Characterisation of phenotypic determinants in rat models of growth retardation

In this thesis, I describe work on the physiological basis of dwarfism in both established (dw/dw) and novel (78N) rat models. In the dwarf (dw/dw) rat, an unknown mutation causes a severe, sub-total growth hormone (GH) deficiency associated with somatotroph hypoplasia, and an increase in lactotroph numbers and prolactin (PRL) storage. It has, recently, been shown that the dw/dw pituitary contains a unique, morphologically distinct lactotroph, the 'intermediate' lactotroph, which has morphological features between those of the type I and II lactotroph subtypes. These 'intermediate' lactotrophs also show functional features of somatotrophic lineage, releasing PRL in response to ghrelin. This female-specific induction of PRL secretion is oestrogen-dependent. The present study reveals that in pregnancy the population of 'intermediate' lactotrophs increases in parallel with the lactotrophic lineage. We have taken two approaches to investigate whether elevated GH releasing factor (GRF) gives rise to the 'intermediate' lactotroph in the dw/dw pituitary. The absence of the 'intermediate' lactotroph in the GRF-insensitive little (lit/lit) mouse, and a possible reduction in 'intermediate' lactotrophs in monosodium glutamate (MSG)-treated dw/dw rats, suggest that the presence of this unique lactotroph subtype in dw/dw pituitary is regulated, in part by GRF. Although small numbers of 'intermediate' lactotrophs are present in pregnant normal female rats, they do not appear to play a significant role. Pregnancy is also associated with an increase in type I lactotrophs, but no change in somatotrophs. The accompanying increase in baseline circulating GH does not elevate plasma insulin like growth factor I (IGF-I) levels, or stimulate skeletal growth, and may be derived from a placental GH-like protein. A novel transgenic rat line---78N---was previously generated in our laboratory. It is a presumed insertional-mutant that exhibits a pleiotropic phenotype associated with neonatal male lethality. Females exhibit juvenile-onset growth retardation. Initial investigation of the cause of the growth retardation showed that the skeletal impairment is GH-independent. In the present study, parallel postmortem examination has revealed kidney abnormalities in both male and female mutants. Morphological analysis showed alteration of the normal cortico-medullary architecture. Molecular analysis of the neonatal male kidney transcriptome revealed numerous differentially expressed genes including kidney androgen-regulated protein (KAP) and neural precursor cells expressed developmentally down-regulated 4 (Nedd4) WW domain binding protein (N4WBP4). The 78N line may be a useful model of idiopathic short stature (ISS) associated with idiopathic nephritic syndrome---minimal change nephropathy (MCD) or disease

Fetus-derived DLK1 is required for maternal metabolic adaptations to pregnancy and is associated with fetal growth restriction.

Pregnancy is a state of high metabolic demand. Fasting diverts metabolism to fatty acid oxidation, and the fasted response occurs much more rapidly in pregnant women than in non-pregnant women. The product of the imprinted DLK1 gene (delta-like homolog 1) is an endocrine signaling molecule that reaches a high concentration in the maternal circulation during late pregnancy. By using mouse models with deleted Dlk1, we show that the fetus is the source of maternal circulating DLK1. In the absence of fetally derived DLK1, the maternal fasting response is impaired. Furthermore, we found that maternal circulating DLK1 levels predict embryonic mass in mice and can differentiate healthy small-for-gestational-age (SGA) infants from pathologically small infants in a human cohort. Therefore, measurement of DLK1 concentration in maternal blood may be a valuable method for diagnosing human disorders associated with impaired DLK1 expression and to predict poor intrauterine growth and complications of pregnancy.M.A.M.C. was supported by a PhD studentship from the Cambridge Centre for Trophoblast Research. Research was supported by grants from the MRC (MR/J001597/1 and MR/L002345/1), the Medical College of Saint Bartholomew's Hospital Trust, a Wellcome Trust Investigator Award, EpigeneSys (FP7 Health-257082), EpiHealth (FP7 Health-278414), a Herchel Smith Fellowship (N.T.) and NIH grant RO1 DK89989. The contents are the authors' sole responsibility and do not necessarily represent official NIH views. We thank G. Burton for invaluable support, and M. Constância and I. Sandovici (University of Cambridge) for the Meox2-cre mice. We are extremely grateful to all of the participants in the Pregnancy Outcome Prediction study. This work was supported by the NIHR Cambridge Comprehensive Biomedical Research Centre (Women's Health theme) and project grants from the MRC (G1100221) and Sands (Stillbirth and Neonatal Death Charity). The study was also supported by GE Healthcare (donation of two Voluson i ultrasound systems for this study) and by the NIHR Cambridge Clinical Research Facility, where all research visits took place.This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/ng.369

