Molecular characterization of MC3R and evaluation of its potential role as a modifier of lung function in cystic fibrosis

Cystic fibrosis (CF), one of the most common lethal autosomal recessive disorder among Caucasians, affects approximately 30,000 individuals in the United States. CF is caused by loss of function mutations in the CF transmembrane conductance regulator (CFTR) gene. Once the disease-causing gene for CF was identified, numerous studies attempted to correlate CFTR mutations with specific CF phenotypes because genotype-phenotype correlations can predict a course of the disease and lead to the design of a genotype-specific therapeutic strategy. Unfortunately, distilling correlation has been challenging for several reasons. First, CF is a multi-system disease that involves different organs and its phenotype is analyzed in context of its various clinical components. Second, patients exhibit a wide range of disease severity although CF is considered a classic Mendelian disorder. In fact, the degree of variability observed in 293 individuals with the identical CFTR mutations suggests that factors other than the CFTR genotype contribute to lung function variation. Given that obstructive lung disease is the cause of death in 90% of CF patients, there has been continued interest in determining these factors that influence the severity of pulmonary disease in CF patients. MC3R has been identified as a compelling candidate for modifying CF lung disease. Before exploring its role as a modifier, we aimed to understand the molecular organization of MC3R. Using 5’ RACE, we discovered a novel upstream exon that extends the length of the 5’ UTR in MC3R without changing the ORF. The full-length 5’ UTR directs utilization of an evolutionarily conserved second in-frame ATG as the primary translation start site. MC3R synthesized from the second ATG is localized to apical membranes of polarized Madin Darby Canine Kidney (MDCK) cells, consistent with its function as a cell surface mediator of melanocortin signaling. Expression of MC3R causes re-localization of a known accessory factor for MC2R, MRAP2, to the apical membrane, which is coincident with the location of MC3R. In contrast, protein synthesized from MC3R cDNAs lacking the 5’ UTR displayed diffuse cytosolic distribution and no effect upon the distribution of MRAP2. Our findings demonstrate that a previously unannotated 5’ exon directs translation of MC3R protein that localizes to apical membranes of polarized cells. To define the mechanism of lung disease in CF that underlies the linkage signal at chr20q13.2, we evaluated MC3R as a potential modifier of lung function. Three rare variants in MC3R were significantly associated with lung function in individuals with CF, suggesting that variation in MC3R contibutes to CF lung disease severity. In silico prediction tools and in vitro studies were performed to understand the functional effect of the rare variants. We demonstrated that antagonist (siRNA) or agonist ([D-Trp8]-γ-MSH) of MC3R can modulate pro-inflammatory cytokine expression. Evidence for the presence of MC3R in primary airway epithelial cells of the lungs further supported our hypothesis that MC3R is a modifier of lung function in CF. These studies support the role of MC3R as a mediator of the inflammatory response and a potential therapeutic target in the treatment of CF

