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Steroid 5β-reductase (AKR1D1) plays important role in hepatic bile acid synthesis and glucocorticoid clearance. Bile acids and glucocorticoids are potent metabolic regulators, but whether AKR1D1 controls metabolic phenotype in vivo is unknown. Akr1d1–/– mice were generated on a C57BL/6 background. Liquid chromatography/mass spectrometry, metabolomic and transcriptomic approaches were used to determine effects on glucocorticoid and bile acid homeostasis. Metabolic phenotypes including body weight and composition, lipid homeostasis, glucose tolerance and insulin tolerance were evaluated. Molecular changes were assessed by RNA-Seq and Western blotting. Male Akr1d1–/– mice were challenged with a high fat diet (60% kcal from fat) for 20 weeks. Akr1d1–/– mice had a sex-specific metabolic phenotype. At 30 weeks of age, male, but not female, Akr1d1–/– mice were more insulin tolerant and had reduced lipid accumulation in the liver and adipose tissue yet had hypertriglyceridemia and increased intramuscular triacylglycerol. This phenotype was associated with sexually dimorphic changes in bile acid metabolism and composition but without overt effects on circulating glucocorticoid levels or glucocorticoid-regulated gene expression in the liver. Male Akr1d1–/– mice were not protected against diet-induced obesity and insulin resistance. In conclusion, this study shows that AKR1D1 controls bile acid homeostasis in vivo and that altering its activity can affect insulin tolerance and lipid homeostasis in a sex-dependent manner.

The ATP-sensitive K+ (KATP) channel controls blood glucose levels by coupling glucose metabolism to insulin secretion in pancreatic β-cells. E23K, a common polymorphism in the pore-forming KATP channel subunit (KCNJ11) 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. KATP-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.

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. NEW & NOTEWORTHY 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.

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.

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., Summary: ARL15 (GTPase ARF family) is associated with adipose traits. ARL15 is palmitoylated, localised to Golgi in preadipocytes and translocated to other Golgi compartments during differentiation. ARL15 interacts with ER-localised ARL6IP5.

Introduction: The C57BL/6J and C57BL/6N are two of the most commonly used mouse strains in biological research. We recently discovered C57BL/6N males develop a modest dilated cardiomyopathy (DCM) by 12 months, which is absent from the closely related C57BL/6J. We identified a variant (Chr8:85365179A>T, c.-5T>A) in myosin light chain kinase 3 ( Mylk3 ) in the 5’ UTR of the primary isoform resident to C57BL/6N substrains, which abolishes translation of this isoform. Myosin light chain 2 (MYL2) is the primary target of MYLK3, and its phosphorylation is vital for survival. Hypothesis: Loss of MYLK3 expression alters phosphorylation of key cardiac proteins. Results: Surprisingly, we found no difference in MYL2 phosphorylation by western blot between C57BL/6N and C57BL/6J hearts, at any of the reported target residues. Phosphoproteomics corroborated this, but highlighted significantly different phosphorylation at a number of phosphosites in other proteins linked to cardiomyopathies and cardiac development, particularly a reduction of a number of residues in ACTN2. We also detected peptide sequences that mapped to the C-terminus and middle of MYLK3 in the C57BL/6N. Western blotting with a C-terminal specific antibody revealed expression of a shorter isoform, present at lower levels in both strains, that is unaffected by the variant due to a distinct N-terminus. Using this antibody in IHC showed similar spatial expression of MYLK3 in hearts from both strains, localizing to the Z-disc, although the expression was much lower in the C57BL/6N presumed to be due to lack of the more abundant full-length isoform. Using immunoprecipitation, we found both isoforms bound to MYL2 in vivo and in vitro , and both were capable of phosphorylating MYL2 in vitro. However, the shorter isoform was not as readily bound to alpha-actinin-2 (ACTN2), a key sarcomeric protein that is reported to bind full-length MYLK3 isoform by its N-terminus. Conclusions: This data indicates loss of full-length MYLK3 causes DCM via a MYL2-independent mechanism, possibly due to lack of phosphorylation of alternative target(s), such as ACTN2. The expression of the shorter MYLK3 isoform in C57BL/6N mice may explain the persistence of MYL2 phosphorylation in these mice and enable their survival.

The obesity-linked FTO regulatory circuitry shows cross-species molecular, cellular, metabolic, and organismal conservation., 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 Irx3 and Irx5 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.