A Novel Automated System Yields Reproducible Temporal Feeding Patterns in Laboratory Rodents

Background The impact of temporal feeding patterns remains a major unanswered question in nutritional science. Progress has been hampered by the absence of a reliable method to impose temporal feeding in laboratory rodents, without the confounding influence of food-hoarding behavior. Objective The aim of this study was to develop and validate a reliable method for supplying crushed diets to laboratory rodents in consistent, relevant feeding patterns for prolonged periods. Methods We programmed our experimental feeding station to deliver a standard diet [StD; Atwater Fuel Energy (AFE) 13.9% fat] or high-fat diet (HFD; AFE 45% fat) during nocturnal grazing [providing 1/24th of the total daily food intake (tdF/I) of ad libitum–fed controls every 30 min] and meal-fed (3 × 1-h periods of ad libitum feeding) patterns in male rats (Sprague-Dawley: 4 wk old, 72–119 g) and mice [C57/Bl6J wild-type (WT): 6 mo old, 29–37 g], and ghrelin-null littermates (Ghr−/−; 27–34 g). Results Grazing yielded accurate, consistent feeding events in rats, with an approximately linear rise in nocturnal cumulative food intake [tdF/I (StD): 97.4 ± 1.5% accurate compared with manual measurement; R2 = 0.86; tdF/I (HFD): 99.0 ± 1.4% accurate; R2 = 0.86]. Meal-feeding produced 3 nocturnal meals of equal size and duration in StD-fed rats (tdF/I: 97.4 ± 0.9% accurate; R2 = 0.90), whereas the second meal size increased progressively in HFD-fed rats (44% higher on day 35 than on day 14; P < 0.01). Importantly, cumulative food intake in grazing and meal-fed rats was identical. Similar results were obtained in WT mice except that less restricted grazing induced hyperphagia (compared with meal-fed WT mice; P < 0.05 from day 1). This difference was abolished in Ghr−/− mice, with meal initiation delayed and meal duration enhanced. Neither pattern elevated corticosterone secretion in rats, but meal-feeding aligned ultradian pulses. Conclusions We have established a consistent, measurable, researcher-defined, stress-free method for imposing temporal feeding patterns in rats and mice. This approach will facilitate progress in understanding the physiologic impact of feeding patterns

Integrating Diverse Datasets Improves Developmental Enhancer Prediction

Gene-regulatory enhancers have been identified using various approaches, including evolutionary conservation, regulatory protein binding, chromatin modifications, and DNA sequence motifs. To integrate these different approaches, we developed EnhancerFinder, a two-step method for distinguishing developmental enhancers from the genomic background and then predicting their tissue specificity. EnhancerFinder uses a multiple kernel learning approach to integrate DNA sequence motifs, evolutionary patterns, and diverse functional genomics datasets from a variety of cell types. In contrast with prediction approaches that define enhancers based on histone marks or p300 sites from a single cell line, we trained EnhancerFinder on hundreds of experimentally verified human developmental enhancers from the VISTA Enhancer Browser. We comprehensively evaluated EnhancerFinder using cross validation and found that our integrative method improves the identification of enhancers over approaches that consider a single type of data, such as sequence motifs, evolutionary conservation, or the binding of enhancer-associated proteins. We find that VISTA enhancers active in embryonic heart are easier to identify than enhancers active in several other embryonic tissues, likely due to their uniquely high GC content. We applied EnhancerFinder to the entire human genome and predicted 84,301 developmental enhancers and their tissue specificity. These predictions provide specific functional annotations for large amounts of human non-coding DNA, and are significantly enriched near genes with annotated roles in their predicted tissues and lead SNPs from genome-wide association studies. We demonstrate the utility of EnhancerFinder predictions through in vivo validation of novel embryonic gene regulatory enhancers from three developmental transcription factor loci. Our genome-wide developmental enhancer predictions are freely available as a UCSC Genome Browser track, which we hope will enable researchers to further investigate questions in developmental biology. © 2014 Erwin et al

A novel long-range enhancer regulates postnatal expression of Zeb2: implications for Mowat-Wilson syndrome phenotypes

