C/C. Maternal and offspring HF diet results in platelet hyperactivation in male mouse offspring, suggesting a novel 'double-hit' effect.
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http://dx.doi.org/10.1038/s41598-020-80373-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809045PMC
January 2021

Calcilytic NPSP795 Increases Plasma Calcium and PTH in an Autosomal Dominant Hypocalcemia Type 1 Mouse Model.

JBMR Plus 2020 Oct 7;4(10):e10402. Epub 2020 Sep 7.

Academic Endocrine Unit, Radcliffe Department of Medicine Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), University of Oxford Oxford UK.

Calcilytics are calcium-sensing receptor (CaSR) antagonists that reduce the sensitivity of the CaSR to extracellular calcium. Calcilytics have the potential to treat autosomal dominant hypocalcemia type 1 (ADH1), which is caused by germline gain-of-function CaSR mutations and leads to symptomatic hypocalcemia, inappropriately low PTH concentrations, and hypercalciuria. To date, only one calcilytic compound, NPSP795, has been evaluated in patients with ADH1: Doses of up to 30 mg per patient have been shown to increase PTH concentrations, but did not significantly alter ionized blood calcium concentrations. The aim of this study was to further investigate NPSP795 for the treatment of ADH1 by undertaking in vitro and in vivo studies involving mice, which have hypocalcemia in association with a gain-of-function CaSR mutation, Leu723Gln. Treatment of HEK293 cells stably expressing the mutant (Gln723) CaSR with 20nM NPSP795 decreased extracellular Ca-mediated intracellular calcium and phosphorylated ERK responses. An in vivo dose-ranging study was undertaken by administering a s.c. bolus of NPSP795 at doses ranging from 0 to 30 mg/kg to heterozygous and to homozygous mice, and measuring plasma PTH responses at 30 min postdose. NPSP795 significantly increased plasma PTH concentrations in a dose-dependent manner with the 30 mg/kg dose causing a maximal (≥10-fold) rise in PTH. To determine whether NPSP795 can rectify the hypocalcemia of and mice, a submaximal dose (25 mg/kg) was administered, and plasma adjusted-calcium concentrations measured over a 6-hour period. NPSP795 significantly increased plasma adjusted-calcium in mice from 1.87 ± 0.03 mmol/L to 2.16 ± 0.06 mmol/L, and in mice from 1.70 ± 0.03 mmol/L to 1.89 ± 0.05 mmol/L. Our findings show that NPSP795 elicits dose-dependent increases in PTH and ameliorates the hypocalcemia in an ADH1 mouse model. Thus, calcilytics such as NPSP795 represent a potential targeted therapy for ADH1. © 2020 The Authors. published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbm4.10402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574706PMC
October 2020

A lead candidate functional single nucleotide polymorphism within the WARS2 gene associated with waist-hip-ratio does not alter RNA stability.

Biochim Biophys Acta Gene Regul Mech 2020 11 30;1863(11):194640. Epub 2020 Sep 30.

MRC Harwell Institute, Mammalian Genetics Unit, Harwell Campus, Oxfordshire OX11 0RD, UK. Electronic address:

We have prioritised a single nucleotide polymorphism (SNP) rs2645294 as one candidate functional SNP in the TBX15-WARS2 waist-hip-ratio locus using posterior probability analysis. This SNP is located in the 3' untranslated region of the WARS2 (tryptophanyl tRNA synthetase 2, mitochondrial) gene with which it has an expression quantitative trait in subcutaneous white adipose tissue. We show that transcripts of the WARS2 gene in a human white adipose cell line, heterozygous for the rs2645294 SNP, showed allelic imbalance. We tested whether the rs2645294 SNP altered WARS2 RNA stability using three different methods: actinomycin-D inhibition and RNA decay, mature and nascent RNA analysis and luciferase reporter assays. We found no evidence of a difference in RNA stability between the rs2645294 alleles indicating that the allelic expression imbalance was likely due to transcriptional regulation.
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http://dx.doi.org/10.1016/j.bbagrm.2020.194640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695619PMC
November 2020

The American lifestyle-induced obesity syndrome diet in male and female rodents recapitulates the clinical and transcriptomic features of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.

Am J Physiol Gastrointest Liver Physiol 2020 09 5;319(3):G345-G360. Epub 2020 Aug 5.

Oxford Centre for Diabetes, Endocrinology and Metabolism, National Institute for Health Research Oxford Biomedical Research Centre, Churchill Hospital, University of Oxford, Oxford, United Kingdom.