Melanokortiinireseptoreiden merkitys sairaalloisessa lihavuudessa

The prevalence of obesity is increasing at an alarming rate in all age groups worldwide. Obesity is a serious health problem due to increased risk of morbidity and mortality. Although environmental factors play a major role in the development of obesity, the identification of rare monogenic defects in human genes have confirmed that obesity has a strong genetic component. Mutations have been identified in genes encoding proteins of the leptin-melanocortin signaling system, which has an important role in the regulation of appetite and energy balance. The present study aimed at identifying mutations and genetic variations in the melanocortin receptors 2-5 and other genes active on the same signaling pathway accounting for severe early-onset obesity in children and morbid obesity in adults. The main achievement of this thesis was the identification of melanocortin-4 receptor (MC4R) mutations in Finnish patients. Six pathogenic MC4R mutations (308delT, P299H, two S127L and two -439delGC mutations) were identified, corresponding to a prevalence of 3% in severe early-onset obesity. No obesity causing MC4R mutations were found among patients with adult-onset morbid obesity. The MC4R 308delT deletion is predicted to result in a grossly truncated nonfunctional receptor of only 107 amino acids. The C-terminal residues, which are important in MC4R cell surface targeting, are totally absent from the mutant 308delT receptor. In vitro functional studies supported a pathogenic role for the S127L mutation since agonist induced signaling of the receptor was impaired. Cell membrane localization of the S127L receptor did not differ from that of the wild-type receptor, confirming that impaired function of the S127L receptor was due to reduced signaling properties. The P299H mutation leads to intracellular retention of the receptor. The -439delGC deletion is situated at a potential nescient helix-loop-helix 2 (NHLH2) -binding site in the MC4R promoter. It was demonstrated that the transcription factor NHLH2 binds to the consensus sequence at the -439delGC site in vitro, possibly resulting in altered promoter activity. Several genetic variants were identified in the melanocortin-3 receptor (MC3R) and pro-opiomelanocortin (POMC) genes. These polymorphisms do not explain morbid obesity, but the results indicate that some of these genetic variations may be modifying factors in obesity, resulting in subtle changes in obesity-related traits. A risk haplotype for obesity was identified in the ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) gene through a candidate gene single nucleotide polymorphism (SNP) genotyping approach. An ENPP1 haplotype, composed of SNPs rs1800949 and rs943003, was shown to be significantly associated with morbid obesity in adults. Accordingly, the MC3R, POMC and ENPP1 genes represent examples of susceptibility genes in which genetic variants predispose to obesity. In conclusion, pathogenic mutations in the MC4R gene were shown to account for 3% of cases with severe early-onset obesity in Finland. This is in line with results from other populations demonstrating that mutations in the MC4R gene underlie 1-6% of morbid obesity worldwide. MC4R deficiency thus represents the most common monogenic defect causing human obesity reported so far. The severity of the MC4-receptor defect appears to be associated with time of onset and the degree of obesity. Classification of MC4R mutations may provide a useful tool when predicting the outcome of the disease. In addition, several other genetic variants conferring susceptibility to obesity were detected in the MC3R, MC4R, POMC and ENPP1 genes.Lihavuus on yleistynyt jatkuvasti viimeisten vuosikymmenten aikana. Se on yksi merkittävämmistä sairastavuuteen ja ennenaikaiseen kuolleisuuteen johtavista tekijöistä, joka altistaa muun muassa tyypin 2 diabetekselle, sydän- ja verisuonitaudeille, tietyille syöpätyypeille sekä keuhkojen toimintahäiriöille. Lihavuuden esiintyvyys on lisääntynyt huomattavasti myös lasten ja nuorten keskuudessa. Vaikka ympäristötekijöillä onkin keskeinen asema lihavuuden synnyssä, on ihmisellä pystytty osoittamaan harvinaisia lihavuuteen johtavia yhden geenin virheitä ruokahalua säätelevässä leptiini melanokortiinijärjestelmässä. Tämän väitöskirjatutkimuksen tavoitteena oli etsiä suomalaisilta aikuisilta ja lapsilta sairaalloiselle lihavuudelle altistavia geneettisiä variaatioita ja mutaatioita melanokortiinireseptoreista 2 - 5 ja mahdollisista muista kandidaattigeeneistä, jotka sijaitsevat samoilla fysiologisilla säätelyreiteillä. Tässä tutkimuksessa osoitettiin seuraavat melanokortiini- 4 reseptori (MC4R) geenin mutaatiot: aminohappomuutokset S127L ja P299H sekä deleetiot -439delGC ja 308delT. Suomessa nämä mutaatiot selittävät noin 3 % varhaislapsuudessa alkaneesta vaikeasta lihavuudesta. Aikuisina sairaalloisen lihaviksi lihoneilta henkilöiltä MC4R-geenin mutaatiota ei löytynyt. Reseptorin toimintaa selvittävillä solutason tutkimuksilla pystyttiin osoittamaan, että S127L-mutaatio vaikuttaa MC4 reseptorin signalointiaktiivisuuteen. Deleetio 308delT muuttaa proteiinin lukukehystä ja keskeyttää proteiinisynteesin ennenaikaisesti. P299H-mutaatio aiheuttaa reseptorille niin pahan häiriön, ettei sitä kuljeteta lainkaan solukalvolle. MC4R-geenin -439delGC-deleetio sijaitsee säätelyalueella mahdollisessa NHLH2-transkriptiotekijän (nescient helix-loop-helix 2) sitoutumiskohdassa. Solutason kokein osoitettiin, että transkriptiotekijä NHLH2 sitoutuu tähän kyseiseen kohtaan ja näin ollen mahdollisesti vaikuttaa MC4R-geenin aktiivisuuteen. Tässä väitöskirjatyössä tunnistettiin useita perinnöllisiä variaatioita melanokortiini- 3 reseptori (MC3R) ja pro-opiomelanokortiini (POMC) geeneissä. Nämä geenivariaatiot eivät yksin aiheuta sairaalloista lihavuutta, mutta tutkimustulokset viittaavat siihen, että osa näistä geenivarianteista saattaa altistaa lihavuudelle. Lisäksi tässä väitöskirjatyössä on tutkittu usean lihavuuden kandidaattigeenin alueelta yhden emäksen variaatioita (SNP) ja selvitetty näiden SNP:ien yhteyttä lihavuuteen. Tämän menetelmän avulla tunnistettiin ektonukleotidi pyrofosfataasi fosfodiesteraasi 1 (ENPP1) geenissä kahden geenivariaation muodostama riskihaplotyyppi, joka assosioituu sairaalloiseen lihavuuteen aikuisilla. Tämän väitöskirjatutkimuksen tärkeimpiin havaintoihin kuuluu MC4R-geenin mutaatioiden osoitus ja näiden geenivirheiden solutason tutkimukset. MC4R-geenin mutaatiot selittävät noin 3 % varhaislapsuudessa alkaneesta vaikeasta lihavuudesta Suomessa. Maailmanlaajuisesti MC4R-geenin virheet selittävät 1-6 % sairaalloisesta lihavuudesta ja ovat yleisin perinnöllinen syy lihavuuden taustalla. Tämän tutkimuksen tulosten perusteella voidaan todeta, että MC4R-geenin mutaation vaikeusaste on yhteydessä lihavuuden ilmiasuun. Tässä tutkimuksessa tunnistettiin lisäksi useita lihavuudelle altistavia geenivariantteja MC3R-, MC4R-, POMC- ja ENPP1-geeneissä

