published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

Objective:  Obesity has become the leading cause of mortality worldwide; however, the fundamental pathophysiology underlying this association remains unclear. The discovery of adipokines, i.e., cytokines produced by adipose cells (adipocytes), revealed that adipose tissue is a highly endocrine organ, thus opening new lines of investigation. The prototypical adipokine leptin increases in obesity, and leptin receptors are found in vascular cells. However, results are contradictory regarding the role of leptin in vascular and endothelial functions. Leptin has been shown to elicit vasodilatation, but has also been linked with atherosclerotic and thrombotic disease. The main aim of the present thesis was to study the association of circulating levels of leptin with markers of endothelial function, and to analyze the effects of leptin infusion in vivo  on vasomotor function and endogenous fibrinolysis. Material:  Four associative studies and two interventional studies were conducted. The former included DISARM (studies 1 and 2), the PIVUS study (study 3), and the Scottish post-infarction study (study 4). The DISARM studies and study 4, respectively, recruited 20 men and 83 men and women with stable ischemic heart disease. Study 3 included a random sample of 1016 subjects (54% women, 70 years old) living in the community of Uppsala, Sweden. For the interventional studies (studies 5 and 6), 10 healthy men were recruited for each study. Methods:  In all studies, endothelial function was estimated based on forearm blood flow (FBF) as measured by strain-gauge venous occlusion plethysmography, at rest or during infusion of vasodilators. In study 3, additional measurement techniques were used, such as brachial ultrasound flow-mediated dilation (FMD) and the aortic augmentation index (AoAIx) by tonometry in the radial artery. Fibrinolytic status was estimated based on basal and stimulated levels of tissue plasminogen activator antigen (t-PA), and by assessment of the endothelial release of t-PA (net t-PA release). Plasma leptin levels were measured by radioimmunoassay. In the associative studies, endothelial function and fibrinolytic status were related to circulating plasma leptin levels. In the experimental studies, exogenous leptin was administered in the brachial artery and endothelial function was assessed by strain-gauge plethysmography Results:  In elderly men and women, leptin was independently associated with decreased endothelial-dependent and -independent vasodilatation, reflecting disturbed endothelial function in resistance vessels. This association was attenuated after adjustment for BMI, and when analyzed among subjects with high plasma leptin levels. FMD (a measure of endothelial function in conduit vessels) was not associated with leptin. Exogenous leptin infusion did not alter vasomotor tone, but the endothelium-dependent and -independent vasodilatation was impaired during concomitant infusion of leptin and vasodilators. Infused leptin in the forearm did not affect blood pressure or pulse rate. Chronic hyperleptinemia, but not acutely induced hyperleptinemia, was associated with release of endothelial tissue plasminogen activator (net t-PA). Conclusions:  In humans, leptin was associated with impaired vasodilatation. However, this relationship was blunted after adjustment for BMI, suggesting that leptin could be the mediator between obesity and impaired vascular function. Furthermore, the observed lack of association in hyperleptinemic subjects may reflect a state of leptin resistance. The experimental result showing attenuated vascular reactivity following leptin infusion is in accordance with the results of the associative studies. The augmented net t-PA release in patients with chronic hyperleptinemia may indicate a state of “vascular activation,” which was not observed in healthy endothelium during a short period of leptin infusion. This thesis addresses several controversial issues regarding the action of leptin on vascular tissue in humans. The final results indicate that the in vivo action of leptin on vascularity is complex and mediated by several mechanisms. Our findings suggest that leptin is an important mediator between obesity and endothelial dysfunction, and should stimulate further investigation of this matter. / Manuel Cruz Gonzalez