The pathogenesis of nonalcoholic fatty liver disease (NAFLD) and the progression to nonalcoholic steatohepatitis (NASH) and increased risk of hepatocellular carcinoma remain poorly understood. Additionally, there is increasing recognition of the extrahepatic manifestations associated with NAFLD and NASH. We demonstrate that intervention with the American lifestyle-induced obesity syndrome (ALIOS) diet in male and female mice recapitulates many of the clinical and transcriptomic features of human NAFLD and NASH. Male and female C57BL/6N mice were fed either normal chow (NC) or ALIOS from 11 to 52 wk and underwent comprehensive metabolic analysis throughout the duration of the study. From 26 wk, ALIOS-fed mice developed features of hepatic steatosis, inflammation, and fibrosis. ALIOS-fed mice also had an increased incidence of hepatic tumors at 52 wk compared with those fed NC. Hepatic transcriptomic analysis revealed alterations in multiple genes associated with inflammation and tissue repair in ALIOS-fed mice. Ingenuity Pathway Analysis confirmed dysregulation of metabolic pathways as well as those associated with liver disease and cancer. In parallel the development of a robust hepatic phenotype, ALIOS-fed mice displayed many of the extrahepatic manifestations of NAFLD, including hyperlipidemia, increased fat mass, sarcopenia, and insulin resistance. The ALIOS diet in mice recapitulates many of the clinical features of NAFLD and, therefore, represents a robust and reproducible model for investigating the pathogenesis of NAFLD and its progression. Nonalcoholic fatty liver disease (NAFLD) affects 30% of the general population and can progress to nonalcoholic steatohepatitis (NASH) and potentially hepatocellular carcinoma. Preclinical models rely on mouse models that often display hepatic characteristics of NAFLD but rarely progress to NASH and seldom depict the multisystem effects of the disease. We have conducted comprehensive metabolic analysis of both male and female mice consuming a Western diet of trans fats and sugar, focusing on both their hepatic phenotype and extrahepatic manifestations.
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http://dx.doi.org/10.1152/ajpgi.00055.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509261PMC
September 2020

null C57BL/6N mice develop cardiomyopathy, whereas null C57BL/6J mice do not.

Life Sci Alliance 2020 04 25;3(4). Epub 2020 Mar 25.

Centre for Endocrinology, William Harvey Research Institute, Charterhouse Square, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK

The C57BL/6J and C57BL/6N mice have well-documented phenotypic and genotypic differences, including the infamous nicotinamide nucleotide transhydrogenase () null mutation in the C57BL/6J substrain, which has been linked to cardiovascular traits in mice and cardiomyopathy in humans. To assess whether loss alone causes a cardiovascular phenotype, we investigated the C57BL/6N, C57BL/6J mice and a C57BL/6J-BAC transgenic rescuing NNT expression, at 3, 12, and 18 mo. We identified a modest dilated cardiomyopathy in the C57BL/6N mice, absent in the two B6J substrains. Immunofluorescent staining of cardiomyocytes revealed eccentric hypertrophy in these mice, with defects in sarcomere organisation. RNAseq analysis identified differential expression of a number of cardiac remodelling genes commonly associated with cardiac disease segregating with the phenotype. Variant calling from RNAseq data identified a myosin light chain kinase 3 () mutation in C57BL/6N mice, which abolishes MYLK3 protein expression. These results indicate the C57BL/6J -null mice do not develop cardiomyopathy; however, we identified a null mutation in as a credible cause of the cardiomyopathy phenotype in the C57BL/6N.
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http://dx.doi.org/10.26508/lsa.201900593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7103425PMC
April 2020

A novel mutation in the mouse Pcsk1 gene showing obesity and diabetes.

Mamm Genome 2020 02 23;31(1-2):17-29. Epub 2020 Jan 23.

Mammalian Genetics Unit, MRC Harwell Institute, Harwell Campus, Oxfordshire, OX11 0RD, UK.

The proprotein convertase subtilisin/Kexin type 1 (PCSK1/PC1) protein processes inactive pro-hormone precursors into biologically active hormones in a number of neuroendocrine and endocrine cell types. Patients with recessive mutations in PCSK1 exhibit a complex spectrum of traits including obesity, diarrhoea and endocrine disorders. We describe here a new mouse model with a point mutation in the Pcsk1 gene that exhibits obesity, hyperphagia, transient diarrhoea and hyperproinsulinaemia, phenotypes consistent with human patient traits. The mutation results in a pV96L amino acid substitution and changes the first nucleotide of mouse exon 3 leading to skipping of that exon and in homozygotes very little full-length transcript. Overexpression of the exon 3 deleted protein or the 96L protein results in ER retention in Neuro2a cells. This is the second Pcsk1 mouse model to display obesity phenotypes, contrasting knockout mouse alleles. This model will be useful in investigating the basis of endocrine disease resulting from prohormone processing defects.
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http://dx.doi.org/10.1007/s00335-020-09826-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060156PMC
February 2020

Monitoring type 2 diabetes from volatile faecal metabolome in Cushing's syndrome and single Afmid mouse models via a longitudinal study.

Sci Rep 2019 12 11;9(1):18779. Epub 2019 Dec 11.

School of Life, Health & Chemical Sciences, Faculty of Science, Technology, Engineering and Mathematics, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK.

