Visualizing insulin actions on neuronal metabolism and function using fluorescent biosensors

University Of Oklahoma Hlth Sciences Ctr, Oklahoma City OK

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Abstract

ABSTRACT Reduced glucose utilization and energy production in peripheral tissues are major issues contributing to decreases in healthspan with age and represent a major challenge for the healthcare system. Although insulin actions and resistance have been extensively studied in peripheral tissues, the precise role of insulin in brain energy dynamics and brain health remains an enigma. Despite the well-accepted importance of glucose for normal brain function, direct glucose utilization by neurons and its effect on aging and age-related diseases have been highly controversial. Contrary to the popular ‘astrocyte-to-neuron lactate shuttle hypothesis’, we reported that neurons are a site for aerobic glycolysis, revealed by transient elevations in the cytosolic NADH/NAD+ ratio (measured with the genetically encoded fluorescence lifetime biosensor, Peredox). In addition, these metabolic responses increased after inhibiting both astrocytic and neuronal monocarboxylate transporters (MCT), which prevented lactate transfer from astrocytes to neurons. The mechanisms contributing to the mismatch between glucose consumption and oxygen utilization in neurons are not understood and are the focus of this application. These fundamental questions must be resolved before we can advance our understanding of the effects of metabolic diseases and aging on the brain. Thus, the premise of this application is that insulin action at the neuronal level has a key role in regulating glucose supply and oxygen utilization. The overarching goal of my laboratory is to elucidate the regulation of energy metabolism in the brain with age, and this proposal uses adult animals to rigorously define the role of insulin on neuronal metabolism. Using genetically encoded fluorescent biosensors and 2-photon fluorescence lifetime microscopy, we will detect simultaneous readouts of neuronal excitability and metabolism in brain slices. This model preserves the complex 3D organization and circuitry of native brain tissue. Specifically, we hypothesize that glycolysis and oxidative phosphorylation in neurons are directly regulated by insulin signaling, which increases energy metabolism (ATP synthesis), the restoration of cytosolic calcium levels, and synaptic plasticity. In this application, we will focus on the dentate granule cells (DGC) of the mouse hippocampus, a structure required for learning and memory and shows high insulin receptor (IR) expression and phosphorylation. It is also known that insulin can cross-react with the IGF-1 receptor (IGF- 1R), although with less affinity, and that double knockout mice for these receptors in the hippocampus manifest as disrupted spatial learning and memory. However, the individual contribution to cognition of the signaling through each receptor has yet to be determined. The following aims are proposed: Aim 1: Define the mechanisms of insulin signaling on energy metabolism in neurons. Aim 2: Characterize the role of neuronal insulin signaling on cytosolic calcium dynamics and synaptic plasticity in the hippocampus. Aim 3: Investigate the role of neuronal insulin resistance on learning and memory. Our results have the potential to resolve the association between aging, metabolic diseases, and cognitive decline and provide a route to new therapeutic interventions.