Is the Effect of Glucose on Hippocampal Memory Insulin-Dependent?

Insulin is now established as a key regulator of brain mechanisms that include both glucose metabolism and synaptic plasticity, especially within the hippocampus. However, the complex set of signaling cascades mediating these effects is not yet understood. Recent studies, many from our lab, have established that insulin plays multiple roles in the brain: in addition to regulation of energy supply, metabolism, and feeding, our work has shown that hippocampal insulin is a key modulator of learning and memory. Exogneous insulin enhances, while pharmacological blockade of intrahippocampal insulin impairs, both metabolism and cognition. Moreover, when systemic insulin signalling is impaired, such as in Type 2 diabetes, hippocampal function and metabolism are again impaired. Memory processes both in the hippocampus and elsewhere (e.g. amygdala) are well established to be sensitive to glucose supply: performance on memory tasks is limited by glucose availability, and provision of additional glucose supports enhanced task performance. Systemically, insulin regulates glucose transport from the blood into cells; conversely, glucose regulates insulin synthesis and release from the pancreas, so that the two molecules mutually regulate. Although this relationship between insulin and glucose has been well studied, there has been little work on their interaction in the brain. For instance, although we have shown that insulin regulates hippocampal glucose metabolism, it is unknown whether glucose acts to enhance memory via stimulation of insulin release within the hippocampus, or whether insulin's procognitive effects are via stimulation of glucose metabolism or a direct modulation of plasticity. In this study, Glut4, an insulin-dependent glucose transporter found on some hippocampal neurons, was directly blocked. Indinavir, a Glut4 inhibitor, was injected directly into the dorsal hippocampus of rats in the presence or absence of a peritoneal glucose injection in order to assess changes in cognition. It was found that indinavir treatment significantly impaired cognition in spontaneous alternation tasks, reduced anxiety, and, surprisingly, and had no effect on cognitive performance in a novel object recognition task. These data support a novel role for GluT4 as a mediator of hippocampal memory processing and suggest that insulin acts to regulate cognitive function at least in part via GluT4-mediated glucose transport into neurons. In the presence of indinavir, glucose was unable to enhance memory, consistent with this interpretation and suggesting that enhancement of hippocampal memory by glucose may require hippocampal insulin signaling. Post-mortem molecular studies of hippocampal protein expression provided further insight into the molecular impact of both glucose treatment and GluT4 blockade. Introduction: Glucose is well known as the main fuel source of the brain. Cognitive demand leads to decreased glucose concentrations during spatial memory tasks, with glucose demands directly corresponding to the complexity of the task (McNay et al., 2000). This study indicates that the limitation imposed on memory processing is the concentration of glucose, with a specific focus on the hippocampus. Metabolically, it is widely known that upon increase in systematic glucose concentration, following a meal for example, insulin is released to lower glucose concentration; the aim of the body is to maintain homeostasis of fuel supply. In addition to studies linking the effect of glucose to cognition, insulin’s role on similar tasks has been studied as well. Literature focuses on both the physiological disorders involving impaired insulin signaling, as well as on specific alteration of insulin signaling on the cellular level. For instance, diabetes mellitus type 2 (T2DM) is a metabolic disorder where the lack of insulin sensitivity leads to hyperglycemia. Patients suffering from T2DM have been seen to experience progressive cognitive deficits (Cukierman et al., 2005). Further, it has been shown that hyperinsulinemia associated with T2DM makes patients more susceptible to developing Alzheimer’s disease (Kroner, 2009). Excess insulin has been linked to the accumulation of beta amyloid, as the two molecules share a breakdown pathway. Studies on the cellular and molecular levels make a connection between insulin activity and learning and memory processes. Evaluation of insulin signaling in the central nervous system revealed the presence of insulin receptors distributed throughout the hippocampus (Zhao et al., 1999) alluding to a significant role of insulin receptors on memory processing. Further, direct administration of insulin to the hippocampus was shown to enhance cognitive function in spatial memory tasks. The same study showed that administration of increasing concentrations of insulin versus cognitive function yields a graph shaped like an inverted U, indicating an optimal level of insulin for cognition. Alteration from this optimal level leads to cognitive impairments (McNay et al., 2010). Furthermore, intranasal insulin administration in humans improved memory performance, suggesting a potential treatment for Alzheimer’s disease (Benedic et al., 2004). Insulin signaling is a complex pathway that has yet to be completely understood. It is known that the activation of insulin receptors via the dimerization of tyrosine residues involves an increase of P13K activity, a kinase involved in cell growth, proliferation, and differentiation. Moreover, it has been recently shown that using insulin-like growth factor affects the phosphorylation of CREB, which is correlated to memory enhancement (Alberini and Chen, 2012). Another key player in insulin signaling is GluT4. In contrast to various other cellular glucose transporters, GluT4 is an insulin-dependent glucose transporter found in the hippocampus. Recently, the activation of GluT4 is thought to be the connection between insulin’s effects on cognitive function as described above. GluT4 is the main molecular target in the present study. Using Indinavir, a drug previously used for the treatment of HIV/AIDS, and a GluT4 antagonist, this study directly alters hippocampal insulin signaling and measures its effect on cognitive function. Glucose and insulin both play significant roles in cognitive processes, as indicated by the memory enhancement produced separately by the administration of each molecule into the hippocampus. Given their interdependent actions systematically, it is imperative then to further our understanding of the way these molecules work together to affect cognition. One may ask two questions; is the effect of glucose on memory a product of insulin signaling? Or, on the contrary, is the effect of insulin on memory mediated by the presence of glucose? This current study tackles the first question: in order to investigate if memory is dependent on insulin signaling within the brain, subjects were divided into two groups either receiving a microinjection of indinavir or aECF directly into the dorsal hippocampus. Each group was further divided into glucose or saline receiving groups, where the treatment was co-administered by peritoneal microinjection. Using this method, this study indirectly tests the dependency of insulin on the effect of glucose on memory by ceasing the uptake of glucose into the cell. In order to measure cognition, three behavioral tasks were performed: spontaneous alternation, novel object recognition, and open field task. Administration of indinavir resulted in a significant decrease in percent alternation in a four-armed maze, and a reduction in anxious behavior in comparison to subjects receiving aECF. The administration of glucose did not significantly affect behavior in spontaneous alternation and novel object recognition tasks. However, glucose administration in open field tasks seemed to ameliorate the effect of indinavir. The effects of co-administration of glucose and indinavir were not significantly different from that of glucose and saline co-administration with aECF. Collectively, this data indicates that GluT4, and hence insulin signaling, plays a key role in cognition. In regards to glucose administration, the data presented in this study is inconsistent with previous studies; it was expected that subjects receiving glucose would show enhancement in memory performance in comparison to the aECF/saline-administered groups, and to the two groups receiving the indinavir treatment. The lack of differences between the indinavir-administered groups indicates the need for further studies. Molecular studies of essential hippocampal proteins involved in insulin signaling and learning and memory are currently in progress.