Das metabolische Syndrom trägt über die Reduktion der Insulinsensitivität entscheidend zur Entstehung von Herz-Kreislauf- und Stoffwechsel-Krankheiten bei. Bei Adipösen sind die Serumkonzentrationen der Adipozytokine Adiponectin und Leptin so verschoben, dass kardiovaskuläre Risikofaktoren resultieren. Immer mehr Betriebe versuchen krankheitsbedingte Ausfallkosten über Gesundheitsförderungsprogramme einzusparen und dadurch die betriebliche Produktivität und Qualität zu steigern. Zudem sollen die volkswirtschaftlichen Kosten der Therapie von Lifestyle-Erkrankungen gesenkt werden. 24 Mitarbeiterinnen und Mitarbeiter der Würzburger Verkehrsbetriebe (WVV) (46,4 ± 7,6 Jahre; body mass index, BMI 37,4 ± 5,9; waist to hip ratio WHR 0,98 ± 0,07) führten ein dreimonatiges Programm mit Ernährungstherapie sowie Ausdauer- und Krafttraining durch. Zu Beginn (T0) und nach Ablauf (T1) der Interventionen wurde ein Oraler Glucose Toleranztest durchgeführt, sowie die Lipidwerte und die Hormone Adiponectin und Leptin bestimmt. Nach weiteren sechs Monaten selbständigem Training (T2) wurden die Messungen wiederholt. Bei T1 lag der mittlere BMI um 5,6%, der mittlere WHR um 3,1% niedriger als bei T0. Die Lipidwerte wurden insgesamt gesenkt, die Insulinsensitivität wieder verbessert. Die Leptinwerte normalisierten sich signifikanter als die Adiponectinwerte. Bei T2 konnten 63% der Teilnehmer ihre körperliche Konstitution beibehalten oder sogar noch verbessern. Die Lipidwerte sowie die Adipozytokine blieben konstant. Die Parameter der Insulinsensitivität konnten stabilisiert oder sogar noch verbessert werden. Mit unserer Studie konnten wir zeigen, dass den Beschäftigten durch ein umfangreiches innerbetriebliches Gesundheitsförderungsprogramm der Einstieg zur Veränderung des Lebensstils erleichtert werden kann. Zudem ist im Hinblick auf die Prävention von kardiovaskulären und metabolischen Komplikationen eine Reduzierung der Risikofaktoren gelungen. Auch konnte der positive Einfluss einer gesünderen Lebensweise auf die Adipozytokine und die Insulinsensitivität nachgewiesen werden. / The metabolic syndrom plays an important factor in developing cardiovascular diseases. Obese people show different serum concentrations of adiponectin and leptin, so that the possibility to develop cardiovascular disease increases. More and more companies offer health promotions to their employees to decrease their costs. 24 employees of the WVV (46,4 ± 7,6 Jahre; body mass index, BMI 37,4 ± 5,9; waist to hip ratio WHR 0,98 ± 0,07) participated a dietic and exercise treatment over 3 month. Before (T0) and after (T1) the interventions lipids, adiponectin and leptin were determined and an OGTT was made. After further 6 month (T2) without official dietic and exercise treatment the analyses were repeated. We could show that an workplace health promotions can help the employees to change thier life-style and to reduce risk factors for cardiovascular diseases. In addition the insulinsensitivity and the adipozytokines were positivily influenced.

O Objetivo do presente trabalho foi investigar os efeitos do aumento da sensibilidade à leptina sobre a ingestão alimentar, o balanço energético e modulação da homeostase glicêmica. Para tanto, camundongos com deleção do gene SOCS3 em células que expressam o receptor de leptina (LepR SOCS3 KO) foram submetidos, iniciamente, à obesidade induzida pela dieta. Observamos que a deleção condicional do SOCS3 não foi capaz de prevenir o ganho de peso induzido pela dieta, mas aumentou a sensibilidade à ação da insulina. A seguir, acompanhamos outro grupo de animais a restrição alimentar e, na sequencia, a hiperfagia e peso corporal. Constatamos que a ausência do SOCS3 em células responsivas à leptina promoveu redução da hiperfagia alimentar e menor ganho de peso no período de realimentação; no entanto, foi observado comprometimento nos mecanismos de controle da glicemia durante o período de restrição alimentar, particularmente, através da modulação no sistema nervoso simpático. Nossos resultados ajudam os efeitos metabólicos da leptina em diferentes condições fisiológicas. Por fim, nossos dados apontam possíveis efeitos colaterais de futuras estratégias que visam aumentar a sensibilidade à leptina como alternativa para o tratamento da obesidade. / The objective of this study was to investigate the effects of leptin sensitivity on food intake, energy balance and modulation of glycemic homeostasis. We produced mice lacking SOCS3 only in leptin receptor-expressing cells (LepR SOCS3 KO mice). Mice received high-fat diet to induce obesity. Inactivation of SOCS3 only in LepR-expressing cells protected against leptin resistance induced by diet-induced obesity (DIO), but did not prevent weight gain. However, LepR SOCS3 KO mice were protected from insulin resistance induced by DIO. Next, another group of animals were subjected to a 48 hours fasting, followed by refeeding. LepR SOCS3 KO mice showed attenuated food intake and weight regain after a 48 h fasting. Post-restriction hyperleptinemia was also prevented in LepR SOCS3 KO mice. Remarkably, LepR SOCS3 KO mice showed impaired glucose control during fasting, leading to hypoglycemia. To investigate the mechanisms of action, we showed that increased leptin sensibility modulates the sympathetic nervous system and can be harmful to glucose homeostasis during fasting. In conclusion, our results help to understand the effects of leptin to prevent obesity, but also highlight possible side effects from strategies that increase leptin sensitivity.

