Insulin Receptors and Intracellular Ca 2+ Form a Double-Negative Regulatory Feedback Loop Controlling Insulin Sensitivity

Since the discovery of insulin and insulin receptors (IR) in the brain in 1978, numerous studies have revealed a fundamental role of IR in the central nervous system and its implication in regulating synaptic plasticity, long-term potentiation and depression, neuroprotection, learning and memory, and energy balance. Central insulin resistance has been found in diverse brain disorders including Alzheimer’s disease (AD). Impaired insulin signaling in AD is evident in the activation states of IR and downstream signaling molecules. This is mediated by Aβ oligomer-evoked Ca 2+ influx by activating N-methyl-D-aspartate receptors (NMDARs) with Aβ oligomers directly, or indirectly through Aβ-induced release of glutamate, an endogenous NMDAR ligand. In the present opinion article, we highlight evidence that IR activity and free intracellular Ca 2+ concentration [Ca 2+] i form a double-negative regulatory feedback loop controlling insulin sensitivity, in which mitochondria play a key role, being involved in adenosine triphosphate (ATP) synthesis and IR activation. We found recently that the glutamate-evoked rise in [Ca 2+] i inhibits activation of IR and, vice versa, insulin-induced activation of IR inhibits the glutamate-evoked rise in [Ca 2+] i. In theory, such a double-negative regulatory feedback loop predicts that any condition leading to an increase of [Ca 2+] i may trigger central insulin resistance and explains why central insulin resistance is implicated in the pathogenesis of AD, with which glutamate excitotoxicity is a comorbid condition. This model also predicts that any intervention aiming to maintain low [Ca 2+] i may be useful for treating central insulin resistance.


Introduction
Since the discovery of insulin 1 and insulin receptors (IR) 2 in the brain in 1978, numerous studies have revealed a fundamental role of IR in the central nervous system (CNS). IR-mediated signaling is implicated in the regulation of diverse functions in the CNS, including synaptic plasticity, long-term potentiation and depression, neuroprotection, learning and memory, and energy balance 3 . Central insulin resistance has been found in neurodegenerative diseases such as Alzheimer's disease (AD) 4,5 and Parkinson's disease (PD) 6 , stroke, and traumatic brain injury (TBI) 7 . Impaired insulin signaling in AD is evident in the activation states of IR and downstream signaling molecules 5 . Compared with control cases, insulin in AD brains induced 24-58% less activation at the level of IR and 90% less activation of insulin receptor substrate 1 (IRS-1) 5 . It has been presumed 5 that the inhibition of IR activation is mediated by Aβ oligomer-triggered Ca 2+ influx, in part by activating N-methyl-D-aspartate receptors (NMDARs) 8 , followed by a rise in Akt1 pS 473 9 , which can inhibit insulin-induced IR activation through Thr phosphorylation of the IR β subunit 10 . Aβ oligomers may activate the NMDAR-gated Ca 2+ influx directly 11 or indirectly through the intermediate release of glutamate, a ligand of NMDAR 11-15 . This suggests that the rise in intracellular free Ca 2+ concentration ([Ca 2+ ] i ), evoked by either Aβ oligomers or glutamate, leads to dysfunctional activation of IR in AD. In the present opinion article, we highlight evidence that IR and [Ca 2+ ] i form a double-negative regulatory feedback loop controlling insulin sensitivity, and mitochondria have a key role in this feedback loop, being involved in adenosine triphosphate (ATP) synthesis and IR activation.

