Investigating the Role of Arcuate Pirt Neurons in Energy Homeostasis

Abstract

The arcuate nucleus (ARC) is a critical hypothalamic region for the control of metabolism and feeding. Previous studies show that a subgroup of inhibitory ARC neurons, which express the vesicular inhibitory amino acid transporter gene ( Slc32a1) and the leptin receptor gene ( Lepr), control metabolism but not feeding. However, the identity of these neurons remains elusive. We identified a candidate neuron subtype in the ARC, marked by the expression of the phosphoinositide interacting regulator of the transient receptor potential ( Pirt) gene. We hypothesize, based on their molecular profile, that PirtARC neurons are activated by feeding to drive thermogenesis. We used the Pirt-Cre transgenic mouse to investigate the role of PirtARC neurons in energy homeostasis. To validate Cre activity, we expressed a reporter virus (AAV8-hSyn-FLEX-TVA-P2A-GFP-2A-oG) in the ARC of male and female Pirt-Cre mice (n=4; 6 injections, 50 nL each) and compared GFP (green fluorescent protein) fluorescence to Pirt expression by RNA fluorescence in situ hybridization (RNA FISH). In the absence of Cre expression, we failed to detect GFP expression, confirming the Cre dependency of the AAV. However, in Pirt-Cre mice, we found that 77% (380 of 495) of ARC Pirt RNA+neurons were GFP+ and that 79% (380 of 483) of GFP+ ARC neurons were Pirt RNA+, thereby validating the Pirt-Cre mouse for genetically targeting PirtARC neurons. Next, to investigate whether PirtARC neurons respond to metabolic states, we subjected male and female C57BL/6J mice to ad libitum feeding, 18-hour fasting, and 18-hour fasting then 2-hour refeeding (n=4 mice per condition). Using RNA FISH, we assessed the co-localization of Pirt mRNA with Fos mRNA (neuronal activity marker) across the metabolic states. We found that the percentage of PirtARC neurons co-expressing Fos mRNA is higher in ad libitum-fed mice than in fasted mice (p=0.0135; one-way ANOVA followed by Tukey’s multiple comparison test), though neither group differed significantly from post-fast re-fed mice (p > 0.05). This indicates that PirtARC neurons are more active in fed mice than fasted ones, consistent with our hypothesis. Finally, to investigate whether PirtARC neurons regulate food intake, we expressed the intersectional chemogenetics (AAV-CreON/FlpON-hM3Dq-HA) in the ARC of male and female Pirt-Cre::Slc32a1-Flp mice (n=6; 6 injections, 100 nL each) to activate PirtARC neurons. We intraperitoneally injected the artificial muscarinic receptor ligand, clozapine N-oxide (CNO; 1 mg/kg), or saline vehicle in ad libitum-fed mice during the 12-hour light cycle and measured food intake after 30 minutes, 1 hour, 2 hours, and 4 hours on alternating days (CNO, 0.11 ± 0.05 g food/4 hrs.; saline, 0.11 ± 0.04 g food/4 hrs.; Two-way ANOVA, CNO vs saline, p > 0.05 for all time points). Unlike other ARC inhibitory neurons, we found that activating PirtARC neurons does not increase food intake. Together, our results show that PirtARC neurons are a novel subtype of feeding-activated, non-orexigenic neurons in the arcuate hypothalamus. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.