Genetic obesity:Disorders and diagnostics

Obesity is a common disease with serious consequences for the health and well-being of patients. In a small proportion of people with excessive weight, a change in genetic material is the main cause of the obesity. In this thesis, the results of genetic testing for these rare obesity disorders are described. Because of the high prevalence of obesity, it is currently impossible to perform genetic diagnostics in all people with obesity. An improved insight in the clinical phenotype of patients with a genetic obesity disorder is therefore needed to determine which patients should undergo genetic testing. Moreover, the impact of diagnosing these disorders is described in this thesis. Increased knowledge about the underlying mechanisms offers great opportunities for the development of novel drug therapy for obesity

Rare mendelian forms of obesity and diabetes and their implications for treatment outcomes

Obesity and diabetes are becoming epidemic health issues worldwide. In recent decades, a considerable amount of work has been done to study the pathogenicity underlying those diseases, which has led to valuable insights into the genetic basis, treatment and prevention of obesity and diabetes. Nevertheless, despite our more detailed pathophysiological understanding of the rare forms of diabetes and obesity than of more common polygenic forms, we still know little about their prevalence and implications outside specialised genetics services. In the present work, I have explored the contribution of Mendelian forms of obesity in individuals with severe obesity. Initial segregation analyses of families with an obese proband, led to the identification of an oligogenic mode of inheritance for obesity. This was followed by re-analysis of pre-existing whole exome sequencing data from 91 individuals with extreme obesity, which revealed an additional 21 possible causative variants in known monogenic/syndromic obesity genes and three further cases of oligogenic inheritance. In addition, 11 candidate variants were identified in genes suggested by rodent models of obesity and/or diabetes, but not previously reported in humans. To further expand the analysis, a unique custom genotyping array focusing on, obesity and diabetes mellitus (T2D, and monogenic forms of diabetes) was designed to be applied to a larger number of samples (N=2068). Application of the array led to the identification of a total of 161 potential causative variants in 40 monogenic obesity/syndromic obesity genes, with a putative diagnostic yield of 11%. Initial analysis suggests that having one of these putative Mendelian forms of obesity resulted in no statistical difference in percentage weight loss at 2 years post-surgery and diabetes remission. Our first analysis on obesity indicates that the use of a custom-designed genotyping array for specific rare diseases may be an advantageous first level screening strategy in terms of cost and time. The work presented here also suggests that the true prevalence of Mendelian forms of obesity among bariatric surgery patients is likely to be high - this presents a significant unmet need for genetic analysis and follow-up.  Open Acces