The analysis of volatile organic compounds (VOCs) as a non-invasive method for disease monitoring, such as type 2 diabetes (T2D) has shown potential over the years although not yet set in clinical practice. Longitudinal studies to date are limited and the understanding of the underlying VOC emission over the age is poorly understood. This study investigated longitudinal changes in VOCs present in faecal headspace in two mouse models of T2D - Cushing's syndrome and single Afmid knockout mice. Longitudinal changes in bodyweight, blood glucose levels and plasma insulin concentration were also reported. Faecal headspace analysis was carried out using selected ion flow tube mass spectrometry (SIFT-MS) and thermal desorption coupled to gas chromatography-mass spectrometry (TD-GC-MS). Multivariate data analysis of the VOC profile showed differences mainly in acetic acid and butyric acid able to discriminate the groups Afmid and Cushing's mice. Moreover, multivariate data analysis revealed statistically significant differences in VOCs between Cushing's mice/wild-type (WT) littermates, mainly short-chain fatty acids (SCFAs), ketones, and alcohols, and longitudinal differences mainly attributed to methanol, ethanol and acetone. Afmid mice did not present statistically significant differences in their volatile faecal metabolome when compared to their respective WT littermates. The findings suggested that mice developed a diabetic phenotype and that the altered VOC profile may imply a related change in gut microbiota, particularly in Cushing's mice. Furthermore, this study provided major evidence of age-related changes on the volatile profile of diabetic mice.
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http://dx.doi.org/10.1038/s41598-019-55339-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906526PMC
December 2019

The development of a high throughput drug-responsive model of white adipose tissue comprising adipogenic 3T3-L1 cells in a 3D matrix.

Biofabrication 2019 12 11;12(1):015018. Epub 2019 Dec 11.

OxSyBio Ltd, Building R27, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire, OX11 0QX, United Kingdom.

Adipose models have been applied to mechanistic studies of metabolic diseases (such as diabetes) and the subsequent discovery of new therapeutics. However, typical models are either insufficiently complex (2D cell cultures) or expensive and labor intensive (mice/in vivo). To bridge the gap between these models and in order to better inform pre-clinical studies we have developed a drug-responsive 3D model of white adipose tissue (WAT). Here, spheroids (680 ± 60 μm) comprising adipogenic 3T3-L1 cells encapsulated in 3D matrix were fabricated manually on a 96 well scale. Spheroids were highly characterised for lipid morphology, selected metabolite and adipokine secretion, and gene expression; displaying significant upregulation of certain adipogenic-specific genes compared with a 2D model. Furthermore, induction of lipolysis and promotion of lipogenesis in spheroids could be triggered by exposure to 8-br-cAMP and oleic-acid respectively. Metabolic and high content imaging data of spheroids exposed to an adipose-targeting drug, rosiglitazone, resulted in dose-responsive behavior. Thus, our 3D WAT model has potential as a powerful scalable tool for compound screening and for investigating adipose biology.
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http://dx.doi.org/10.1088/1758-5090/ab56feDOI Listing
December 2019

Abcc5 Knockout Mice Have Lower Fat Mass and Increased Levels of Circulating GLP-1.

Obesity (Silver Spring) 2019 08;27(8):1292-1304

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.

Objective: A previous genome-wide association study linked overexpression of an ATP-binding cassette transporter, ABCC5, in humans with a susceptibility to developing type 2 diabetes with age. Specifically, ABCC5 gene overexpression was shown to be strongly associated with increased visceral fat mass and reduced peripheral insulin sensitivity. Currently, the role of ABCC5 in diabetes and obesity is unknown. This study reports the metabolic phenotyping of a global Abcc5 knockout mouse.

Methods: A global Abcc5 mouse was generated by CRISPR/Cas9. Fat mass was determined by weekly EchoMRI and fat pads were dissected and weighed at week 18. Glucose homeostasis was ascertained by an oral glucose tolerance test, intraperitoneal glucose tolerance test, and intraperitoneal insulin tolerance test. Energy expenditure and locomotor activity were measured using PhenoMaster cages. Glucagon-like peptide 1 (GLP-1) levels in plasma, primary gut cell cultures, and GLUTag cells were determined by enzyme-linked immunosorbent assay.

Results: Abcc5 mice had decreased fat mass and increased plasma levels of GLP-1, and they were more insulin sensitive and more active. Recombinant overexpression of ABCC5 protein in GLUTag cells decreased GLP-1 release.

Conclusions: ABCC5 protein expression levels are inversely related to fat mass and appear to play a role in the regulation of GLP-1 secretion from enteroendocrine cells.
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http://dx.doi.org/10.1002/oby.22521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658130PMC
August 2019

Mice with a Brd4 Mutation Represent a New Model of Nephrocalcinosis.

J Bone Miner Res 2019 07 4;34(7):1324-1335. Epub 2019 Mar 4.

Academic Endocrine Unit, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

Nephrolithiasis (NL) and nephrocalcinosis (NC), which comprise renal calcification of the collecting system and parenchyma, respectively, have a multifactorial etiology with environmental and genetic determinants and affect ∼10% of adults by age 70 years. Studies of families with hereditary NL and NC have identified >30 causative genes that have increased our understanding of extracellular calcium homeostasis and renal tubular transport of calcium. However, these account for <20% of the likely genes that are involved, and to identify novel genes for renal calcification disorders, we investigated 1745 12-month-old progeny from a male mouse that had been treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for radiological renal opacities. This identified a male mouse with renal calcification that was inherited as an autosomal dominant trait with >80% penetrance in 152 progeny. The calcification consisted of calcium phosphate deposits in the renal papillae and was associated with the presence of the urinary macromolecules osteopontin and Tamm-Horsfall protein, which are features found in Randall's plaques of patients with NC. Genome-wide mapping located the disease locus to a ∼30 Mbp region on chromosome 17A3.3-B3 and whole-exome sequence analysis identified a heterozygous mutation, resulting in a missense substitution (Met149Thr, M149T), in the bromodomain-containing protein 4 (BRD4). The mutant heterozygous (Brd4 ) mice, when compared with wild-type (Brd4 ) mice, were normocalcemic and normophosphatemic, with normal urinary excretions of calcium and phosphate, and had normal bone turnover markers. BRD4 plays a critical role in histone modification and gene transcription, and cDNA expression profiling, using kidneys from Brd4 and Brd4 mice, revealed differential expression of genes involved in vitamin D metabolism, cell differentiation, and apoptosis. Kidneys from Brd4 mice also had increased apoptosis at sites of calcification within the renal papillae. Thus, our studies have established a mouse model, due to a Brd4 Met149Thr mutation, for inherited NC. © 2019 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658219PMC
July 2019

