Dongsheng Cai, M.D., Ph.D. - US grants

The hypothalamus critically controls body weight and energy homeostasis. The Insulin and leptin pathways are most pivotal in the hypothalamic regulation of energy balance and therefore critical in the prevention against excessive energy accumulation and the associated obesity and type 2 diabetes (T2D). Recent advances show that persistent overnutrition induces hypothalamic insensitivity to insulin and leptin; however, the involved molecular basis remains unclear. Following our previous discovery that pro-inflammatory nuclear transcription factor NF-κB and its upstream activator IKKβ are activated in peripheral tissues by overnutrition leading to local insulin resistance, this proposal will investigate the role of IKKβ/NF-κB in the hypothalamic dysregulation of energy balance, and in particular, in relation to the loss of insulin and leptin sensitivity in the hypothalamus. Preliminary results show that a high-fat diet activates IKKβ/NF-κB in mouse hypothalamus, while activation of IKKβ/NF-κB induces hypothalamic insulin resistance. Animal tests further show that activation of IKKβ/NF-κB in the mediobasal hypothalamus (MBH) induced weight gain, while the ablation of IKKβ in a subtype of insulin/leptin-sensitive hypothalamic neurons protected against the dietary induction of obesity. Therefore, this proposal hypothesizes that chronic overnutrition activates IKKβ/NF-κB in the hypothalamus, desensitizes hypothalamic neurons to insulin and leptin, and causes energy imbalance leading to obesity-T2D. This hypothesis predicts that suppressing hypothalamic IKKβ could reverse these diseases. The 3 specific aims which will be pursued are as follows: (1) depict the effect of overnutrition on NF-κB in the insulin/leptinsensitive MBH neurons; (2) study the role of IKKβ/NF-κB on hypothalamic insulin and leptin resistance; (3) assess the metabolic outcomes of ablating IKKβ site-specifically in the MBH neurons or cell-specifically in the most relevant subtypes of insulin/leptin sensitive neurons ¿ AGRP and POMC neurons. To achieve these aims, a series of our established mouse models and approaches of site-directed gene manipulations will be empoyed to analyze NF-κB, insulin/leptin signaling, and the metabolic phenotypes. The completion of this study will advance our knowledge about the brain pathogenesis of obesity-T2D, provide a molecular basis for developing new therapeutic and preventive strategies, and also establish a new model to study the nutritioninflammation axis in the brain underlying nutritional diseases.

DESCRIPTION (provided by applicant): Aging is not only an important biological phenomenon but a core basis in many deleterious aging-related diseases. Having appreciated that IKKb/NF-kB-dependent inflammation mediates hypothalamic mechanism of aging, the long-term objective of this research is to study the involved neural types and molecular cascades, in order to develop strategies for treating aging-related diseases. In preliminary studies, hypothalamic astroglia have been targeted, using mouse models of astroglia-specific IKKb/NF-kB activation or inhibition. Preliminary data have demonstrated IKKb/NF-kB activation or inhibition in hypothalamic astroglia was sufficient to lead to aging acceleration or retardation, respectively. Hence, supported by these data, the hypothesis of this project is that astroglial IKKb/NF-kB is activated during early aging to induce neuronal inflammation and thus mediates the hypothalamic mechanism of aging. This hypothesis predicts that astroglial IKKb/NF-kB inhibition can reduce aging-related hypothalamic neuronal inflammation and therefore provide anti-aging effects. This hypothesis will be examined in 3 Aims: (1) Study the role of astroglial IKKb/NF-kB in aging-related hypothalamic inflammation; (2) Study the role of brain or hypothalamic astroglial IKKb/NF-kB in aging physiology and lifespan; (3) Study the neuronal mechanism in astroglial IKKb/NF-kB -mediated aging development. Experiments in these Aims will be carried out by using mouse models of site- and cell type-specific IKKb/NF-kB activation or inhibition. A list of molecular methods will be used to analyze astroglial and neuronal inflammatory changes as well as the inhibitory impacts on anti-aging molecular markers such as SIRTs and FOXOs. Also, a battery of physiological and histological approaches will be used to analyze aging of mouse models. Overall, successful completion of this project can yield new insights into the hypothalamic mechanism of aging, and enlighten a potential of targeting the hypothalamus for managing healthy aging and counteracting deadly aging-related diseases.