Gout and Metabolic Disease: Investigation of Potential Relationship in the New Zealand Population

Hyperuricaemia, pathologically defined as the presence of higher levels of serum urate, results from a compromise in the delicate balance between the production and excretion of urate primarily in the liver and the kidneys, respectively. Hyperuricaemia is a prerequisite for gout, a painful inflammatory arthritis. The symptoms of gout arise from the body’s immune response to monosodium urate crystals that accumulate in the synovial fluid of the joints. Hyperuricaemia and gout are complex traits. A number of genetic loci confer risk to develop hyperuricaemia. Genome-wide association studies (GWAS), an indispensable tool in population genetics, has identified at least twenty eight genomic loci that contain variants affecting serum urate concentration. Gene-environment interactions also play a significant role in this context. Exogenous factors such as the intake of purine-rich foods increase the frequency of gout flares. Population-specific genetic effects on gout are as evident, if not more, as for other complex phenotypes. The prevalence of gout is much higher in the New Zealand Polynesian population compared to other populations. Approximately 7% of New Zealand Māori and Pacific Island people and 3% of New Zealand Europeans are affected by gout. The coexistence of metabolic conditions with gout, usually called gout-comorbidities, adds another level of complexity. However, not many studies have attempted to address the causal relationship between these traits. In fact, my research project was instigated as an attempt to study the causal associations between gout and its comorbidities and fill in some gap in the scientific literature. The research was, however, limited to three metabolic conditions/comorbidities of gout – imbalanced iron homeostasis, metabolic syndrome and disrupted lipid metabolism. My study shows an association between increased serum ferritin and the risk of gout and seeded an idea that the consumption of iron-rich diet may play a role in increasing the frequency and severity of gout flares. Genetic association analysis using two variants in the HFE gene was done to confirm the association between ferritin and urate, which showed positive association in a smaller dataset and provoked the idea to investigate the causality, if it exists, between gout and iron metabolism. Using the robust ‘Two-sample Mendelian randomisation’ approach and exploiting summary statistics data from two large GWA studies, I was able to find an evidence of a causal effect of iron on urate metabolism, but not urate on iron metabolism. In the context of the metabolic syndrome, the role of variants within/near the ADRB3, MC3R, MC4R and ADTRP genes were investigated. The positive effects identified for these variants supported the possible involvement of obesity and insulin resistance-related genes in gout pathophysiology. With the help of gene sequencing-based rare variant analyses, several novel population-specific association signals were found within the coding regions of two lipid-related genes, LRP2 and A1CF. Polynesian-specific novel genetic effects were identified to be predictive for gout for common variants within the LRP2 gene. Rare variants within the LRP2 gene were also identified and a higher prevalence of non-synonymous polymorphisms that can increase the risk of hyperuricaemia was observed in European individuals compared to Polynesians. These results indicated LRP2 to contribute to the difference in gout prevalence between Māori and Pacific Island individuals compared to the New Zealand European population. Collectively, my study reports a causal role of iron and ferritin in increasing serum urate concentration and the involvement of imbalanced iron homeostasis in hyperuricaemia. Also, positive genetic associations indicated that genes contributing to metabolic syndrome and lipid metabolism can increase the risk of gout, and also have population-specific effects for the Polynesian and European ancestral groups in New Zealand

