Matthew Merrins - US grants

DESCRIPTION (provided by applicant): Secretion of insulin from beta-cells of pancreatic islets is biphasic and pulsatile. Loss of first phase secretion and steady-state plasma insulin oscillations are an early development in Type 2 diabetes mellitus. Despite extensive study of the signaling cascades underlying glucose-stimulated insulin secretion, it remains unclear how oscillations in insulin secretion arise. Our overall hypothesis is that slow oscillations in islet metabolism and insulin secretion reflect slow oscillations in glycolyis. Based on this hypothesis, a computational model has been developed in which slow glycolytic oscillations mediated by phosphofructokinase-1 (PFK1) interact with fast oscillations arising from membrane electrical activity and Ca2+ (the 'Dual Oscillator Model', or DOM). Although the DOM can uniquely account for the diversity of oscillatory patterns observed in islets in vitro and in vivo, direct evidence for beta-cell glycolytic oscillations is lacking, leaving the open question of whether metabolic oscillations are intrinsic, as the DOM predicts, or driven by Ca2+ oscillations, as proposed in competing models. To test the predictions of the DOM at the level of metabolic activity, we propose to examine whether glucokinase and phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB), which are regulators of PFK1, represent oscillatory parameters that modulate metabolism-secretion coupling in islets. Dynamic FRET imaging using novel metabolic sensors will be combined with patch-clamp electrophysiology, mathematical modeling, and optical measurements of Ca2+ and NAD(P)H, to address the following Specific Aims: 1. Define the spatial and temporal properties of glucokinase activation, including regulation via its binding partner PFKFB, and assess their respective contributions to the modulation of metabolic oscillations. 2. Modify the Dual Oscillator Model to incorporate changes in glycolytic flux and islet oscillatory behavior induced by glucokinase/PFKFB interaction and fructose-2,6-bisphosphate production by PFKFB. 3. Determine whether Ca2+ drives changes in metabolism, metabolism drives Ca2+, or both occur.

DESCRIPTION (provided by applicant):Type 2 diabetes involves reduced ?-cell mass and impaired insulin secretion, but the relationship between mass and secretion is not well understood. Cyclin dependent kinases (Cdks) regulate cell cycle machinery and ?-cell proliferation and could be possible targets for new drugs to increase ?-cell mass in patients. Our new data, however, shows that interrupting Cdk2 signaling unexpectedly disrupts ?-cell function long before changes in ?-cell mass occur, suggesting Cdk2 might be an integrator of ?-cell proliferation and secretory function. We hypothesize that Cdk2 affects ?-cell function by interacting with ion channels, the insulin receptor signaling pathway, and ?-cell fuel metabolism. We will take advantage of a mouse model lacking Cdk2 in its ?-cells and use novel metabolic sensors combined with patch-clamp electrophysiology and insulin secretion assays to (1) determine the targets of Cdk2 in the ?-cell secretory pathway, (2) determine the role of Cdk2 in controlling ?-cell bioenergetics, and (3) establish the Cdk2 pathway in human islets and test whether Cdk2 expression rescues ?-cell function in diabetic human islets. To accomplish these aims, my mentor, Dr. Les Satin, will provide support for the electrophysiology, and my co-mentor Dr. Rane and collaborator Dr. Bernal- Mizrachi will provide training in the use of more advanced diabetes models. The collaborative environment at the Brehm Diabetes Center will provide access to reagents and expertise that will support this work and enable the creation of other mouse models to acutely delete Cdk2. The access to resources and training we propose will allow me to establish an independent line of research and will promote my development, productivity, and independence. In addition, the results obtained will be important for the development of new treatments for increasing ?-cell mass and secretory function in Type 2 diabetics.

? DESCRIPTION (provided by applicant): Advancing age is associated with an increased risk of impaired glucose tolerance and the development of type 2 diabetes. Because these disorders are themselves risk factors for diseases associated with significant morbidity and mortality, new approaches to maintain glucose homeostasis in the aged are urgently needed. Although beta cell dysfunction has been implicated as a contributing factor to disordered glucose homeostasis in the aged, the nature of the beta cell defects that emerge in older individuals has not been carefully studied. Thus, the objective of the proposed study is to identify the precise nature of the beta cell defects that emerge with age and obesity, and gain insight as to whether these defects can be mitigated by known interventions that extend healthspan.

? DESCRIPTION (provided by applicant): Advancing age is associated with an increased risk of impaired glucose tolerance and the development of type 2 diabetes. Because these disorders are themselves risk factors for diseases associated with significant morbidity and mortality, new approaches to maintain glucose homeostasis in the aged are urgently needed. Although beta cell dysfunction has been implicated as a contributing factor to disordered glucose homeostasis in the aged, the nature of the beta cell defects that emerge in older individuals has not been carefully studied. Thus, the objective of the proposed study is to identify the precise nature of the beta cell defects that emerge with age and obesity, and gain insight as to whether these defects can be mitigated by known interventions that extend healthspan.

Abstract Over 375 million people suffer from type 2 diabetes (T2D), a disease defined by the failure of pancreatic ?-cells. Insulin secretion and maintenance of endoplasmic reticulum (ER) Ca²+ levels are two essential ?-cell functions known to fail in T2D. Therefore, uncovering the metabolic regulatory pathways critical to these processes will allow the development of new therapies for T2D. We propose that modulation of the glycolytic enzyme pyruvate kinase (PK) has strong potential to be of protective and therapeutic value. We have discovered that of the three PK isoforms in the ?-cell, activation of PKM2 both potentiates insulin secretion and raises ER Ca²+ levels. The objective of the research described in this application is to use novel metabolic imaging methods to identify the specific steps in the ?-cell metabolic and secretory pathways that are controlled by PKM2, and to determine the extent to which PKM2 activation can protect/restore insulin secretion and ER Ca²+ during diabetes progression. To do so, we will: 1) Determine how PKM2 regulates the triggering and metabolic amplifying pathways of insulin secretion, 2) Determine the role of PKM2 in ER Ca²+ homeostasis and the neurohormonal amplification of insulin secretion, and 3) Assess the contribution of impaired PKM2 activity to diabetes pathophysiology and the therapeutic potential of PKM2 activation. Completion of these aims will heighten our understanding of nutrient signaling in the pancreatic ?-cell, and evaluate promising new targets for the prevention and therapy of T2D.