White adipose tissue (WAT) plays a central role in metabolism, with multiple diseases and genetic mutations causing its remodeling. Quantitative analysis of white adipocyte size is of great interest to understand physiology and disease, but previous studies of H&E histology have been limited to a subsample of whole depot cross-sections. In this paper, we present the deep learning pipeline DeepCytometer, that can segment mouse and human whole slides (≃40,000 cells per mouse slide on average) using an adaptive tiling method, correct for cell overlap and reject non-white adipocytes from the segmentation. Using quantile colour maps we show intra- and inter-depot cell size heterogeneity with local correlation; quantile estimates also suggest significant differences in population estimates from 75 whole slides compared to smaller data sets. We propose three linked levels (body weight BW, depot weight DW and cell area quartiles) for exploratory analysis of mouse Klf14 phenotypes in gonadal and subcutaneous depots. We find a rich set of phenotypes when stratifying by sex, depot and three genotype strata: (1) WTs/Hets with a Het father (Controls), (2) WTs with a Het mother, and (3) Hets with a Het mother (functional KOs or FKOs). Namely, at BW level, mean difference testing suggests that female FKOs are similar to Controls, but WTs with a Het mother are significantly larger. At DW and cell levels, linear models with interaction terms and BW or DW covariates, respectively, reveal phenotypes not shown by difference of means tests. For example, at DW level, gonadal and subcutaneous models are similar, and female FKOs have lower fat percentage than Controls due to both an offset and the DW/BW slope in the linear model. Meanwhile, female WTs with a Het mother have on average similar fat percentage to Controls, but as their slopes are close to zero, their DWs are uncorrelated to BW, suggesting that larger female WTs with a Het mother have lower fat percentage than smaller ones. In contrast to depot level, at cell level female gonadal phenotypes diverge from subcutaneous ones. Furthermore, male Controls and FKOs have similar average area values in subcutaneous depots, but area~DW slope flattening in FKOs suggests that larger DWs could be caused by cell size increase in Controls and by cell count increase in FKOs. Thus, DeepCytometer and associated exploratory analysis reveal new insights into adipocyte heterogeneity and phenotyping.

BackgroundSteroid 5β-reductase (AKR1D1) plays important roles in hepatic glucocorticoid clearance and bile acid synthesis. Glucocorticoids and bile acids are potent metabolic regulators, but whether AKR1D1 controls metabolic phenotype in vivo is unknown.MethodsAkr1d1-/-mice were generated on a C57BL/6 background. Liquid chromatography / mass spectrometry, metabolomic and transcriptomic approaches were used to determine effects on glucocorticoid and bile acid homeostasis. Metabolic phenotypes including body weight and composition, lipid homeostasis, glucose tolerance and insulin sensitivity were evaluated. Molecular changes were assessed by RNASeq and western blotting. Male Akr1d1-/-mice were challenged with a 60% high fat diet.ResultsAkr1d1-/-mice had a sex specific metabolic phenotype. At 30-weeks of age male, but not female, Akr1d1-/-mice were more insulin sensitive and had reduced lipid accumulation in the liver and adipose tissue, concomitant with hypertriglyceridemia and increased intramuscular triacylglycerol. This phenotype was underpinned by sexually dimorphic changes in bile acid metabolism and composition, but without overt effects on glucocorticoid action. Male Akr1d1-/-mice were not protected against diet induced obesity and insulin resistance.ConclusionThis study shows that AKR1D1 controls bile acid homeostasis in vivo and that altering its activity can affect insulin sensitivity and lipid homeostasis in a sex dependent manner.

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.

Summary 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., Graphical abstract, Highlights • 3q21 variants are associated with type 2 diabetes and increased bone mineral density • 3q21 variants affect chromatin accessibility in mesenchymal cells • rs56371916 is a 3q21 causal variant and ADCY5 its target in adipocytes and osteoblasts • ADCY5 and rs56371916 affect lipid oxidation processes in adipocytes and osteoblasts, Nasa Sinnott-Armstrong and colleagues identify a pleiotropic risk locus at 3q21 that is associated with type 2 diabetes (T2D) and greater bone mineral density (BMD) and its associated cell-autonomous mechanisms in adipocytes and osteoblasts. Together, these findings provide a possible explanation for the perplexing finding that individuals with T2D have higher BMD but greater susceptibility to bone fracture.

