Shin-ichiro Imai - US grants

DESCRIPTION (provided by applicant): Our long-term objectives in this proposal are to understand the physiological significance of a novel metabolic network comprised of systemic NAD biosynthesis as a driver and Sirt1 as a mediator in the regulation of metabolism and aging in mammals. We have focused particularly on the role of Sirt1 in pancreatic b cells and demonstrated that Sirt1 promotes glucose-stimulated insulin secretion in b cells by analyzing pancreatic beta cell-specific Sirt1-overexpressing (BESTO) transgenic mice. Interestingly, this Sirt1-mediated enhancement of b cell function is blunted in aged BESTO mice, partly due to an age-associated decline in NAD biosynthesis. Furthermore, our extensive expression profiling performed in islets from BESTO and calorically restricted mice suggests that CR enhances Sirt1 activity in b cells, possibly due to augmented NAD biosynthesis at a systemic level. These findings set the stage for a novel avenue of exciting research concerning the connection between NAD biosynthesis and Sirt1 in the regulation of metabolism and aging. We have previously demonstrated that nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the NAD biosynthetic pathway from nicotinamide (a form of vitamin B3), plays an important role in the regulation of Sirt1 activity and glucose-stimulated insulin secretion in 2 cells. Together, these findings led us to the hypothesis that a dynamic alteration in Nampt-mediated NAD biosynthesis in aging and caloric restriction (CR) causes significant changes in Sirt1 activity at a systemic level and thereby induces age-associated and CR-responsive metabolic changes in multiple tissues, such as 2 cells and hepatocytes. To address this hypothesis, we propose the following specific aims: 1) To further establish the physiological significance of Nampt-mediated NAD biosynthesis in the regulation of Sirt1 activity in b cells and hepatocytes, NAD content, Sirt1 target gene expression, and physiological functions will be examined in primary islets and hepatocytes by manipulating Nampt and Sirt1 activities genetically and pharmacologically, 2) to investigate whether Nampt-mediated NAD biosynthesis and Sirt1 activity decline with age in b cells and hepatocytes, Nampt protein levels, Nampt and Sirt1 activities, and b cell and hepatic functions will be examined in the process of aging, and 3) to analyze whether CR augments Nampt-mediated NAD biosynthesis and Sirt1 activity in b cells and hepatocytes, the metabolic effects of CR on Nampt-mediated NAD biosynthesis and Sirt1 activity will be analyzed in various genetic and pharmacological models. These studies should provide new insight into the physiological significance and therapeutic applications of this novel metabolic network comprised of systemic NAD biosynthesis and Sirt1 for age-associated complications in humans. Public Health Relevance: The proposed study should provide the following important outcomes: First, this study will identify a previously unrecognized metabolic network that plays a critical role in the regulation of metabolism and aging in mammals. Second, this study will provide insights into the mechanisms for age-associated changes in tissue function, especially in pancreatic b cells and liver, and anti-aging effects of caloric restriction. Lastly, the anticipated outcome of this study will provide important insight into the development of new preventive/therapeutic interventions for important age-associated metabolic complications, such as impaired glucose tolerance and type 2 diabetes in humans.