Glutamate-evoked rise in [Ca 2+ ] i causes inhibition of IR activation
Glutamate serves as the major excitatory neurotransmitter in the CNS. Its excessive accumulation in a synaptic cleft can trigger excitotoxicity, a pathologic process leading to neuronal cell death. Glutamate-induced activation of the NMDAR-gated Ca 2+ influx is generally considered central to the development of excitotoxicity 16 . Prolonged glutamate exposure causes a rapid initial increase in the [Ca 2+ ] i , followed by a larger secondary [Ca 2+ ] i increase concomitant with a decrease in the mitochondrial inner membrane potential (ΔΨ m ) 17-19 . We recently found that on Ca 2+ -induced mitochondrial depolarization, insulin induced 48% less activation of IR (assessed by pY 1150/1151 ) compared with control 20 . Earlier, we showed that a decrease in ΔΨ m can abrogate IR activation 18 , since the ΔΨ m -dependent hydrogen peroxide (H 2 O 2 ) mitochondrial signal at complex II is critically involved in the activation of IR in neurons 21-23 . Thus, the glutamate-evoked increase in [Ca 2+ ] i , followed by the drop in ΔΨ m , leads to the inhibition of insulin-induced activation of IR (Figure 1a).

Insulin prevents glutamate-evoked rise in [Ca 2+ ] i
Normally, the NMDAR-gated Ca 2+ influx is counterbalanced with Ca 2+ efflux, which is governed by plasma membrane Ca 2+ ATPase (PMCA) and the Na + /Ca 2+ exchanger (NCX) 24,25 . NCX-mediated Ca 2+ efflux is also ATP-dependent, since NCX exchanges one Ca 2+ for three Na + , and the three Na + are then pumped out by the Na + /K + ATPase at the expense of one ATP. In excitotoxicity, prolonged stimulation with glutamate leads to ATP depletion and an abnormal rise in [Ca 2+ ] i , since the massive Ca 2+ influx is no longer counterbalanced by Ca 2+ efflux 26 . Therefore, maintenance of ATP production is crucial for preventing the rise in [Ca 2+ ] i in excitotoxicity. We found recently that pre-treatment with insulin prevents neurons from glutamate-evoked ATP depletion due to its protective effect on spare respiratory capacity (SRC), a measure that relates to the amount of extra ATP that can be produced via oxidative phosphorylation in case of increased energy demand 19 . The effect of insulin on SRC relates to its action on mitochondrial metabolism. It has long been known that the tricarboxylic acid cycle is the intracellular site of insulin action and that insulin acutely stimulates succinate oxidation at mitochondrial complex II 26,27 . Succinate oxidation at mitochondrial complex II has been identified recently as the main source of SRC 28 . In line with this, insulin prevented the glutamate-evoked rise in [Ca 2+ ] i in our experiments with glutamate excitotoxicity 19 (Figure 1b). .

IR and [Ca 2+ ] i form a double-negative feedback loop controlling insulin sensitivity
Collectively, this evidence suggests that a double-negative regulatory feedback loop exists between IR activity and [Ca 2+ ] i . The glutamate-evoked rise in [Ca 2+ ] i inhibits activation of IR and, vice versa, insulin-induced activation of IR inhibits the glutamate-evoked rise in [Ca 2+ ] i (Figure 1c).
This double-negative feedback loop model predicts that any condition leading to an increase in [Ca 2+ ] i may trigger insulin resistance. It appears to explain why central insulin resistance

Amendments from Version 1
The manuscript has been revised in accordance with reviewer's notes that (a) bistability is not a necessary consecuence of a double negative regulatory feedback loop and (b) Figure 1 will be more useful when signal transduction pathways will be shown. Sentences related to "bistability" have been removed from the abstract, main text, and reference list. Figure 1 has been revised and includes now pathways described in the text of the first version.
Any further responses from the reviewers can be found at the end of the article is implicated in the pathogenesis of disorders such as AD 4,5 , PD 6 , stroke, and TBI 7 , with which glutamate excitotoxicity is a comorbid condition 29 . The model also predicts that any intervention aiming to prevent Ca 2+ influx of or enhance efflux of Ca 2+ from neurons, thereby maintaining low [Ca 2+ ] i , may be useful for treating central insulin resistance. Given that Ca 2+ efflux is ATP-dependent, any intervention directed to enhance ATP production in neurons may be especially useful to improve insulin sensitivity in the brain.

