Insulin Activates Hepatic-Related Neurons in the Paraventricular Nucleus of the Hypothalamus via mTOR Signaling

Academic Background

Glucose homeostasis is a critical physiological process for sustaining life, with the liver playing a central role in this regulation. The liver maintains blood glucose levels by modulating glycogen production and glucose release. Although insulin can directly act on liver tissue to inhibit glucose production, recent studies have shown that the central nervous system (CNS), particularly the hypothalamus, also plays a significant role in regulating glucose metabolism. The paraventricular nucleus of the hypothalamus (PVN) is a heterogeneous nucleus responsible for integrating autonomic and metabolic regulation, especially autonomic output related to liver function. PVN neurons express insulin receptors and are connected to the liver via the vagus nerve, making PVN a key central site for insulin-mediated regulation of liver function.

However, the mechanisms by which insulin regulates liver function through the CNS remain unclear. Specifically, how insulin influences vagal output via PVN neurons to regulate hepatic glucose production is still an unresolved question. To address this, researchers conducted this study to uncover the neural circuitry mechanisms by which insulin regulates liver function through PVN neurons.

Source of the Paper

This research paper was co-authored by Karoline Martins dos Santos, Sandy E. Saunders, Vagner R. Antunes, and Carie R. Boychuk. The authors are affiliated with University of Texas Health San Antonio, University of São Paulo, and University of Missouri, respectively. The paper was first published on December 12, 2024, in the Journal of Neurophysiology, with the DOI 10.1152/jn.00284.2024.

Research Process and Results

1. Experimental Design and Procedure

a) Pseudorabies Virus (PRV) Retrograde Tracing

To investigate the neural circuitry between PVN neurons and the liver, researchers used pseudorabies virus (PRV-152) for retrograde tracing. PRV-152 is a modified virus capable of transsynaptic spread, allowing it to label neural circuits related to the liver. The specific steps were as follows:

1. Liver Injection of PRV-152: PRV-152 was injected into the liver of mice, and the animals were euthanized at 48 hours, 72 hours, and 96 hours post-injection to observe the number of PRV-152-labeled neurons in PVN and the dorsal motor nucleus of the vagus (DMV) at different time points.
2. Vagotomy Experiment: To confirm the relationship between PVN neurons and the vagus nerve, researchers performed a left vagotomy before injecting PRV-152 and observed changes in labeled neurons in PVN and DMV.

b) Immunohistochemical Phenotyping

To determine the neuropeptide phenotype of PVN hepatic-related neurons (PVNhepatic), researchers performed immunohistochemical staining on PVN neurons, detecting three major neuropeptides: oxytocin (OT), vasopressin (VP), and corticotropin-releasing hormone (CRH).

c) Electrophysiological Recordings

To study the effects of insulin on PVNhepatic neuronal activity, researchers used patch-clamp techniques to record the electrical activity of PVNhepatic neurons. The specific steps were as follows:

1. Brain Slice Preparation: Brain slices containing PVN were extracted from mice and incubated in artificial cerebrospinal fluid (ACSF).
2. Insulin Treatment: During neuronal activity recording, insulin was added to the brain slices to observe its effects on neuronal firing frequency.
3. mTOR Signaling Pathway Inhibition Experiment: To investigate the molecular mechanisms of insulin action, researchers pretreated the slices with the mTOR inhibitor rapamycin before adding insulin, observing its effects on insulin-induced changes in neuronal firing frequency.

2. Key Results

a) PRV Retrograde Tracing Results

• DMV Neuron Labeling: A small number of labeled neurons appeared in DMV 48 hours after PRV-152 injection, with a significant increase in labeled neurons at 72 and 96 hours. Vagotomy nearly eliminated labeled neurons in DMV, indicating that DMV neurons are connected to the liver via the vagus nerve.
• PVN Neuron Labeling: PVN neurons began to show labeling 72 hours after PRV-152 injection, with a significant increase in labeled neurons at 96 hours. Vagotomy nearly eliminated labeled neurons in PVN, indicating that PVN neurons are connected to the liver via the vagus nerve.

b) Immunohistochemical Phenotyping Results

PVNhepatic neurons did not show co-labeling with OT, VP, or CRH, suggesting that these neurons may not express these neuropeptides.

c) Electrophysiological Recording Results

• Effects of Insulin on PVNhepatic Neurons: Insulin significantly increased the firing frequency of PVNhepatic neurons. Unlabeled PVN neurons showed weaker responses to insulin, indicating the specificity of insulin’s action.
• Role of the mTOR Signaling Pathway: Pretreatment with rapamycin inhibited insulin-induced increases in PVNhepatic neuronal firing frequency, indicating that insulin’s action depends on the mTOR signaling pathway.

3. Conclusions and Significance

This study revealed the neural circuitry mechanism by which insulin activates PVNhepatic neurons via the mTOR signaling pathway, thereby regulating liver function through the vagus nerve. Specifically, insulin activates PVNhepatic neurons, increasing their firing frequency, which in turn inhibits hepatic glucose production via the vagus nerve. This discovery not only deepens our understanding of the role of the CNS in glucose homeostasis but also provides new potential targets for the treatment of metabolic diseases such as diabetes.

4. Research Highlights

• Novel Neural Circuitry Mechanism: This study is the first to reveal the neural circuitry mechanism by which insulin regulates liver function through PVNhepatic neurons.
• Key Role of the mTOR Signaling Pathway: The study clarified the critical role of the mTOR signaling pathway in insulin-mediated regulation of PVNhepatic neuronal activity.
• Specific Labeling Techniques: Through PRV retrograde tracing and immunohistochemistry, researchers successfully labeled and identified PVNhepatic neurons.

5. Other Valuable Information

• Rigorous Experimental Design: Researchers ensured the reliability and specificity of the results through vagotomy experiments and mTOR inhibitor experiments.
• Potential Applications: This study provides a theoretical foundation for developing CNS-targeted therapeutic strategies for diabetes.