DESCRIPTION (provided by applicant): Obesity and type-2 diabetes (T2D) are two epidemic problems, but partly due to limited understandings on the underlying mechanisms, currently there are inadequate interventional approaches to treat or prevent these diseases. Having appreciated that IKKß/NF-?B-dependent inflammation mediates neuronal dysregulations in the brain and particularly the hypothalamus, the long-term objective of this research is to study the involved neural connections, molecular pathways, and physiological basis which can eventually lead to new strategies in treating and preventing these diseases. Preliminary studies have focused on astroglia which account for majority of brain cells, and using astroglia-specific mouse models with IKKß/NF-?B activation or inhibition, it was revealed that IKKß/NF-?B activation in astroglia causes neuronal inflammation leading to energy imbalance that promotes obesity development. Conversely, IKKß/NF-?B inhibition in astroglia can protect neuronal functions to provide anti-obesity benefits. Hence, this project hypothesizes that astroglial IKKß/NF-?B is sensitively activated by overnutrition to adversely affect neurons and cause metabolic dysregulations that underlie the development of obesity and T2D; conversely, IKKß/NF-?B inhibition in astroglia help improve the micro-environment of neurons leading to improved neuronal functions and thus counteraction against these diseases. Three Specific Aims are propsoed: (1) Study the action of astroglial IKKß/NF-?B in inducing neuronal inflammation; (2) Study the role of astroglial IKKß/NF-?B in affecting neuronal metabolic regulation; (3) Study the disease relevance of astroglial IKKß/NF-?B and its crosstalk with neurons. A combined genetic and gene delivery appraoches together with relevant neural analyses will be used to carry out these Aims. All key animal models, reagents and techniques have been established, and supportive preliminary results have been obtained. Overall, successful completion of this project will significantly clarify the brain mechanism of obesity and T2D, and enlighten the development of interventional strategies against these diseases.

DESCRIPTION (provided by applicant): Obesity and hypertension (HT) are two epidemic health problems globally. Unfortunately, these two problems are often coupled to form obesity-related HT (OHT), but due to the poor understanding about the underlying mechanism, currently there is an almost complete lack of effective interventions against OHT. Interestingly, recent research from this project has led to the discovery that the mechanism of OHT involves sympathetic upregulation arising from obesity-induced activation of proinflammatory IKK¿ in POMC neurons localized in the hypothalamus. Having appreciated that IKK¿ in POMC neurons represents a converging point between obesity and HT, the long-term objective of this research is to study the involved physiological, molecular and neural circuitry basis which can eventually lead to interventional strategies in treating and preventing OHT. Preliminary studies have revealed that IKK¿ activation in POMC neurons can promote glutamate release, and importantly, IKK¿ and glutamate signaling in POMC neurons can similarly mediate the activation of downstream presympathetic circuitry to induce HT. Conversely, inhibition of IKK¿ or glutamate signaling in POMC neurons can similarly counteract against OHT. Hence, this project hypothesizes that IKK¿ in POMC neurons employs glutamate signaling from these neurons to activate downstream presympathetic neurons and drive the sympathetic induction of OHT. Three Specific Aims are proposed to test this hypothesis: (1) Study the hypertensive role of IKK¿ and glutamate in POMC neurons; (2) Study the sympathetic basis of OHT involving IKK¿ and glutamate in POMC neurons; (3) Study the POMC neuron-related neural circuitry in relation to OHT. A combined genetic and gene delivery approaches together with relevant cardiovascular and neural analyses will be used to carry out these Aims. All key animal models, reagents and techniques have been established in the studies which generated preliminary results, making the proposal feasible. Overall, successful completion of this project will significantly clarify the brain mechanism of OHT, and enlighten the development of interventional strategies against OHT.

DESCRIPTION (provided by applicant): Energy balance and body weight control are regulated by hypothalamic neurons through sensing nutrients; however, the underlying molecular basis is still much unclear. Hypoxia-inducible factor (HIF) has recently been known to be activated by normoxic metabolic signals, and our recent publication showed that hypothalamic HIF2?/HIF? responds to glucose and HIF knockout in hypothalamic neurons are obssogenic. Therefore, the objective of this research is to study hypothalamic nutrient-sensing mechanism and its importance in metabolic physiology or disease. Our recent preliminary studies further revealed that physiological levels of glucose or leucine both activated hypothalamic HIF2? to be causally associated with diet-induced metabolic responses. Hence, the hypothesis of this proposal is that hypothalamic HIF is important for diet-induced metabolic control in terms of appetite and energy expenditure (including the forms of diet-induced spontaneous physical activity and thermogenesis). This hypothesis leads to prediction that while hypothalamic HIF inhibition causes energy imbalance and obesity, hypothalamic HIF gene delivery can improve energy balance to counteract dietary obesity. Three Specific Aims are propsoed to: (1) study the effects of nutrients on hypothalamic HIF and the involved molecular mechanism; (2) study the effects of hypothalamic HIF manipulations in appetite and energy expenditure control (including diet-induced spontaneous physical activity and diet-induced thermogenesis), and also study the molecular mediators and the sympathetic nervous system-directed physiological mechanism; (3) study effects of hypothalamic HIF inhibition or activation on dietary obesity. Experimental approaches will be significantly based on analyses of metabolic and behavioral physiology as well as HIF pathway in normal mice and the models with hypothalamic HIF manipulations in relation with various nutrient deliveries. Success of this project can lead to a new paradigm of energy balance control and a new target for treating obesity.