A Wars2 Mutant Mouse Model Displays OXPHOS Deficiencies and Activation of Tissue-Specific Stress Response Pathways.

Cell Rep 2018 12;25(12):3315-3328.e6

MRC Harwell Institute, Mammalian Genetics Unit and Mary Lyon Centre, Harwell Campus, Oxfordshire OX11 0RD, UK. Electronic address:

Mutations in genes essential for mitochondrial function have pleiotropic effects. The mechanisms underlying these traits yield insights into metabolic homeostasis and potential therapies. Here we report the characterization of a mouse model harboring a mutation in the tryptophanyl-tRNA synthetase 2 (Wars2) gene, encoding the mitochondrial-localized WARS2 protein. This hypomorphic allele causes progressive tissue-specific pathologies, including hearing loss, reduced adiposity, adipose tissue dysfunction, and hypertrophic cardiomyopathy. We demonstrate the tissue heterogeneity arises as a result of variable activation of the integrated stress response (ISR) pathway and the ability of certain tissues to respond to impaired mitochondrial translation. Many of the systemic metabolic effects are likely mediated through elevated fibroblast growth factor 21 (FGF21) following activation of the ISR in certain tissues. These findings demonstrate the potential pleiotropy associated with Wars2 mutations in patients.
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http://dx.doi.org/10.1016/j.celrep.2018.11.080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315286PMC
December 2018

An N-Ethyl-N-Nitrosourea (ENU)-Induced Tyr265Stop Mutation of the DNA Polymerase Accessory Subunit Gamma 2 (Polg2) Is Associated With Renal Calcification in Mice.

J Bone Miner Res 2019 03 14;34(3):497-507. Epub 2018 Dec 14.

Academic Endocrine Unit, Oxford Centre for Diabetes, Endocrinology and Metabolism, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

Renal calcification (RCALC) resulting in nephrolithiasis and nephrocalcinosis, which affects ∼10% of adults by 70 years of age, involves environmental and genetic etiologies. Thus, nephrolithiasis and nephrocalcinosis occurs as an inherited disorder in ∼65% of patients, and may be associated with endocrine and metabolic disorders including: primary hyperparathyroidism, hypercalciuria, renal tubular acidosis, cystinuria, and hyperoxaluria. Investigations of families with nephrolithiasis and nephrocalcinosis have identified some causative genes, but further progress is limited as large families are unavailable for genetic studies. We therefore embarked on establishing mouse models for hereditary nephrolithiasis and nephrocalcinosis by performing abdominal X-rays to identify renal opacities in N-ethyl-N-nitrosourea (ENU)-mutagenized mice. This identified a mouse with RCALC inherited as an autosomal dominant trait, designated RCALC type 2 (RCALC2). Genomewide mapping located the Rcalc2 locus to a ∼16-Mbp region on chromosome 11D-E2 and whole-exome sequence analysis identified a heterozygous mutation in the DNA polymerase gamma-2, accessory subunit (Polg2) resulting in a nonsense mutation, Tyr265Stop (Y265X), which co-segregated with RCALC2. Kidneys of mutant mice (Polg2 ) had lower POLG2 mRNA and protein expression, compared to wild-type littermates (Polg2 ). The Polg2 and Polg2 mice had similar plasma concentrations of sodium, potassium, calcium, phosphate, chloride, urea, creatinine, glucose, and alkaline phosphatase activity; and similar urinary fractional excretion of calcium, phosphate, oxalate, and protein. Polg2 encodes the minor subunit of the mitochondrial DNA (mtDNA) polymerase and the mtDNA content in Polg2 kidneys was reduced compared to Polg2 mice, and cDNA expression profiling revealed differential expression of 26 genes involved in several biological processes including mitochondrial DNA function, apoptosis, and ubiquitination, the complement pathway, and inflammatory pathways. In addition, plasma of Polg2 mice, compared to Polg2 littermates had higher levels of reactive oxygen species. Thus, our studies have identified a mutant mouse model for inherited renal calcification associated with a Polg2 nonsense mutation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jbmr.3624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446808PMC
March 2019

An -Ethyl--Nitrosourea (ENU) Mutagenized Mouse Model for Autosomal Dominant Nonsyndromic Kyphoscoliosis Due to Vertebral Fusion.

JBMR Plus 2018 May 8;2(3):154-163. Epub 2018 Mar 8.

Academic Endocrine Unit Radcliffe Department of Medicine University of Oxford Oxford Centre for Diabetes, Endocrinology and Metabolism Churchill Hospital Headington UK.