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.

M.Sc.(Med.), Faculty of Health Sciences, University of the Witwatersrand, 2009. / Leptin is a substance that is released from adipose tissue and although it is primarily employed to modify body size, it also targets a number of other tissues, including the myocardium. Although plasma leptin concentrations may predict cardiovascular risk beyond conventional measurements, it is uncertain whether this may be explained by an independent effect on left ventricular mass (LVM) and geometry. Previous clinical studies evaluating the independent relationship between plasma leptin concentrations and LVM have been conducted in either small study samples (n=31-55), in severely obese participants only, in select subgroups (with insulin resistance) or in population samples with a relatively low mean body mass index (BMI). In the present dissertation I therefore assessed whether plasma leptin concentrations are associated with LVM and LV mean wall thickness independent of adiposity indices in 378 adults of African descent randomly recruited from a population sample with ~63% of people whom were either overweight or obese. LVM was determined using two-dimensional directed M-mode echocardiography and indexed to height2.7 (LVMI). ~28% of the sample had LV hypertrophy. Marked differences in plasma leptin concentrations were noted between men and women. Thus, multivariate regression analysis was employed to identify independent relations between plasma leptin concentrations and either LVMI or LV mean wall thickness in sex-specific groups. Before adjustments for potential confounders, plasma leptin concentrations were associated with LVMI in both women (r=0.25, p<0.0001) and in men (r=0.20, p=0.017) as well as with LV mean wall thickness in both women (r=0.22, p<0.001) and in men (r=0.27, p=0.002). Moreover, participants with LV hypertrophy defined as an LVM index of >51 g/m2 had markedly greater plasma leptin concentrations than those participants without LV hypertrophy. However, plasma leptin concentrations were also associated with age, conventional systolic blood pressure and with adiposity indices (p<0.0001), factors that had robust relationships with LVMI and LV mean wall thickness. In multivariate regression models with plasma leptin concentrations, adiposity indices, age, systolic blood pressure and a number of alternative potential confounders in the same regression model, although adiposity indices were strong independent predictors of both LVMI and LV mean wall thickness in both women and men (p<0.002-p<0.0001), plasma leptin concentrations were not independently related to either LVMI (p=0.32-0.96), or LV mean wall thickness (p=0.33-0.81). In conclusion, plasma leptin concentrations, although associated with, are not independent predictors of LVMI beyond adiposity indices and other related factors in a population sample with a high prevalence of excess adiposity. Therefore, plasma leptin concentrations are unlikely to predict cardiovascular risk beyond conventional risk measurements because of an impact on LVM.

Secreted by adipose tissue, leptin acts as a signal of energy reserve status, and acts in the brain as a negative feedback mechanism to suppress food intake and increase energy expenditure, the net effect of which is maintenance of energy homeostasis. In addition to its role as a satiety factor, leptin has widespread autonomic effects, increasing sympathetic tone to a variety of tissues, including those involved in arterial pressure regulation. Thus, leptin has been implicated as a critical link between obesity and hypertension. However, the specific mechanisms whereby leptin elicits its diverse effects are not fully understood. This is further complicated by the many sites of leptin action within the brain, as well as its diverse intracellular effects. Here, we investigate the possibility that distinct aspects of leptin function are controlled by different neuronal populations and/or molecular signaling cascades. Specifically, we identify unique roles for leptin action on POMC and AgRP neurons in differentially mediating the regional sympathetic effects of leptin. Furthermore, we show that leptin action via mTORC1 is required for the cardiovascular sympathetic but not the metabolic effects. Together, these findings point to complex neuroanatomical and molecular differences in the mechanisms underlying leptin’s effects on different physiological processes, with important implications for future research into obesity-associated hypertension.