GENETICS OF OBESITY IN STARR COUNTY, TEXAS MEXICAN AMERICANS

Currently, over two-thirds of Americans are classified as over-weight or obese. Obesity increases risk for many other diseases including type 2 diabetes, heart disease, stroke, and cancer, making obesity the largest public health problem in America and most other Westernized nations. Hispanics have a higher rate of both obesity and type 2 diabetes, making them a particularly interesting population in which to study obesity. For the last 33 years, the Starr County Health Studies has collected an array of phenotypes and biological samples from residents of Starr County, along Texas-Mexico border. This study includes 825 subjects who were not known to have diabetes at ascertainment. These subjects have now been seen a second time, on average 8.5 years later. At both visits we measured several aspects of obesity including BMI, bioimpedance to estimate percent body fat, and waist, hip, and arm circumferences. By using multivariate approaches to leverage the array of obesity measures, we have better captured both the amount of adipose tissue and the location of fat deposits. To assess association of obesity related traits with genetic variation from both genome-wide array data imputed to 1000 Genomes Phase 1 integrated dataset and exome sequencing, both gene-based and single variant tests were conducted. Through these single variant tests, we identified an association with waist to hip ratio and low frequency variants, in two adjacent GABA receptor subunit genes, GABRB2 and GABRA6, including a nonsynonymous variant in GABRA6. Additional associations include an association with a composite measure of adiposity that encompasses degree of adiposity and location of excess fat above or below the waist and TREK1, a gene responsible for trafficking the GABAA receptor to the cell membrane. Gene based tests of rare variants yielded associations between central versus peripheral adiposity and ACSL1, a gene involved in triglyceride biosynthesis. Further replication is required to confirm these associations. While the importance of neuronal signaling pathways in body fat distribution has long been known, many aspects of these pathways are poorly understood. Better understanding of these pathways may identify potential pharmaceutical targets

Functional characterisation of genetic variants influencing human food preferences using bioinformatics and in vivo models

Chronic diseases place a huge burden on society and are the leading cause of death worldwide. Obesity, which is currently a worldwide epidemic, is a risk factor for chronic disease. Understanding drivers of food choices may help to address the escalating obesity problem. Many people are at an increased risk of obesity as they carry genetic variants linked to particular eating behaviours. Identification of causal variants can help to understand which genes and biological mechanisms underlie food preference. Previous work used UK Biobank data from ~500,000 individuals to identify 302 loci associated with food consumption. This research project aimed to understand which genes were responsible for these genetic associations, prioritise genes for functional validation study, and understand the function of these genes through in vivo food preference studies. It is well-known that there can be intake-related bias in food frequency questionnaire responses due to participants over- or under-reporting dietary intake and this makes nutritional research difficult. Mendelian randomization was used to statistically identify reporting bias in the UK Biobank food frequency questionnaire responses and these were subsequently corrected by the group using a bespoke method. A 6-stage gene prioritisation strategy was developed to identify a candidate gene for functional validation in vivo. Genes were allocated points based on three main components: gene expression in humans and mice, conservation of gene product across species using pairwise protein alignments, and knockout phenotype details from available mouse models. Genes were ranked in order of preference for functional follow-up study. The top two genes identified were IER5L (associated with meat and fat) and DCAF12 (associated with salt). It was difficult to determine a coherent role for IER5L in food preference. The top SNP mapped to IER5L was found to be in an eQTL containing two other genes: CRAT and PPP2R4. It was not possible to exclude any of these genes from the meat/fat association, but there were clear links in the literature between CRAT and food intake. CRAT was selected for functional follow up, along with the DCAF12 gene associated with adding salt to food. However, it was not possible to follow up the CRAT gene due to logistical reasons. The project instead progressed to the in vivo stage using the well-known MC4R appetite regulatory gene to develop a model of food preference that could be used with Crat–/– mice in the future. A novel 3-food preference model was developed using Mc4r–/– mice and WT mice. Mice were given a choice of three diets: protein-enriched diet (to model the CRAT meat association), fat-enriched diet (to model the CRAT fat association) and standard chow. Mice preferred to eat a completely fat diet or a mixed choice diet regardless of genotype. Mc4r–/– mice were heavier than WT mice but were not hyperphagic as was expected from previous studies. As expected, Mc4r–/– mice demonstrated hyperinsulinemia compared to WT mice but there were no differences in glucose or triglyceride levels found between Mc4r–/– and WT mice. In vivo modelling of the DCAF12 salt association was carried out using Dcaf12–/– and WT mice to investigate salt preference. Mice were given a choice of two bottles of water followed by a choice of 0.4% (75mM) salt solution and water. This was repeated using a higher concentration 0.8% (150mM) salt solution. Dcaf12–/– mice did not show a preference for the salt solution as expected, and preference reduced as salt concentration increased. Unexpectedly, mice demonstrated a right-hand side preference during the water bottle choice period. Furthermore, mice consumed less food as salt concentration increased. This salt-preference study highlighted a number of complexities that can be associated with translating human effects into in vivo models. Research into the genetics of eating behaviour can help to identify and understand the impact of genetic variants on dietary pathways. If we can understand biological drivers of individual food choices, this information may be used to guide people towards a healthier lifestyle and reduce levels of obesity

