The Identification of a Leptin Dependent Neural Pathway Regulating Adipose Tissue Innervation

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

Mechanism of AADACL1 Regulation of PKC Isoforms in Human Platelets

Our lab discovered a novel enzyme, arylacetamide deacetylase-like 1 (AADACL1) that regulates lipid metabolism and platelet activation in human platelets. Inhibition of AADACL1 by a small molecule inhibitor JW480 decreases platelet aggregation in response to platelet agonists such as collagen. Our work suggests that AADACL1 regulates platelet activation through its lipid substrate, 2-acetyl monoalkylglycerol ether (2­acetyl MAGE), which we hypothesize directly modulates protein kinase C (PKC) function. The lipid 2-acetyl MAGE binds a PKC lipid binding domain with high affinity, similar to the PKC activator, diacylglycerol and decreases PKC kinase activity in vitro. Consistent with this, inhibition of AADACL1 also correlates with decreased PKC phosphorylation in human platelets. These data provide evidence that AADACL1 closely regulates PKC activation in platelets and reveal the drug target potential of AADACL1.Bachelor of Scienc

A Current of Anti-Japanese Sentiments : Anti-Japanese sentiment of Liberal Criticism

It was not only the intellectuals who had congregated in All-China Federation of National Salvation Association that called for the realization of resistance to Japan and democracy before "7・7" (i.e. "The Marco Polo Bridge Incident"). In the past studies, however, the public anti-Japanese movements, which had gathered into All-China Federation of National Salvation Association, have been mainly discussed. In the present work I would like to analize the anti-Japanese sentiments of Lo Lung-chi (羅隆基) and his fellows, who had close relation to The National Socialist Party (中国国家社会党), using Liberal Criticism (『自由評論』) as the basic material. The period investigated is that from the winter of 1935 to the autumn of 1936. The aim of this thesis is as follows: (1) to make clear the community between the anti-Japanese sentiments of All-China Federa tion of National Salvation Association and those of the intellectuals of the Liberal Criticism faction, and to demonstrate that this community brought about the gradual consolidation of several anti-Japanese movements into one after "9・18" (i.e. "The Manchurian Incident"); (2) to clarify the difference in opinion between All-China Federation of National Salvation Association and the intellectuals of the Liberal Criticism faction, and to point out that this explains the essential difference in their views as to the Chinese Revolution after the anti-Japanese resistance was over