The zinc-finger, E-box-binding homeobox-2 (Zeb2) gene encodes a SMAD-interacting transcription factor that has diverse roles in development and disease. Mutations at the hZeb2 locus cause Mowat–Wilson syndrome (MWS), a genetic disorder that is associated with mental retardation and other, case- and sex-dependent clinical features. Recent studies have detailed microRNA-mediated control of Zeb2, but little is known about the genomic context of this gene or of enhancer sequences that may direct its diverse functions. Here, we describe a novel transgenic rodent model in which Zeb2 regulatory sequence has been disrupted, resulting in a postnatal developmental phenotype that is autosomal dominant. The phenotype exhibits a genotype-by-sex interaction and manifests primarily as an acute attenuation of postnatal kidney development in males. Other aspects of embryonic and neonatal development, including neuronal, are unaffected. The transgene insertion site is associated with a 12 kb deletion, 1.2 Mb upstream of Zeb2, within a 4.1 Mb gene desert. A conserved sequence, derived from the deleted region, enhanced Zeb2 promoter activity in transcription assays. Tissue and temporal restriction of this enhancer activity may involve postnatal changes in proteins that bind this sequence. A control human/mouse VISTA enhancer (62 kb upstream of Zeb2) also up-regulated the Zeb2 promoter, providing evidence of a string of conserved distal enhancers. The phenotype arising from deletion of one copy of the extreme long-range enhancer indicates a critical role for this enhancer at one developmental stage. Haploinsufficiency of Zeb2 in this developmental context reflects inheritance of MWS and may underlie some sex-dependent, non-neural characteristics of this human inherited disorder

The intermediate lactotroph: A morphologically distinct, ghrelin-responsive pituitary cell in the dwarf (dw/dw) Rat

Profound somatotroph hypoplasia in the dwarf (dw/dw) rat is accompanied by an estrogen-dependent induction of prolactin secretion by the GH secretagogue, GHRP-6. Using electron microscopy, we demonstrated that the reduction in the somatotroph population in the dw/dw pituitary is accompanied by the presence of a morphologically distinct lactotroph subpopulation. In these cells, which did not coexpress GH, the size, shape, and number of the secretory granules were between those of the type I and type II lactotrophs. We therefore called these cells intermediate lactotrophs. The intermediate lactotrophs accounted for up to 30% of the total prolactin-positive cell population in dw/dw males and up to 12% in females. Using tannic acid to quantify the fusion of secretory granules, we have shown that the intermediate lactotrophs are unresponsive to either GH-releasing factor (GRF) or TRH but exhibit a sexually dimorphic secretory response to acute ghrelin treatment, granular fusions being 4-fold higher in females. No cell matching the morphology of the novel lactotroph subpopulation was observed in the pituitary of the GRF-insensitive lit/lit mouse. However, ablation of GRF neurons with neonatal monosodium glutamate treatment had no effect on the population of intermediate lactotrophs in the dw/dw rat. Thus, the presence of the intermediate lactotrophs in the dw/dw pituitary appears to be independent of the function of the GRF neurons.Icnelia Huerta-Ocampo, Helen C. Christian, Nichola M. Thompson, Muna M. El-Kasti, and Timothy Well

Urinary peptide profiling identifies a panel of putative biomarkers for diagnosing and staging endometriosis

Objective: To identify a potential diagnostic endometriosis marker using matrix-enhanced laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based urinary proteomics. Design: Prospective randomized pilot study. Setting: University hospital, tertiary referral center for endometriosis. Patient(s): 53 women undergoing laparoscopic surgery for pain and/or infertility comprising 30 women without endometriosis and 23 with endometriosis. Intervention(s): Laparoscopy and urine specimens. Main Outcome Measure(s): Urinary peptide profiles. Result(s): We observed distinct patterns of peptide profiles in the urine samples of women presenting with typical clinical symptoms of endometriosis. Six statistically significant putative peptide markers were identified (four during the periovulatory phase and two during the luteal phase) by comparing controls with moderate/severe endometriosis patients. The periovulatory peptide mass of 1,767.1 Da and the luteal peptide mass of 1,824.3 Da both showed a sensitivity of 75% and a specificity of 85% and 71%, respectively. Also detected were seven peptide markers (two during the periovulatory phase and five during the luteal phase) by comparing the urinary peptide profiles of patients with minimal/mild to moderate/severe endometriosis. The periovulatory peptide mass of 3,280.9 Da and the luteal peptide mass of 1,933.8 Da showed a sensitivity of 82% and 75% and a specificity of 88% and 75%, respectively. Conclusion(s): Urinary proteomic analysis may provide a novel method of diagnosing and staging endometriosis. © 2011 American Society for Reproductive Medicine, Published by Elsevier Inc

Urinary peptide profiling identifies a panel of putative biomarkers for diagnosing and staging endometriosis.

