Title: The Role of Leptin in Body Weight Regulation
Description: 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.