Molecular Basis of Inherited Diseases in Companion Animals

This book includes a collection of publications describing the molecular etiology of inherited diseases and conditions in companion animals (dogs and cats). In addition to contributing to the health of companion animals, this research also benefits humans that have similar types of diseases

Genetic analysis of extreme obesity

Obesity is an increasing health problem worldwide as a result of the changing environment, with calorie-dense food and a sedentary lifestyle. However, numerous twin studies indicate that genetics plays a significant role in determining which individuals become obese or stay lean while sharing the same ‘obesogenic’ environment. Genetic research in obesity has two major goals: the first being to elucidate the pathophysiological basis of obesity, and the second is to provide an evidential foundation for personalised medicine. An obesity cohort was established, consisting of over a thousand severely-obese individuals (mean BMI 48.1 kg/m2 [± 8.67 SD]) undergoing bariatric surgery, as the basis for genetic and phenotypic analysis of the severely obese. The relatively high proportion of metabolically-healthy, but severely-obese individuals in this cohort illustrated some of the many unknown pathophysiological mechanisms, while a consistent increase of public distress among the more severely obese confirmed the ongoing stigmatisation of the obese in UK society. Screening for the most common form of Mendelian obesity, MC4R deficiency identified a lower than anticipated prevalence (0.77%) in the bariatric cohort, but analysis of treatment outcomes indicated that bariatric surgery both (RYGB and VSG) is effective for the individuals affected. Lifestyle intervention for children with MC4R deficiency, on the other hand, appeared to have less beneficial outcomes long term. Using whole exome sequencing on 40 super-obese bariatric participants, a higher than anticipated prevalence of putative Mendelian obesity (20.5%) was found, including several novel disruptive variants in known obesity genes. Finally, a novel Mendelian obesity and diabetes syndrome was detected, in a consanguineous family with a complex obesity phenotype, caused by a homozygous truncating mutation of the CPE gene (c.76_98del; p.Glu26ArgfsX68).Open Acces