Background: Maternal over-nutrition increases the risk of diabetes and cardiovascular events in offspring. While prominent effects on cardiovascular health are observed, the impact of this on platelet physiology has not been studied. Here, we sought to determine whether maternal high-fat diet (HF) ingestion can affect the platelet function in offspring.Methods: C57BL6/N mice dams were given a HF or control (C) diet for 8 weeks prior to and during pregnancy. Male offspring also received either C or HF diets for 26 weeks. Experimental groups were: C/C, dam and offspring fed chow; C/HF dam fed chow and offspring fed high-fat diet; HF/C and HF/HF. Various phenotypic (including body weight and % of body fat) and metabolic (glycaemia, triglyceridemia) tests were performed and blood collected for platelet studies. Results: Compared to C/C, HF/HF animals were obese, with fat accumulation, hyperglycaemia, insulin resistance and low respiratory exchange rate. HF/HF, but not C/HF mice also showed hypertriglyceredaemia and higher mean platelet volume. These platelets were hyperreactive, displaying higher fibrinogen binding after stimulation with different agonists. They also showed increased platelet adhesion and spreading on collagen. Maternal obesity led to an overall effect of increased platelet reactivity in offspring. Both maternal and offspring HF groups presented decreased levels of collagen receptor GPVI with increased oxidative stress. Western blotting experiments in stimulated platelets showed increased phosphorylation of PKC substrates, total tyrosine and AKT at Ser473, whilst response to nitric oxide donor PAPA-NONOate was unchanged compared to C/C.Conclusions: Maternal HF diet ingestion programmes platelet hyperactivation in male mouse offspring, whilst HF in both dams and offspring resulted in a 'double-hit' effect of increased serum triglyceride levels, large platelets and increased reactivity. This involved enhanced Tyr phosphorylation, ROS production and decreased GPVI expression. Since platelet function can be programmed by early developmental periods, it is possible to use this window of intervention to reduce the risk of thrombotic events.

The ATP-sensitive K

Obesity is a major risk factor for a plethora of metabolic disturbances including diabetes and cardiovascular disease. Accumulating evidence is showing that there is an adipose tissue depot-dependent relationship with obesity-induced metabolic dysfunction. While some adipose depots, such as subcutaneous fat, are generally metabolically innocuous, others such as visceral fat, are directly deleterious. A lesser known visceral adipose depot is the pericardial adipose tissue depot. We therefore set out to examine its transcriptional and morphological signature under chow and high-fat fed conditions, in comparison with other adipose depots, using a mouse model. Our results revealed that under chow conditions pericardial adipose tissue has uncoupling-protein 1 gene expression levels which are significantly higher than classical subcutaneous and visceral adipose depots. We also observed that under high-fat diet conditions, the pericardial adipose depot exhibits greatly upregulated transcript levels of inflammatory cytokines. Our results collectively indicate, for the first time, that the pericardial adipose tissue possesses a unique transcriptional and histological signature which has features of both a beige (brown fat-like) but also pro-inflammatory depot, such as visceral fat. This unique profile may be involved in metabolic dysfunction associated with obesity.

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.

Individual risk of type 2 diabetes (T2D) is modified by perturbations of adipose mass, distribution and function. To investigate mechanisms responsible, 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, expression of >400 genes. We demonstrate that, in human cellular studies, reduced KLF14 expression increases pre-adipocyte proliferation but disrupts lipogenesis, and, in mice, adipose-specific deletion of Klf14 partially recapitulates the human phenotype of insulin resistance, dyslipidemia and T2D. We show that KLF14 T2D risk-allele carriers shift body fat from gynoid to abdominal stores, and display a marked increase in adipocyte cell size: these effects on fat distribution, and the T2D-association, are female-specific. Metabolic risk associated with variation at this imprinted locus depends on both the sex of the subject, and of the parent from whom the risk-allele derives The replicated genome-wide significant T2D association signal at chr7q32.3 maps to a 45kb recombination interval, extending from 3kb to 48kb upstream of KLF141,2 (Figure 1a-c). In previous work based on microarray-derived RNA expression data, KLF14, which encodes an imprinted transcription factor, was exposed as the likely cis-effector gene for this locus in subcutaneous adipose tissue1 and revealed to be a trans-regulator of a programme of adipose tissue expression3. The KLF family of zinc-finger binding proteins have wide-ranging regulatory roles in biological processes such as proliferation, differentiation and growth4,5. However, little is known about KLF14, a single exon gene whose transcription is limited to the maternally inherited chromosome in embryonic, extra-embryonic, and adult tissue in humans and mice6.

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 N-ethyl-N-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 Hvf. 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 Hvf 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. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

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. OtpR108W/+ 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., Highlights • A mouse Otp mutation alters hypothalamic neuropeptide expression leading to increased food intake, obesity and anxiety. • In severe early onset obesity, we found a heterozygous LOF variant in a patient with attention deficit disorder features. • These studies show for the first time that mutations in the Otp/OTP gene cause obesity.