OBJECTIVE: To identify a potential diagnostic endometriosis marker using matrix-enhanced laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based urinary proteomics. DESIGN: Prospective randomized pilot study. SETTING: University hospital, tertiary referral center for endometriosis. PATIENT(S): 53 women undergoing laparoscopic surgery for pain and/or infertility comprising 30 women without endometriosis and 23 with endometriosis. INTERVENTION(S): Laparoscopy and urine specimens. MAIN OUTCOME MEASURE(S): Urinary peptide profiles. RESULT(S): We observed distinct patterns of peptide profiles in the urine samples of women presenting with typical clinical symptoms of endometriosis. Six statistically significant putative peptide markers were identified (four during the periovulatory phase and two during the luteal phase) by comparing controls with moderate/severe endometriosis patients. The periovulatory peptide mass of 1,767.1 Da and the luteal peptide mass of 1,824.3 Da both showed a sensitivity of 75% and a specificity of 85% and 71%, respectively. Also detected were seven peptide markers (two during the periovulatory phase and five during the luteal phase) by comparing the urinary peptide profiles of patients with minimal/mild to moderate/severe endometriosis. The periovulatory peptide mass of 3,280.9 Da and the luteal peptide mass of 1,933.8 Da showed a sensitivity of 82% and 75% and a specificity of 88% and 75%, respectively. CONCLUSION(S): Urinary proteomic analysis may provide a novel method of diagnosing and staging endometriosis

The 5-item frailty index predicts 30-day morbidity and mortality in radical nephrectomy patients: A propensity matched analysis

PURPOSE: To assess the ability of the 5-item frailty index (5-IFi) score to predict 30-day morbidity and mortality post-radical nephrectomy (RN). METHODS: ACS-NSQIP database was used to select patients who underwent RN from 2011 to 2020. 5-IFi score was calculated by assigning a point for each of the following comorbidities: chronic obstructive pulmonary disease or pneumonia, congestive heart failure, dependent functional status, hypertension, and diabetes. Patients were divided into 3 frailty groups 0, 1, and ≥2. Patient demographics, medical comorbidities, prolonged length of stay, and prolonged operative time were compared between different groups; mortality and morbidity using the Clavien-Dindo classification (CVD). Multivariable logistic regression models and propensity score matching were performed as a sensitivity analysis to control for possible confounders. RESULTS: Cohort consisted of 36,682 patients whereby 5-IFi class 0, 1, and ≥2 included 11,564 (31.5%), 16,571 (45.2%), and 8,547 (25.3%) patients respectively. A multivariable analysis and propensity score matching revealed that patients with 5-IFi classes 1 and ≥ 2 were more likely to have a prolonged length of stay (OR = 1.11, 1.3, respectively) and to mortality (OR = 1.85 for frailty class ≥2); in addition to CVD class 1 & 2 (OR = 1.51, OR = 1.13, respectively), and CVD ≥ 4 (OR = 1.41, 1.86, respectively) as compared to 5-IFi class 0 (P \u3c 0.001). CONCLUSION: The 5-IFi score was found to be an independent predictor of prolonged length of stay, morbidity, and mortality after RN. This tool can play a major role in preoperative risk assessment and patient counseling based on individualized risks

Adiposity profile in the dwarf (dw/dw) rat: an unusually lean model of profound growth hormone deficiency

This study describes the previously uncharacterized ontogeny and regulation of truncal adipose reserves in the profoundly GH-deficient dwarf (dw/dw) rat. We show that, despite normal proportionate food intake, dw/dw rats develop abdominal leanness and hypoleptinemia (circulating leptin halved in dw/dw males, P < 0.05) during puberty. This contrasts with the hyperleptinemia seen in moderately GH-deficient Tgr rats (circulating leptin doubled at 6 wk of age, P < 0.05) and in GH receptor-binding protein (GHR/BP)-null mice (circulating leptin doubled; P < 0.05). This lean/hypoleptinemic phenotype was not completely normalized by GH treatment, but dw/dw rats developed abdominal obesity in response to neonatal MSG treatment or maintenance on a high-fat diet. Unlike Tgr rats, dw/dw rats did not become obese with age; plasma leptin levels and fat pad weights became similar to those in wild-type rats. In contrast with truncal leanness, tibial marrow adiposity was normal in male and doubled in female dwarves (P < 0.01), this increase being attributable to increased adipocyte number (P < 0.01). Neonatal MSG treatment and high-fat feeding elevated marrow adiposity in dw/dw rats by inducing adipocyte enlargement (P < 0.05). These results demonstrate that, despite lipolytic influence of GH, severe GH deficiency in dw/dw rats is accompanied by a paradoxical leanness. This lean/hypoleptinemic phenotype is not solely attributable to reduced GH signaling and does not appear to result from a reduction in nutrient intake or the ability of dw/dw adipocytes to accumulate lipid. Disruption of preadipocyte differentiation or adipocyte proliferation in the dw/dw rat may lead to the development of this unusually lean/hypoleptinemic phenotype