Abstract In 2018, health statistics revealed that, despite the many preventive measures established, cancer was the second leading cause of death in the United States and the leading cause of death in 22 states, exceeded only by heart disease. With obesity/leptin levels reaching pandemic levels worldwide, and cancer having a well-known association with obesity, both chronic diseases represent a large proportion of public health challenges. Guided by the social ecological model, the purpose of this cross-sectional, quantitative study was to examine if a significant difference exists in leptin levels among adults with different types of obesity-associated and common cancers and those without cancer. Further, using secondary data from the third National Health and Nutrition Examination Survey, the correlation of cancer risk factors with leptin levels among a multiethnic sample of adults living in the United States was also examined. ANCOVA and multiple linear regression analysis revealed that a significant difference exists in leptin levels among individuals with different types of cancer. A correlation also exists between cancer risk factors and leptin levels in adults with different types of cancer. The results further revealed that those with cancer had higher leptin levels than those without cancer after adjusting for related covariates. Health professional and educators worldwide working together to increase awareness and health literacy to empower not only the current study population, but all populations in adopting healthier lifestyles that will hopefully aid in reducing the risk, incidence, and mortality rates of obesity and cancer at the individual, community, societal and national levels may ultimately lead to positive social change.

Leptin is primarily an adipose-derived hormone that acts in the hypothalamus to regulate body fat levels by suppressing appetite and increasing metabolic rate. Pregnancy is characterised by increased food intake and fat mass to meet the metabolic demands of this physiological state. Leptin concentrations also increase during pregnancy, but this does not prevent the pregnancy-induced hyperphagia, suggesting a state of leptin resistance. The aims of this thesis were to measure hypothalamic leptin responsiveness during pregnancy and to investigate the potential mechanisms underlying pregnancy-induced leptin resistance. The satiety response to intracerebroventricular (i.c.v) leptin was measured in fasted non-pregnant (diestrous), early pregnant (day 7), and mid-pregnant (day 14) rats. Serial blood samples collected from another group of rats demonstrated that despite initial elevated plasma leptin concentrations in pregnant rats, fasting significantly decreased leptin concentrations so that pregnant and non-pregnant groups had similar, low leptin concentrations. Leptin treatment significantly reduced food intake in non-pregnant and early pregnant rats but not in mid-pregnant rats. In addition, there was no post-fasting hyperphagic response in the pregnant rats. These results indicate that pregnant rats become resistant to the satiety action of leptin. To investigate the mechanisms underlying pregnancy-induced leptin resistance, leptin-induced activation of hypothalamic leptin-target neurons was examined. Signal transducer and activator of transcription 3 (STAT3) phosphorylation was measured in non- pregnant and mid-pregnant rats following i.c.v. administration of leptin. Western blot and immunohistochemistry analysis indicated that leptin-induced STAT3 phosphorylation was significantly reduced in the ventromedial nucleus of the hypothalamus (VMH) during pregnancy. A suppression in the amount of leptin-induced STAT3 activation was observed in the arcuate nucleus during pregnancy, yet there was no overall change in the number of leptin responsive neurons compared to non-pregnant rats. This raises the possibility of a decrease in the degree of responsiveness of arcuate nucleus neurons to leptin during pregnancy. Using double-labelled immuno-histochemistry for alpha-melanocyte stimulating hormone (α-MSH) and leptin-induced pSTAT3 it was demonstrated that pro-opiomelanocortin (POMC) neurons remain responsive to leptin during pregnancy. In the VMH, consistent with the reduced pSTAT3, pregnancy also induced a 2-fold reduction in mRNA for the long form of the leptin receptor (Ob-Rb), the only isoform with full signal transduction capabilities. Expression of mRNA for one of the short forms of the leptin receptor (Ob-Ra) in the choroid plexus was decreased in early and late pregnancy, suggesting that reduced leptin transport into the brain may contribute to pregnancy-induced leptin resistance. CSF/plasma leptin concentration ratios did not differ between pregnant and non-pregnant rats however, suggesting unimpaired leptin transport during pregnancy. These results indicate that pregnancy is a state of hypothalamic leptin resistance and is associated with impaired activation of the leptin-induced JAK/STAT3 signalling pathway in the VMH and arcuate nucleus, and reduced expression of Ob-Rb mRNA in the VMH. This state of leptin resistance represents an important adaptation of the maternal brain allowing increased food intake and fat mass so that the maternal body can meet the metabolic demands of pregnancy and prepare for the subsequent demands of lactation.

Thesis (Ph. D.)--University of Virginia, 2006. / Includes bibliographical references. Also available online through Digital Dissertations.