Data availability
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Zhen Deng
Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou, China This model predicts that any disease that causes elevated [Ca 2+ ] i may trigger central insulin resistance, and explains why central insulin resistance is related to the pathogenesis of AD, and glutamate excitotoxicity is a comorbidity. The model also predicts that any intervention aimed at maintaining low [Ca 2+ ] i can be used to treat central insulin resistance.
It is an interesting theory between brain insulin resistance and AD. Although not much experimental data support the theory directly, it is worth following. In the latter case, several laboratories have shown mGLUR5 acts as a scaffolding complex for amyloid accumulation causing receptor clustering at the membrane surface and results in elevated intracellular calcium levels.

Are all factual statements correct and adequately supported by citations? Yes
Additionally, the manuscript could be improved if a discussion on how their model might vary across different CNS disorders such as AD, Parkinson's TBI, etc. in relation to healthy functional activity. The authors neglect to take into account factors that also play a role in modulating insulin signaling. These include: Inflammation. This is prominent in numerous disease states and can negatively impact insulin signaling, while exacerbating mechanisms associated with multiple neurodegenerative disorders.
○ Inhibitory feedback regulation. Insulin signaling is tightly controlled to prevent perturbations in metabolism as well as control the specificity of the signal on multiple downstream effectors. Several phosphatases are responsible for this, not just at the receptor, but also on effector enzymes.The current model does not take into consideration this tightly controlled feedback loop.

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Receptor internalization. Upon insulin binding, the insulin receptor becomes internalized as another means to control the strength and duration of the signal. This internalization is more prominent in hyperinsulinemia and may account for the resulting insulin resistance. How would this model change during stages of insulin resistance, which are hypothesized to initiate the cognitive decline observed in AD? ○ Peripheral insulin signaling.Insulin produced in the pancreas is able to enter the CNS.How does the proposed model take into consideration fluctuations during normal periods of food consumption? ○ Are the authors proposing a similar mechanism for the structurally analogous insulin-like growth factor-1?
Minor concerns: The glutamate pathway in Figure 1 should have a different color scheme to make it easier to differentiate from Calcium concentration.
○ Figure 1B is slightly confusing. The red line makes it seem that low levels of glutamate give rise to high levels of Calcium instead of showing just the rise in calcium. A way to incorporate glutamate activation of NMDA receptors in Fig 1B & C might help to conceptualize the model.

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The abstract is lengthy in relation to the article. I would suggest this is shortened and made more succinct.
○ insulin action that precedes all other signaling events and effects of insulin.
In our opinion article we selected only two measurable parameters, namely [Ca 2+ ]i and insulin receptor activation state defined as Tyr1150/1151 phosphorylation, but not downstream molecules of IR signaling pathway such as IRS-1 or others. Therefore, our opinion relates only to the activation of insulin receptor, and not to downstream molecules or events. This approach relates directly to insulin sensitivity, since the activation of the receptor with insulin is the only stage at which insulin sensitivity can be measured directly. In our studies [references 19 and 20] that underlie the our opinion we used glia-free cortical neurons and directly measured ATP levels.
Our opinion relates to link between [Ca 2+ ]i and insulin receptor activation state, and amyloid-NMDA relationship are out of scope of our opinion.
Our opinion is limited to only insulin receptor activation, but not to more broad "insulin signaling". According to current knowledge, inflammation affects insulin signaling, but downstream of insulin receptor at IRS-1 level. This is out of scope of our opinion.
Our opinion does not relate to any signaling events downstream of insulin receptor, such phosphatase action and receptor internalization. We did not propose the same mechanism for IGF-1 in our opinion aticle, since we have no supportive evidence. However, it is likely that there is a link between [Ca 2+ ]i and activation state of IGFR1.
In a conclusion, the model proposed in the opinion article relates only to relationship beteen two measurable parameters, namely intracellular Ca2+ concentrations and insulin receptor activation state. Therefore, all other downstream elements of insulin signaling system and factors affecting the downstream elements are out of scope of this opinion. The opinion is completely based on our experimental results previously published, references 19 and 20 of the opinion article.