The Role of Leptin in Body Weight Regulation

Leptin is an adipocyte-derived hormone which circulates in concentrations that are closely correlated with amounts of body fat. It provides a chronic signal to the central nervous system (CNS) regarding quantity of stored body fat and as such it is involved in the regulation of long term energy homeostasis. Leptin also declines abruptly when negative energy is imposed, providing a signal of incipient threats to the adequacy of fat stores. Humans and mice maintain body weight (fat) at remarkably stable levels without conscious effort to adjust food intake or energy expenditure. Changes in body weight induced by either overfeeding or dietary restriction are rapidly reversed when free feeding is resumed, indicating that altered body weight is accompanied by physiological adjustments that oppose this change. The “set-point” that is being defended depends on individuals’ genetic makeup and developmental environment during the perinatal period. Several aspects of leptin physiology were investigated in the work presented in this dissertation including: the effects of transient hyperleptinemia at specific developmental periods on subsequent body weight set point in mice; regulation of body weight in the absence of leptin in mice; genetic contributors to circulating leptin concentrations in human and mice, and; the efficacy of an MC4R agonist – a downstream target of leptin – on maintenance of reduced body weight in mice. Chapter 2 and 3. The effects of transient hyperleptinemia at specific developmental periods on subsequent body weight set point in mice To assess whether leptin per se influences the body weight set point and whether there is a critical time window for such effects, we generated a transgenic mouse in which non-invasive induction of transient hyperleptinemia is dissociated from adiposity. This transgenic mouse uses a TET-ON system in which transgenic (CMV-driven) leptin expression is regulated by exposure to doxycycline (dox) in a dose-responsive manner that can be rapidly turned on and off. Circulating leptin concentrations can be elevated to those in a high fat-fed obese mouse within one day and either sustained indefinitely or restored to baseline concentrations within 24 hours. Acute overexpression of leptin in the adult transgenic mice reduces food intake and causes transient weight loss – confirming that the transgenic leptin is bioactive and capable of triggering anticipated physiological responses. This leptin transgenic mouse enables reversible increases in circulating leptin to virtually any level at any point in development. Using this system we investigated the physiological consequences of developmentally timed transient hyperleptinemia on subsequent apparent set point for adiposity. Specifically, we evaluated the physiological effects of elevated leptin during adulthood, “adolescence” and the immediate postnatal period on the defense of body weight (adiposity) later in life and on the susceptibility to gain weight when offered a highly palatable diet ad libitum. We showed that inducing chronic hyperleptinemia in adult or “adolescent” mice does not increase the set point of defended body weight when excess leptin is removed; however, transient elevation of circulating leptin in the immediate postnatal period increases the hyperphagic response of the offspring to a highly palatable diet 7 weeks later, and renders animals more susceptible to obesity as adults. We demonstrated that leptin per se is capable of influencing the susceptibility of mice to gain weight on high fat diet; however, these effects are restricted to a critical time window which we identified to be the immediate postnatal period. Chapter 4. Regulation of body weight in the absence of leptin in mice Leptin-deficient Lepob/ob mice show metabolic compensation for lost weight and they appear to defend body fat by leptin-independent mechanisms. We attempted to identify mechanisms involved in leptin-independent regulation of body weight. Lepob/ob mice were either fed ad libitum or calorie restricted to lose 20% of body weight. Calorie-restricted mice reduced energy expenditure and, when released to ad libitum feeding, regained fat and lean mass (to the levels of ad libitum controls) within 5 weeks. Calorie-restricted mice did so while their ad libitum caloric intake was equal to that of the control animals. These results confirm that, in congenitally leptin deficient animals, leptin is not required for compensatory reduction in energy expenditure accompanying weight loss, but suggest that the hyperphagia of the weight-reduced state is leptin-dependent. Chapter 5. Genetic contributors to circulating leptin concentrations in human and mice While circulating leptin concentrations correlate closely with body fat, at any given level of adiposity, there is substantial variation in circulating leptin. We collaborated with Dr. Ruth Loos – professor of Environmental Medicine & Public Health at Icahn School of Medicine at Mount Sinai – and her associates who carried out a genome-wide association study of circulating leptin concentrations adjusted for body mass and composition, and identified five loci associated with reduced circulating leptin concentrations [1]. The aim of the study was to identify and functionally assess potentially causal gene(s) within each implicated region. Our aim was to identify genes that modify leptin production/release in a manner that might account for reduced circulating leptin concentrations and hence predisposition to obesity. We developed an assay to directly measure effects of the candidate genes in ex vivo adipose tissue explants on production and secretion of leptin. Using siRNAs, we knocked down expression of these genes in perigonadal adipose tissue explants from mice fed high fat diet and demonstrated that Adig, located in the SLC32A1 locus, modulates leptin production and secretion [1]. These studies provide a prototype for the functional deconvolution of groups of genes identified by genome-wide association studies in which a specific cell type can be implicated. Chapter 6. The efficacy of an MC4R agonist – a downstream target of leptin – on maintenance of reduced body weight in mice Finally, we investigated the efficacy of an MC4R agonist in maintenance of reduced body weight in mice [2]. Weight loss is difficult to maintain due to physiological adaptations in energy expenditure and drive to eat that accompany this state. Exogenous leptin sufficient to restore circulating levels to those preceding weight (fat) loss reverses many of the relevant phenotypes. MC4R is a downstream target of leptin signaling and is central in energy homeostasis. In collaboration with scientists at AstraZeneca, we studied the effectiveness of a novel peptide MC4R agonist in maintenance of reduced body weight compared to its use in inducing weight loss. In the weight reduced state, 5x lower doses of the same molecule were comparably efficacious to a higher dose in the ad libitum state [2]. This protocol provides a model for evaluating the mechanisms and quantitative efficacy of weight-maintenance strategies and agents. These data support the concept that the pharmacology of the weight reduced state may be more tractable than that designed to induce weight loss. Overall, the major conclusions from these studies are that: transient hyperleptinemia during the postnatal period can influence the susceptibility of mice to diet-induced obesity in adulthood; factors other than leptin contribute to body weight regulation in leptin deficient mice; functional, biological assays can be used to identify causal genes in genome-wide association study identified loci, and; pharmacological agents to maintain reduced weight may be a tractable target for treatment of obesity