Kyphosis and scoliosis are common spinal disorders that occur as part of complex syndromes or as nonsyndromic, idiopathic diseases. Familial and twin studies implicate genetic involvement, although the causative genes for idiopathic kyphoscoliosis remain to be identified. To facilitate these studies, we investigated progeny of mice treated with the chemical mutagen -ethyl--nitrosourea (ENU) and assessed them for morphological and radiographic abnormalities. This identified a mouse with kyphoscoliosis due to fused lumbar vertebrae, which was inherited as an autosomal dominant trait; the phenotype was designated as hereditary vertebral fusion (HVF) and the locus as . Micro-computed tomography (μCT) analysis confirmed the occurrence of nonsyndromic kyphoscoliosis due to fusion of lumbar vertebrae in HVF mice, consistent with a pattern of blocked vertebrae due to failure of segmentation. μCT scans also showed the lumbar vertebral column of HVF mice to have generalized disc narrowing, displacement with compression of the neural spine, and distorted transverse processes. Histology of lumbar vertebrae revealed HVF mice to have irregularly shaped vertebral bodies and displacement of intervertebral discs and ossification centers. Genetic mapping using a panel of single nucleotide polymorphic (SNP) loci arranged in chromosome sets and DNA samples from 23 HVF (eight males and 15 females) mice, localized to chromosome 4A3 and within a 5-megabase (Mb) region containing nine protein coding genes, two processed transcripts, three microRNAs, five small nuclear RNAs, three large intergenic noncoding RNAs, and 24 pseudogenes. However, genome sequence analysis in this interval did not identify any abnormalities in the coding exons, or exon-intron boundaries of any of these genes. Thus, our studies have established a mouse model for a monogenic form of nonsyndromic kyphoscoliosis due to fusion of lumbar vertebrae, and further identification of the underlying genetic defect will help elucidate the molecular mechanisms involved in kyphoscoliosis. © 2018 The Authors. is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbm4.10033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124210PMC
May 2018

Molecular reductions in glucokinase activity increase counter-regulatory responses to hypoglycemia in mice and humans with diabetes.

Mol Metab 2018 11 13;17:17-27. Epub 2018 Aug 13.

Wellcome Trust/ MRC Institute of Metabolic Science and Department of Medicine, University of Cambridge, United Kingdom. Electronic address:

Objective: Appropriate glucose levels are essential for survival; thus, the detection and correction of low blood glucose is of paramount importance. Hypoglycemia prompts an integrated response involving reduction in insulin release and secretion of key counter-regulatory hormones glucagon and epinephrine that together promote endogenous glucose production to restore normoglycemia. However, specifically how this response is orchestrated remains to be fully clarified. The low affinity hexokinase glucokinase is found in glucose-sensing cells involved in glucose homeostasis including pancreatic β-cells and in certain brain areas. Here, we aimed to examine the role of glucokinase in triggering counter-regulatory hormonal responses to hypoglycemia, hypothesizing that reduced glucokinase activity would lead to increased and/or earlier triggering of responses.

Methods: Hyperinsulinemic glucose clamps were performed to examine counter-regulatory responses to controlled hypoglycemic challenges created in humans with monogenic diabetes resulting from heterozygous glucokinase mutations (GCK-MODY). To examine the relative importance of glucokinase in different sensing areas, we then examined responses to clamped hypoglycemia in mice with molecularly defined disruption of whole body and/or brain glucokinase.

Results: GCK-MODY patients displayed increased and earlier glucagon responses during hypoglycemia compared with a group of glycemia-matched patients with type 2 diabetes. Consistent with this, glucagon responses to hypoglycemia were also increased in I366F mice with mutated glucokinase and in streptozotocin-treated β-cell ablated diabetic I366F mice. Glucagon responses were normal in conditional brain glucokinase-knockout mice, suggesting that glucagon release during hypoglycemia is controlled by glucokinase-mediated glucose sensing outside the brain but not in β-cells. For epinephrine, we found increased responses in GCK-MODY patients, in β-cell ablated diabetic I366F mice and in conditional (nestin lineage) brain glucokinase-knockout mice, supporting a role for brain glucokinase in triggering epinephrine release.

Conclusions: Our data suggest that glucokinase in brain and other non β-cell peripheral hypoglycemia sensors is important in glucose homeostasis, allowing the body to detect and respond to a falling blood glucose.
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http://dx.doi.org/10.1016/j.molmet.2018.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197723PMC
November 2018

Regulatory variants at KLF14 influence type 2 diabetes risk via a female-specific effect on adipocyte size and body composition.

Nat Genet 2018 04 9;50(4):572-580. Epub 2018 Apr 9.

Department of Medicine, Huddinge, Karolinska Institutet, Stockholm, Sweden.