DESCRIPTION (provided by applicant): Strong evidence has implicated the molecular circadian clock as a key integrator of behavior and physiology, and both genetic and epigenetic perturbation of circadian systems has been associated with obesity and cardiovascular disease. Conversely, we have found that high-fat diet leads to disruption of circadian behavioral and molecular rhythms. Interestingly, we have also observed that animals provided high-fat diet only during the dark period gain less weight than those fed only during the light. Collectively, these observations underscore interconnections between overnutrition, circadian disruption, and cardiometabolic pathologies. Recently, we have made the discovery that Nampt, the rate-limiting enzyme in NAD+ biosynthesis, is a clock-controlled gene that produces 24 hr oscillations in levels of NAD+. NAD+ is also an essential cofactor in hepatic lipid and carbohydrate metabolism and may function as an oscillating nutrient sensor coupling circadian and metabolic pathways. Indeed, both Nampt and NAD+ levels are low in Clock19 and Bmal1-/- mice (and increased in Cry1-/-/Cry2-/-animals). In turn, alterations in Nampt/NAD+ modulate the nutrient-responsive deacetylase SIRT1, which we and others have found to inhibit transcription of the clock repressor Per2. Thus the overarching goal of this proposal is to test the hypothesis that high-fat diet, together with alterations in feeding time induced by high-fat intake, disrupts synchrony between cycles of energy storage and utilization in fat and liver and leads to alterations in the nutrient-responsive feedback loop comprised of CLOCK/BMAL1 and NAMPT/NAD+/SIRT1. Taken together, our recent combined findings on cardiometabolic, energy balance, and circadian clock networks have put us in position to test novel hypotheses regarding the mechanisms by which circadian coupled cellular processes regulate cardiometabolic function and energy balance. The Specific Aims are as follows: Specific Aim 1: To test the hypothesis that high-fat diet disrupts circadian control of metabolic physiology due to (a) changes in feeding time and/or (b) due to changes in dietary nutrient composition. Specific Aim 2: To test the hypothesis that high-fat diet disrupts properties of the cell autonomous circadian oscillator either (a) due to changes in feeding time and/or (b) due to changes in nutrient composition of diet. Specific Aim 3: To test the hypothesis that high-fat diet disrupts the novel circadian-metabolic feedback loop involving NAD+ biogenesis and the NAD+-dependent deacetylase SIRT1. (End of Abstract)

DESCRIPTION (provided by applicant): How physiological responses to alterations in dietary intake affect the process of aging and longevity is a fundamental question to understand the systemic regulation of the complex connection between metabolism and aging. Diet restriction (DR), the single, most reliable regimen known to retard aging and extend lifespan in a variety of organisms, has provided a unique model to address this important question. This research proposal aims to understand molecular mechanisms underlying physiological adaptive responses to DR, particularly the central adaptive response, in mammals. We have been interested in the evolutionarily conserved SIR2 (silent information regulator 2) family of NAD-dependent deacetylases/ADP-ribosyltransferases, also called sirtuins, as a critical regulator that coordinates physiological responses to DR. Our new study has recently demonstrated a novel function of the mammalian SIR2 ortholog SIRT1 in the hypothalamus, particularly in the dorsomedial and lateral hypothalamic nuclei (DMH and LH, respectively), as a key mediator that controls the orexin type 2 receptor (OX2R)-mediated signaling in response to peripheral signals including ghrelin, an orexigenic hormone secreted from stomach, induced by DR. Therefore, in this proposal, we hypothesize that SIRT1 controls central adaptive responses to DR, including the augmentation of physical activity and the maintenance of body temperature, through the up-regulation of the Ox2r expression and the neural activation in the DHM and LH. To address this hypothesis, we will examine 1) how SIRT1 up-regulates the transcription of the Ox2r gene through a newly identified target homeodomain transcription factor in response to DR, 2) whether stereotactic injection of lentiviruses expressing shRNA against Sirt1 or the SIRT1 target transcription factor into the DMH and/or LH abrogates the central adaptive response to DR, 3) how SIRT1 activity is augmented by DR in the DMH and LH, and 4) whether SIRT1-mediated central adaptive response is also important for the control of longevity in mice. Because very little has been known about the central adaptive mechanism for DR, the proposed study will provide critical insights into the physiological mechanism that orchestrates responses to DR and may assure longevity in mammals.