ABSTRACT/SUMMARY Obesity-related hypertension (OHT), an etiologically prevalent disorder accounting for ~75% of patients with hypertension, is however hard to control, and this clinical difficulty is related to the specific yet much unclear mechanism of OHT. Having recently appreciated that the hypothalamic neuronal basis of OHT is significantly due to obesity-induced IKK?/NF-?B-dependent hypothalamic inflammation, the long-term objective of this research is to study hypothalamic neural types and molecular cascades that mediate OHT. In preliminary studies, using mouse models with hypothalamic astrocytic IKK?/NF-?B activation or inhibition, supportive evidence has been obtained suggesting that hypothalamic astrocytes have site-specific multiple programs in causing hypertension. Hence, the hypothesis of this proposal is, under chronic high-fat diet feeding, hypothalamic astrocytic IKK?/NF-?B is activated to cause OHT, mechanistically mediated by (a) converging effects of adipokines and consequent hypothalamic ER stress due to blood-brain barrier (BBB) breakdown, (b) activation of POMC neurons due to astrocytic cytokine-induced dysfunction of arcuate DA neurons, and (c) excess of ?-MSH and glutamate in the PVN due to impaired astrocytic clearance. Three Aims are proposed to: (1) study the neuroanatomic and BBB breakdown basis for the role of astrocytic IKK?/NF-?B in OHT; (2) study altered MBH astrocyte-neuron relationship in obesity and the contribution to OHT; and (3) study altered PVN astrocyte-neuron relationship in obesity and the contribution to OHT. Experiments will be carried out using mouse models of site- and cell-specific IKK?/NF-?B manipulations as well as manipulations of the downstream molecular pathways in astrocytes and relevant neurons. Blood pressure phenotypes will be examined in these models in the context of dietary obesity or genetic manipulations of proposed mechanisms. Astrocytic changes, downstream programs, and further downstream neuronal alterations will be rigorously analyzed. Overall, successful completion of this project can yield new insights into the hypothalamic mechanism of OHT and enlighten the strategy of targeting hypothalamic astrocytic programs in combating this disease.

ABSTRACT/SUMMARY Having appreciated the role of pro-inflammatory IKK?/NF-?B pathway in the hypothalamic mechanism of obesity, the objective of this research is to study how hypothalamic neurons and astrocytes are programmatically involved. Through recent studies including preliminary experiments on mice with hypothalamic astrocytic IKK?/NF-?B activation or inhibition, supportive evidence was obtained to suggest that astrocytes in the mediobasal hypothalamus (MBH) have multiple programs through IKK?/NF-?B for being responsible for metabolic imbalance. Hence, the hypothesis of this proposal is, sustained activation of astrocytic IKK?/NF-?B in the MBH under chronic high-fat diet feeding leads to altered astrocyte-neuron relationships in the MBH, at least due to an impairment in astrocyte-dependent GABA clearance and also an enhancement in astrocytic cytokine production ? which employ GABA and cytokine receptor signaling to influence the responsible neurons, resulting in multiple forms of neuronal dysregulation on metabolic balance which underlie obesity and pre-diabetic development. Three Aims are designed to study (1) astrocytic IKK?/NF-?B?autophagy axis in linking hypothalamic neurons to metabolic dysregulation, (2) IKK?/NF-?B- dependent astrocytic clearance of GABA in linking POMC neuronal inhibition to obesity, and (3) IKK?/NF-?B- driven astrocytic cytokines in linking other responsive hypothalamic neurons to disease development. A combination of cellular, molecular, genetic, physiological and pharmacological approaches will be used to analyze astrocytic biology and functions, the overall and local influences on neurons, and their mechanistic roles for obesity and pre-diabetes. Successful completion of this project will be important for understanding the hypothalamic basis of obesity and diabetes and provide new paradigms in designing interventional strategies.