Individual risk of type 2 diabetes (T2D) is modified by perturbations to the mass, distribution and function of adipose tissue. To investigate the mechanisms underlying these associations, we explored the molecular, cellular and whole-body effects of T2D-associated alleles near KLF14. We show that KLF14 diabetes-risk alleles act in adipose tissue to reduce KLF14 expression and modulate, in trans, the expression of 385 genes. We demonstrate, in human cellular studies, that reduced KLF14 expression increases pre-adipocyte proliferation but disrupts lipogenesis, and in mice, that adipose tissue-specific deletion of Klf14 partially recapitulates the human phenotype of insulin resistance, dyslipidemia and T2D. We show that carriers of the KLF14 T2D risk allele shift body fat from gynoid stores to abdominal stores and display a marked increase in adipocyte cell size, and that these effects on fat distribution, and the T2D association, are female specific. The metabolic risk associated with variation at this imprinted locus depends on the sex both of the subject and of the parent from whom the risk allele derives.
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http://dx.doi.org/10.1038/s41588-018-0088-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5935235PMC
April 2018

N-ethyl-N-nitrosourea-Induced Adaptor Protein 2 Sigma Subunit 1 () Mutations Establish Loss-of-Function Mice.

JBMR Plus 2017 May;1(1):3-15

Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology, and Metabolism (OCDEM), Churchill Hospital, Oxford, UK.

The adaptor protein-2 sigma subunit (AP2σ), encoded by , forms a heterotetrameric complex, with AP2α, AP2β, and AP2μ subunits, that is pivotal for clathrin-mediated endocytosis, and AP2σ loss-of-function mutations impair internalization of the calcium-sensing receptor (CaSR), a G-protein-coupled receptor, and cause familial hypocalciuric hypercalcemia type-3 (FHH3). Mice with AP2σ mutations that would facilitate investigations of the in vivo role of AP2σ, are not available, and we therefore embarked on establishing such mice. We screened >10,000 mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) for mutations and identified 5 variants, comprising 2 missense (Tyr20Asn and Ile123Asn) and 3 intronic base substitutions, one of which altered the invariant donor splice site dinucleotide to . Three-dimensional modeling and cellular expression of the missense variants did not reveal them to alter AP2σ structure or CaSR-mediated signaling, but investigation of the donor splice site variant revealed it to result in an in-frame deletion of 17 evolutionarily conserved amino acids (del17) that formed part of the AP2σ α1-helix, α1-β3 loop, and β3 strand. Heterozygous mutant mice ( ) were therefore established, and these had AP2σ haplosufficiency but were viable with normal appearance and growth. mice, when compared with mice, also had normal plasma concentrations of calcium, phosphate, magnesium, creatinine, urea, sodium, potassium, and alkaline phosphatase activity; normal urinary fractional excretion of calcium, phosphate, sodium, and potassium; and normal plasma parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D (1,25(OH)) concentrations. However, homozygous mice were non-viable and died between embryonic days 3.5 and 9.5 (E3.5-9.5), thereby indicating that AP2σ likely has important roles at the embryonic patterning stages and organogenesis of the heart, thyroid, liver, gut, lungs, pancreas, and neural systems. Thus, our studies have established a mutant mouse model that is haplosufficient for AP2σ.
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http://dx.doi.org/10.1002/jbm4.10001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5824975PMC
May 2017

FTO demethylase activity is essential for normal bone growth and bone mineralization in mice.

Biochim Biophys Acta Mol Basis Dis 2018 Mar 2;1864(3):843-850. Epub 2017 Dec 2.

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK. Electronic address:

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http://dx.doi.org/10.1016/j.bbadis.2017.11.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798602PMC
March 2018

Disruption of the homeodomain transcription factor orthopedia homeobox (Otp) is associated with obesity and anxiety.

Mol Metab 2017 11 24;6(11):1419-1428. Epub 2017 Aug 24.

MRC Harwell Institute, Mammalian Genetics Unit and Mary Lyon Centre, Harwell Campus, Oxfordshire, OX11 0RD, UK. Electronic address:

Objective: Genetic studies in obese rodents and humans can provide novel insights into the mechanisms involved in energy homeostasis.

Methods: In this study, we genetically mapped the chromosomal region underlying the development of severe obesity in a mouse line identified as part of a dominant N-ethyl-N-nitrosourea (ENU) mutagenesis screen. We characterized the metabolic and behavioral phenotype of obese mutant mice and examined changes in hypothalamic gene expression. In humans, we examined genetic data from people with severe early onset obesity.

Results: We identified an obese mouse heterozygous for a missense mutation (pR108W) in orthopedia homeobox (Otp), a homeodomain containing transcription factor required for the development of neuroendocrine cell lineages in the hypothalamus, a region of the brain important in the regulation of energy homeostasis. Otp mice exhibit increased food intake, weight gain, and anxiety when in novel environments or singly housed, phenotypes that may be partially explained by reduced hypothalamic expression of oxytocin and arginine vasopressin. R108W affects the highly conserved homeodomain, impairs DNA binding, and alters transcriptional activity in cells. We sequenced OTP in 2548 people with severe early-onset obesity and found a rare heterozygous loss of function variant in the homeodomain (Q153R) in a patient who also had features of attention deficit disorder.

Conclusions: OTP is involved in mammalian energy homeostasis and behavior and appears to be necessary for the development of hypothalamic neural circuits. Further studies will be needed to investigate the contribution of rare variants in OTP to human energy homeostasis.
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http://dx.doi.org/10.1016/j.molmet.2017.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681237PMC
November 2017

Cinacalcet corrects hypercalcemia in mice with an inactivating Gα11 mutation.

JCI Insight 2017 10 19;2(20). Epub 2017 Oct 19.

Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.