DESCRIPTION (provided by applicant): Recent studies have suggested that systemic interplay between multiple tissues regulates aging and longevity in model organisms. In mammals, however, the complexity of tissue interplay is multiplied, and a systemic network for mammalian aging/longevity control has been poorly understood. Our long-term goal is to understand such a systemic regulatory network for aging/longevity control in mammals, and to translate that knowledge into an effective intervention to prevent and treat age-associated pathophysiology in humans. To achieve this goal, we have particularly focused on the tissue-specific functions of the mammalian NAD+-dependent protein deacetylase SIRT1, and NAD+ biosynthesis mediated by nicotinamide phosphoribosyltransferase (NAMPT). We have recently demonstrated that the hypothalamus, particularly the dorsomedial and lateral hypothalamic nuclei (DMH and LH, respectively), is the critical place where SIRT1 regulates aging and longevity in mice. Thus, understanding how hypothalamic NAD+ levels are regulated is critical to better understand the system dynamics of mammalian aging/longevity control. Most recently, we have found that adipose tissue plays an important role in modulating NAD+ production in the hypothalamus through the SIRT1-mediated secretion of extracellular NAMPT (eNAMPT). Therefore, we hypothesize that adipose tissue-secreted eNAMPT regulates hypothalamic SIRT1 function, particularly in the DMH and LH, and also that the imbalance between eNAMPT and hypothalamic SIRT1 functions is developed during aging, causing functional defects in the hypothalamus and thereby affecting age-associated pathophysiology in mammals. To address this hypothesis, we will 1) investigate the physiological relevance of eNAMPT in the regulation of hypothalamic SIRT1 function, using loss- and gain-of-function mouse models; 2) confirm the biochemical function of eNAMPT as a systemic NAD+ biosynthetic enzyme by manipulating the equilibrium of the eNAMPT enzymatic reaction in vivo; and 3) assess possible causes for the imbalance between adipose tissue and the hypothalamus during aging by examining changes in eNAMPT enzymatic activity, hypothalamic uptake/utilization of NMN, and SIRT1 protein levels in hypothalamic nuclei, in young, middle age, and old mice. Preliminary results presented in this proposal provide strong support to our hypothesis. Thus, elucidating the physiological importance of this unprecedented intertissue communication mechanism between adipose tissue and the hypothalamus will further advance our understanding of a systemic regulatory network for aging and longevity in mammals and contribute to the development of a possible intervention to achieve better health span in our aging society.

Recent studies demonstrate that the hypothalamus functions as a high-order ?control center of aging?, counteracting age-associated pathophysiological changes and thereby promoting longevity in mammals. Our group demonstrated that the mammalian NAD+-dependent protein deacetylase SIRT1 in the hypothalamus, particularly the dorsomedial and lateral hypothalamic nuclei (DMH and LH, respectively), is critical to counteract age-associated physiological decline and promote longevity in mice. In the DMH, SIRT1 and its binding partner Nkx2-1 highly colocalize, allowing us to identify a specific subset of DMH neurons, namely, SIRT1/Nkx2-1-double positive neurons. Recently, we have identified a set of genes specifically expressed in these SIRT1/Nkx2-1-double positive DMH neurons. One of these genes is Prdm13, which encodes a member of the PR domain family of transcriptional regulators. Prdm13 is one of the downstream target genes regulated by SIRT1 and Nkx2-1 in the DMH. DMH-specific Prdm13-knockdown mice exhibit decreased sleep quality, increased adiposity, and reduction in adipose Nampt, a key systemic NAD+ biosynthetic enzyme secreted from adipose tissue to remotely regulate hypothalamic function. On the other hand, we found that the DMH- specific knockdown of the thyrotoropin-releasing hormone (Trh) gene, another gene highly and selectively expressed in the SIRT1/Nkx2-1-double positive DMH neurons, caused defects in skeletal muscle mitochondrial gene expression, specific myokine expression, and physical activity. These results suggest that SIRT1/Nkx2-1-double positive DMH neurons contain at least two functionally distinct neuronal subpopulations, namely, Prdm13- and Trh-positive neurons, and that each subpopulation regulates distinct inter-tissue feedback loops between the hypothalamus and adipose tissue or skeletal muscle. In this research proposal, we will extensively investigate the physiological importance of these two inter-tissue feedback loops. We will also examine whether maintaining the activity of these feedback loops can counteract age-associated pathophysiological changes and possibly extend lifespan in mice. The anticipated outcome from the proposed research will make a significant impact to our understanding of the systemic regulation of aging and longevity in mammals.