The adaptor protein-2 sigma subunit (AP2σ), encoded by AP2S1, 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 Ap2s1 mutations and identified 5 Ap2s1 variants, comprising 2 missense (Tyr20Asn and Ile123Asn) and 3 intronic base substitutions, one of which altered the invariant donor splice site dinucleotide gt to gc. Three-dimensional modeling and cellular expression of the missense Ap2s1 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 (Ap2s1+/del17 ) were therefore established, and these had AP2σ haplosufficiency but were viable with normal appearance and growth. Ap2s1+/del17 mice, when compared with Ap2s1+/+ 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)2) concentrations. However, homozygous Ap2s1del17/del17 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.

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.

As bone is used in a dynamic mechanical environment, understanding the structural origins of its time-dependent mechanical behaviour – and the alterations in metabolic bone disease – is of interest. However, at the scale of the mineralized fibrillar matrix (nanometre-level), the nature of the strain-rate dependent mechanics is incompletely understood. Here, we investigate the fibrillar- and mineral-deformation behaviour in a murine model of Cushing’s syndrome, used to understand steroid induced osteoporosis, using synchrotron small- and wide-angle scattering/diffraction combined with in situ tensile testing at three strain rates ranging from 10-4 to 10-1 s-1. We find that the effective fibril- and mineral-modulus and fibrillar-reorientation show no significant increase with strain-rate in osteoporotic bone, but increase significantly in normal (wild-type) bone. By applying a fibril-lamellar two-level structural model of bone matrix deformation to fit the results, we obtain indications that altered collagen-mineral interactions at the nanoscale – along with altered fibrillar orientation distributions – may be the underlying reason for this altered strain-rate sensitivity. Our results suggest that an altered strain-rate sensitivity of the bone matrix in osteoporosis may be one of the contributing factors to reduced mechanical competence in such metabolic bone disorders, and that increasing this sensitivity may improve biomechanical performance.

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.

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss., Random mutagenesis can uncover novel genes involved in phenotypic traits. Here the authors perform a large-scale phenotypic screen on over 100 mouse strains generated by ENU mutagenesis to identify mice with age-related diseases, which they attribute to specific mutations revealed by whole-genome sequencing.

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., Highlights • SNP rs2645294 was identified by PPA as one potential functional SNP for WHRadjBMI associated with the TBX15/WARS2 locus • SNP rs2645294 has an expression QTL for the WARS2 gene • Transcripts containing SNP rs2645294 showed allelic expression imbalance in a heterozygous human white adipose cell line • SNP rs2645294 was a potential WARS2 3’UTR gene regulatory SNP • No difference in allelic RNA stability was found, imbalance likely originating from transcriptional regulatory mechanisms

Genetic differences between C57BL/6 substrains lead to different cardiovascular traits; a null mutation in Mylk3 likely causes cardiomyopathy in C57BL/6N mice, whereas C57BL/6J Nnt-null mice do not develop cardiomyopathy., The C57BL/6J and C57BL/6N mice have well-documented phenotypic and genotypic differences, including the infamous nicotinamide nucleotide transhydrogenase (Nnt) null mutation in the C57BL/6J substrain, which has been linked to cardiovascular traits in mice and cardiomyopathy in humans. To assess whether Nnt 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 (Mylk3) mutation in C57BL/6N mice, which abolishes MYLK3 protein expression. These results indicate the C57BL/6J Nnt-null mice do not develop cardiomyopathy; however, we identified a null mutation in Mylk3 as a credible cause of the cardiomyopathy phenotype in the C57BL/6N.

In recent years, genome-wide association studies (GWAS) have identified hundreds of loci and thousands of single-nucleotide polymorphisms (SNPs) associated with type 2 diabetes mellitus (T2DM) and obesity traits [such as body mass index (BMI) and waist–hip ratio (WHR)] in the human population (1–4). The vast majority of these SNPs are in non-coding regions of the genome and distal to promoters, suggesting they act through gene regulation which makes their functional interpretation challenging (5). Collectively, comparing the epigenetic landscape between mouse and human has established new pathways involved in obesity and diabetes, and in fact, inter-species conservation has successfully been used as criteria in finding functional and disease-relevant elements (6–8). By contrast, genome-wide comparative analysis of the mouse and human epigenome across tissues has highlighted the presence of cis-regulatory divergence (9, 10). New mouse engineering approaches together with bioinformatics dissection of trait-associated regions, for example, epigenetic modifications and genome interactions hold great promise to fully understand the underlying mechanisms of human disease-associated non-coding variants in T2DM and obesity

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.