Summary Age-related diseases are the major causes of morbidity and mortality in Western society, and aging is a significant risk factor for the development of Alzheimer's disease (AD). Calorie restriction (CR), a dietary intervention which extends lifespan while delaying or preventing age-related disease, can slow or prevent AD in animal models, but reduced-calorie diets are notoriously difficult to sustain. Over the past decade, significant progress has been made in identifying small molecules that can mimic some of the benefits of a CR diet and extend lifespan and/or healthspan. There is growing evidence that these geroprotectors may be able to treat or prevent Alzheimer's disease, but significant questions remain. This proposal, which is responsive to PAR-18- 596, will address major outstanding questions surrounding the use of geroprotectors for AD, as we address the high-priority topic of a ?Geroscience Approaches to Alzheimer's Disease.? Here, we will rigorously test four geroprotectors covering a broad range of mechanisms including the inhibition of mTOR, the activation of AMPK, and the induction of sirtuins in two mouse models of AD. As research into geroprotectors thus far has utilized models of disease based on mutations identified in early onset AD, we will perform a comparative study of these geroprotectors in both an early onset mouse model of AD, the 3xTg mouse, and a novel mouse model of late-onset AD recently developed by the MODEL-AD consortium. Late- onset AD represents the majority of human cases of AD, and thus assessing the efficacy of geroprotectors in late-onset models of AD is critical. The risk of late-onset AD is significantly increased by the development of type 2 diabetes, and the prevalence of both obesity and diabetes continues to increase in the elderly. Importantly, many geroprotectors affect metabolic health, altering glycemic control and body composition. We will therefore perform the first ever assessment of geroprotectors on cognition, AD pathology, frailty, and the overall metabolic health of mouse models of AD with diet-induced obesity. Finally, glucose metabolism is disrupted in the brains and neurons of AD patients, and recent work has identified defects in glucose uptake and mitochondrial dysfunction. We will leverage a set of novel metabolic biosensors to identify the precise nature of the metabolic signaling defects in the neurons of early and late-onset mouse models of AD, and further determine if geroprotectors can restore normal metabolism at the level of the single cell. In the long term, the work proposed here will significantly advance the concept of a geroscience approach to AD, improving our understanding of the efficacy of geroprotectors in early and late-onset models of AD, in lean mice and in the context of diet-induced obesity, and at the level of the whole organism and single neuron. Not only will we identify geroprotectors for future clinical evaluation, but we will establish an overall approach that will be invaluable for the preclinical evaluation of strategies to reverse or prevent AD in the future.

Summary Age-related diseases are the major causes of morbidity and mortality in Western society, and aging is a significant risk factor for the development of Alzheimer's disease (AD). Calorie restriction (CR), a dietary intervention which extends lifespan while delaying or preventing age-related disease, can slow or prevent AD in animal models, but reduced-calorie diets are notoriously difficult to sustain. Over the past decade, significant progress has been made in identifying small molecules that can mimic some of the benefits of a CR diet and extend lifespan and/or healthspan. There is growing evidence that these geroprotectors may be able to treat or prevent Alzheimer's disease, but significant questions remain. This proposal, which is responsive to PAR-18- 596, will address major outstanding questions surrounding the use of geroprotectors for AD, as we address the high-priority topic of a ?Geroscience Approaches to Alzheimer's Disease.? Here, we will rigorously test four geroprotectors covering a broad range of mechanisms including the inhibition of mTOR, the activation of AMPK, and the induction of sirtuins in two mouse models of AD. As research into geroprotectors thus far has utilized models of disease based on mutations identified in early onset AD, we will perform a comparative study of these geroprotectors in both an early onset mouse model of AD, the 3xTg mouse, and a novel mouse model of late-onset AD recently developed by the MODEL-AD consortium. Late- onset AD represents the majority of human cases of AD, and thus assessing the efficacy of geroprotectors in late-onset models of AD is critical. The risk of late-onset AD is significantly increased by the development of type 2 diabetes, and the prevalence of both obesity and diabetes continues to increase in the elderly. Importantly, many geroprotectors affect metabolic health, altering glycemic control and body composition. We will therefore perform the first ever assessment of geroprotectors on cognition, AD pathology, frailty, and the overall metabolic health of mouse models of AD with diet-induced obesity. Finally, glucose metabolism is disrupted in the brains and neurons of AD patients, and recent work has identified defects in glucose uptake and mitochondrial dysfunction. We will leverage a set of novel metabolic biosensors to identify the precise nature of the metabolic signaling defects in the neurons of early and late-onset mouse models of AD, and further determine if geroprotectors can restore normal metabolism at the level of the single cell. In the long term, the work proposed here will significantly advance the concept of a geroscience approach to AD, improving our understanding of the efficacy of geroprotectors in early and late-onset models of AD, in lean mice and in the context of diet-induced obesity, and at the level of the whole organism and single neuron. Not only will we identify geroprotectors for future clinical evaluation, but we will establish an overall approach that will be invaluable for the preclinical evaluation of strategies to reverse or prevent AD in the future.