Student Theses and Dissertations

Putianqi Wang

Date of Award

2021

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Friedman Laboratory

Abstract

Leptin, secreted by the adipose tissue, is an afferent signal of a negative feedback loop that regulates body weight balance through its effects on feeding and energy expenditure. Mutations in the leptin gene or its cognate receptor result in severe obesity in both human and mice. My thesis work revealed a leptin-dependent, plastic pathway spanning the central to peripheral nervous system that is responsible for regulating energy homeostasis in mice. Leptin deficient (ob/ob) mice accumulate excessive fat mass due to increased food intake, and decreased energy expenditure partially as a result of defective fat utilization. Chronic leptin delivery to ob/ob mice reverses both phenotypes and leads to drastic fat loss. In contrast, dietary restriction of ob/ob mice fails to increase energy expenditure and results in reduced lean body mass rather than fat mass. These findings indicate that leptin is necessary for mice to efficiently utilize fat as energy source, however, the underlying mechanism is not known. The sympathetic nervous system (SNS) is the major regulator of several critical steps involved in fat utilization, we therefore hypothesized that leptin might regulate the plasticity of SNS innervation of adipose tissue to promote fat usage. We first visualized SNS innervation, using a whole-mount tissue clearing method (Adipo- Clear) paired with light sheet microscopy imaging, in both brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) of wild-type (WT) and ob/ob mice. Surprisingly, we found that ob/ob mice have a profound, around six-fold, reduction of SNS innervation density in both BAT and iWAT compared to age matched WT mice. The same phenotype was also observed in db/db mice which carry a mutation in leptin receptor. Furthermore, we showed that exogenous leptin delivery to adult ob/ob mice for 14 days through a subcutaneous osmotic pump normalizes their SNS innervation levels in both BAT and iWAT to WT level. This effect is independent of leptin-induced anorexia because ob/ob mice pair-fed to their leptin treated littermates fail to show any innervation increase in adipose tissues. These findings demonstrate that leptin regulates SNS innervation plasticity in adipose tissue. We then tested whether restoring adipose tissue innervation structure in ob/ob mice is sufficient to correct their fat utilization defects, such as thermogenesis and lipolysis defects. Thermogenesis is the process that dissipates energy as heat from BAT in cold conditions; and lipolysis is the process that breaks down lipid storage from iWAT to meet energy demand of other organs in times of privation. We first exposed mice to cold challenge and found that ob/ob mice, having BAT innervation density restored but having no leptin in serum, can still activate BAT thermogenesis similarly as WT mice. In contrast, ob/ob mice, not having innervation restored but having high leptin level in serum, fail to activate thermogenesis and succumb to cold just like their ob/ob littermates given no leptin treatment. This experiment led us to the surprising finding that SNS structure, rather than active leptin signaling, is critical for thermogenesis. Additionally, we observed similar trends when we fasted ob/ob mice to induce lipolysis from iWAT. In aggregate, these findings confirm our hypothesis that leptin regulates structural plasticity of SNS in adipose tissue, which in turn promotes fat utilization and energy expenditure. We next uncovered the neural mechanism underlying leptin dependent innervation regulation. We identified LepR expressing neurons in the arcuate nucleus of hypothalamus (ARC) as regulators of SNS innervation. In WT mice, either genetic deletion of LepR in ARC or diet induced leptin signaling loss in ARC leads to dramatic SNS innervation reduction in both BAT and iWAT. There are two major LepR expressing neuron populations in the ARC, agouti related peptide (AGRP) neurons and pro-opiomelanocortin (POMC) neurons. We employed a CRISPRbased gene editing strategy to selectively delete LepR in either AGRP or POMC neurons and found that leptin signaling loss in either population leads to same level of SNS innervation reduction in adipose tissue. Moreover, the magnitude of SNS innervation reduction resulted from ablating LepR in either AGRP or POMC is halved comparing to that from ablating LepR in the entire ARC region. These data suggest that AGRP and POMC neurons act synergistically in regulating leptin dependent SNS innervation. In order to regulate SNS plasticity in adipose tissue, leptin signaling needs to reach sympathetic preganglionic neurons in the spinal cord which act as the conduit between the brain and target-innervating sympathetic neurons in the periphery. However, AGRP and POMC neurons send efferents mostly within the brain, indicating the existence of downstream central populations that mediate leptin’s effects on innervation. We identified a group of brain-derived neurotropic factor (BDNF) expressing neurons in the paraventricular nucleus of hypothalamus (PVH) that 1) project directly to the sympathetic preganglionic neurons in the spinal cord, 2) are activated by leptin signaling and receive inputs from both AGPR and POMC neurons, and 3) are necessary for leptin to regulate SNS innervation. In conclusion, leptin requires downstream BDNF signaling to regulate sympathetic plasticity in adipose tissue. These downstream neurons may present therapeutic potentials for treating obesity associated with leptin resistance. The work mentioned above revealed a novel role of leptin in bidirectionally regulating sympathetic neural plasticity in adipose tissues and its underlying neural mechanism. Large-scale sympathetic neural plasticity is normally only observed during organ development or tissue injuries; therefore, it is important to understand how leptin turns on this process in adult animals. Since little is known about the connectivity between the brain and adipose tissue, we first used retrograde circuit tracing approaches to anatomically characterize the locations of the sympathetic pre- and postganglionic neurons innervating both iWAT and BAT. Interestingly, we found little overlap between iWAT and BAT innervating pre-ganglionic neurons, and zero overlap between post-ganglionic neurons, suggesting that the sympathetic neurons are highly specific to target organs. Furthermore, we revealed that there are similar number of fat innervating postganglionic neurons between WT and ob/ob mice, despite the drastic differences in adipose tissue SNS innervation density between these two mouse lines. These results suggest that the gene expression profiles of the fat innervating postganglionic neurons in WT and ob/ob mice are distinct. Therefore, we are currently conducting single cell sequencing experiments to uncover the molecular identities of the sympathetic postganglionic neurons in WT and ob/ob mice; we also hope to reveal the molecular mechanisms underlying leptin dependent plasticity in these neurons. We believe that targeting fat innervating postganglionic neurons might be a good strategy to treat metabolic diseases, and these experiments might help identify potential therapeutic targets.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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