Loss-of-function mutations of GNA11, which encodes G-protein subunit α11 (Gα11), a signaling partner for the calcium-sensing receptor (CaSR), result in familial hypocalciuric hypercalcemia type 2 (FHH2). FHH2 is characterized by hypercalcemia, inappropriately normal or raised parathyroid hormone (PTH) concentrations, and normal or low urinary calcium excretion. A mouse model for FHH2 that would facilitate investigations of the in vivo role of Gα11 and the evaluation of calcimimetic drugs, which are CaSR allosteric activators, is not available. We therefore screened DNA from > 10,000 mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for GNA11 mutations and identified a Gα11 variant, Asp195Gly (D195G), which downregulated CaSR-mediated intracellular calcium signaling in vitro, consistent with it being a loss-of-function mutation. Treatment with the calcimimetic cinacalcet rectified these signaling responses. In vivo studies showed mutant heterozygous (Gna11+/195G) and homozygous (Gna11195G/195G) mice to be hypercalcemic with normal or increased plasma PTH concentrations and normal urinary calcium excretion. Cinacalcet (30mg/kg orally) significantly reduced plasma albumin-adjusted calcium and PTH concentrations in Gna11+/195G and Gna11195G/195G mice. Thus, our studies have established a mouse model with a germline loss-of-function Gα11 mutation that is representative for FHH2 in humans and demonstrated that cinacalcet can correct the associated abnormalities of plasma calcium and PTH.
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http://dx.doi.org/10.1172/jci.insight.96540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846897PMC
October 2017

Mutant Mice With Calcium-Sensing Receptor Activation Have Hyperglycemia That Is Rectified by Calcilytic Therapy.

Endocrinology 2017 08;158(8):2486-2502

Radcliffe Department of Medicine, Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford OX3 7LE, United Kingdom.

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor that plays a pivotal role in extracellular calcium homeostasis. The CaSR is also highly expressed in pancreatic islet α- and β-cells that secrete glucagon and insulin, respectively. To determine whether the CaSR may influence systemic glucose homeostasis, we characterized a mouse model with a germline gain-of-function CaSR mutation, Leu723Gln, referred to as Nuclear flecks (Nuf). Heterozygous- (CasrNuf/+) and homozygous-affected (CasrNuf/Nuf) mice were shown to have hypocalcemia in association with impaired glucose tolerance and insulin secretion. Oral administration of a CaSR antagonist compound, known as a calcilytic, rectified the glucose intolerance and hypoinsulinemia of CasrNuf/+ mice and ameliorated glucose intolerance in CasrNuf/Nuf mice. Ex vivo studies showed CasrNuf/+ and CasrNuf/Nuf mice to have reduced pancreatic islet mass and β-cell proliferation. Electrophysiological analysis of isolated CasrNuf/Nuf islets showed CaSR activation to increase the basal electrical activity of β-cells independently of effects on the activity of the adenosine triphosphate (ATP)-sensitive K+ (KATP) channel. CasrNuf/Nuf mice also had impaired glucose-mediated suppression of glucagon secretion, which was associated with increased numbers of α-cells and a higher α-cell proliferation rate. Moreover, CasrNuf/Nuf islet electrophysiology demonstrated an impairment of α-cell membrane depolarization in association with attenuated α-cell basal KATP channel activity. These studies indicate that the CaSR activation impairs glucose tolerance by a combination of α- and β-cell defects and also influences pancreatic islet mass. Moreover, our findings highlight a potential application of targeted CaSR compounds for modulating glucose metabolism.
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http://dx.doi.org/10.1210/en.2017-00111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5551547PMC
August 2017

A mouse model for inherited renal fibrosis associated with endoplasmic reticulum stress.

Dis Model Mech 2017 06 21;10(6):773-786. Epub 2017 Mar 21.

Academic Endocrine Unit, University of Oxford, Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Headington, Oxford OX3 7LJ, UK

Renal fibrosis is a common feature of renal failure resulting from multiple etiologies, including diabetic nephropathy, hypertension and inherited renal disorders. However, the mechanisms of renal fibrosis are incompletely understood and we therefore explored these by establishing a mouse model for a renal tubular disorder, referred to as autosomal dominant tubulointerstitial kidney disease (ADTKD) due to missense uromodulin () mutations (ADTKD-). ADTKD-, which is associated with retention of mutant uromodulin in the endoplasmic reticulum (ER) of renal thick ascending limb cells, is characterized by hyperuricemia, interstitial fibrosis, inflammation and renal failure, and we used targeted homologous recombination to generate a knock-in mouse model with an ADTKD-causing missense cysteine to arginine uromodulin mutation (C125R). Heterozygous and homozygous mutant mice developed reduced uric acid excretion, renal fibrosis, immune cell infiltration and progressive renal failure, with decreased maturation and excretion of uromodulin, due to its retention in the ER. The ER stress marker 78 kDa glucose-regulated protein (GRP78) was elevated in cells expressing mutant uromodulin in heterozygous and homozygous mutant mice, and this was accompanied, both and , by upregulation of two unfolded protein response pathways in primary thick ascending limb cells from homozygous mutant mice. However, this did not lead to an increase in apoptosis Thus, we have developed a novel mouse model for renal fibrosis, which will be a valuable resource to decipher the mechanisms linking uromodulin mutations with ER stress and renal fibrosis.
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http://dx.doi.org/10.1242/dmm.029488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5483009PMC
June 2017

Mouse Models of Human GWAS Hits for Obesity and Diabetes in the Post Genomic Era: Time for Reevaluation.

