Expression of a Constitutively Active Human Insulin Receptor in Hippocampal Neurons Does Not Alter VGCC Currents

Abstract

Memory and cognitive decline are the product of numerous physiological changes within the aging brain. Multiple theories have focused on the oxidative, calcium, cholinergic, vascular, and inflammation hypotheses of brain aging, with recent evidence suggesting that reductions in insulin signaling may also contribute. Specifically, a reduction in insulin receptor density and mRNA levels has been implicated, however, overcoming these changes remains a challenge. While increasing insulin receptor occupation has been successful in offsetting cognitive decline, alternative molecular approaches should be considered as they could bypass the need for brain insulin delivery. Moreover, this approach may be favorable to test the impact of continued insulin receptor signaling on neuronal function. Here we used hippocampal cultures infected with lentivirus with or without IRβ, a constitutively active, truncated form of the human insulin receptor, to characterize the impact continued insulin receptor signaling on voltage-gated calcium channels. Infected cultures were harvested between DIV 13 and 17 (48 h after infection) for Western blot analysis on pAKT and AKT. These results were complemented with whole-cell patch-clamp recordings of individual pyramidal neurons starting 96 h post-infection. Results indicate that while a significant increase in neuronal pAKT/AKT ratio was seen at the time point tested, effects on voltage-gated calcium channels were not detected. These results suggest that there is a significant difference between constitutively active insulin receptors and the actions of insulin on an intact receptor, highlighting potential alternate mechanisms of neuronal insulin resistance and mode of activation.

Elevating Insulin Signaling Using a Constitutively Active Insulin Receptor Increases Glucose Metabolism and Expression of GLUT3 in Hippocampal Neurons

“…Routine confirmation of mCherry fluorescence was performed on both control and IRβ dishes using a spectral analysis camera (Nuance, CRi Inc., Boston, MA, United States). The expression rate of mCherry was ∼80% of cells per dish, similar to that reported in our prior IRβ study in this same cell culture model (Frazier et al, 2018). As with our previous study (Frazier et al, 2018), plasmid expression appeared to be limited to only neurons, as no detectable fluorescence was noted in astrocytes.…”

“…This receptor, IRβ, is comprised almost solely of the intracellular catalytic β subunit, yet is still capable of translocating and successfully inserting into the plasma membrane (Lebwohl et al, 1991). The truncation of the α subunit renders the modified receptor constitutively active, as highlighted by our previous work showing that IRβ significantly elevates pAKT levels for at least 48 h in the same cell culture model used here (Frazier et al, 2018). Thus, this technique allows us to reliably increase IR signaling in the absence of exogenous insulin.…”

“…Using a lentiviral delivery system, mixed, primary hippocampal cultures received one of two plasmids: a control plasmid containing a neuron-specific synapsin promoter and a fluorescent marker (mCherry), or an experimental plasmid containing the synapsin promoter, mCherry, and the constitutively active IRβ receptor. Both plasmids were constructed from a pHR-SFFV-KRABdCas9-P2A-Cherry backbone vector (gift from Jonathan Weissman, plasmid #60954, Addgene, Watertown, MA, United States) as described previously (Frazier et al, 2018). Briefly, the synapsin promoter and IRβ sequence were ligated between the AscI and BamHI sites using PCR and standard digestion protocols.…”

“…Additionally, while low nanomolar concentrations of insulin are often employed to selectively bind the IR, the possibility for non-specific activation of other known or unknown receptors (i.e., IGF-I) still remains. For this reason, we opted to induce elevated insulin signaling by expressing a genetically modified human IR using a previously validated molecular approach (Frazier et al, 2018). This receptor, IRβ, is comprised almost solely of the intracellular catalytic β subunit, yet is still capable of translocating and successfully inserting into the plasma membrane (Lebwohl et al, 1991).…”

Evaluation of the potential of organ glucose metabolism by 18F-FDG accumulation with insulin loading in super-aged mice compared with young normal mice

“…In contrast, the level of 18 F-FDG accumulation in the cortex signi cantly decreased after insulin loading in the super-aged group. Aging is associated with reductions in the levels of both insulin and its receptor in the brain, which may even cause the brain to be in the state of insulin resistance [43][44][45][46] . Cholerton et al 44 indicated that chronic high levels of insulin and insulin resistance may exert a negative effect on several body systems, including the central nervous system, for some time prior to the onset of diabetes.…”