Front Endocrinol (Lausanne) 2017 7;8:11. Epub 2017 Feb 7.

Mammalian Genetics Unit, Medical Research Council Harwell Institute , Oxfordshire , UK.

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http://dx.doi.org/10.3389/fendo.2017.00011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5294391PMC
February 2017

G mutation in mice causes hypocalcemia rectifiable by calcilytic therapy.

JCI Insight 2017 02 9;2(3):e91103. Epub 2017 Feb 9.

Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.

Heterozygous germline gain-of-function mutations of G-protein subunit α (Gα), a signaling partner for the calcium-sensing receptor (CaSR), result in autosomal dominant hypocalcemia type 2 (ADH2). ADH2 may cause symptomatic hypocalcemia with low circulating parathyroid hormone (PTH) concentrations. Effective therapies for ADH2 are currently not available, and a mouse model for ADH2 would help in assessment of potential therapies. We hypothesized that a previously reported dark skin mouse mutant () - which has a germline hypermorphic Gα mutation, Ile62Val - may be a model for ADH2 and allow evaluation of calcilytics, which are CaSR negative allosteric modulators, as a targeted therapy for this disorder. Mutant and mice were shown to have hypocalcemia and reduced plasma PTH concentrations, similar to ADH2 patients. In vitro studies showed the mutant Val62 Gα to upregulate CaSR-mediated intracellular calcium and MAPK signaling, consistent with a gain of function. Treatment with NPS-2143, a calcilytic compound, normalized these signaling responses. In vivo, NPS-2143 induced a rapid and marked rise in plasma PTH and calcium concentrations in and mice, which became normocalcemic. Thus, these studies have established mice, which harbor a germline gain-of-function Gα mutation, as a model for ADH2 and have demonstrated calcilytics as a potential targeted therapy.
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http://dx.doi.org/10.1172/jci.insight.91103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291742PMC
February 2017

Mice with an N-Ethyl-N-Nitrosourea (ENU) Induced Tyr209Asn Mutation in Natriuretic Peptide Receptor 3 (NPR3) Provide a Model for Kyphosis Associated with Activation of the MAPK Signaling Pathway.

PLoS One 2016 13;11(12):e0167916. Epub 2016 Dec 13.

Oxford Centre for Diabetes, Endocrinology and Metabolism, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom.

Non-syndromic kyphosis is a common disorder that is associated with significant morbidity and has a strong genetic involvement; however, the causative genes remain to be identified, as such studies are hampered by genetic heterogeneity, small families and various modes of inheritance. To overcome these limitations, we investigated 12 week old progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) using phenotypic assessments including dysmorphology, radiography, and dual-energy X-ray absorptiometry. This identified a mouse with autosomal recessive kyphosis (KYLB). KYLB mice, when compared to unaffected littermates, had: thoraco-lumbar kyphosis, larger vertebrae, and increased body length and increased bone area. In addition, female KYLB mice had increases in bone mineral content and plasma alkaline phosphatase activity. Recombination mapping localized the Kylb locus to a 5.5Mb region on chromosome 15A1, which contained 51 genes, including the natriuretic peptide receptor 3 (Npr3) gene. DNA sequence analysis of Npr3 identified a missense mutation, Tyr209Asn, which introduced an N-linked glycosylation consensus sequence. Expression of wild-type NPR3 and the KYLB-associated Tyr209Asn NPR3 mutant in COS-7 cells demonstrated the mutant to be associated with abnormal N-linked glycosylation and retention in the endoplasmic reticulum that resulted in its absence from the plasma membrane. NPR3 is a decoy receptor for C-type natriuretic peptide (CNP), which also binds to NPR2 and stimulates mitogen-activated protein kinase (MAPK) signaling, thereby increasing the number and size of hypertrophic chondrocytes. Histomorphometric analysis of KYLB vertebrae and tibiae showed delayed endochondral ossification and expansion of the hypertrophic zones of the growth plates, and immunohistochemistry revealed increased p38 MAPK phosphorylation throughout the growth plates of KYLB vertebrae. Thus, we established a model of kyphosis due to a novel NPR3 mutation, in which loss of plasma membrane NPR3 expression results in increased MAPK pathway activation, causing elongation of the vertebrae and resulting in kyphosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167916PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154531PMC
June 2017

Islet Insulin Secretion Measurements in the Mouse.

Curr Protoc Mouse Biol 2016 Sep 1;6(3):256-271. Epub 2016 Sep 1.

Mammalian Genetics Unit, Medical Research Council Harwell, Harwell Science and Innovation Campus, Oxfordshire, United Kingdom.

This article describes detailed protocols for in vitro measurements of insulin function and secretion in isolated mouse islets for the analysis of glucose homeostasis. We specify a method of enzyme digestion and hand picking to isolate and release the greatest number of high quality islets from the pancreas of the mouse. We describe an effective method for generating dynamic measurements of insulin secretion using a perifusion assay including a detailed protocol for constructing a peristaltic pump and tubing assembly. In addition we describe an alternative and simple technique for measuring insulin secretion using static incubation of isolated islets. © 2016 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpmo.14DOI Listing
September 2016
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