Diurnal regulation of metabolism by Gs-alpha in hypothalamic QPLOT neurons

**Background:** Neurons in the hypothalamic preoptic area (POA) regulate thermoregulation, sleep, and metabolism. A subset termed QPLOT neurons (expressing Qrfp, Ptger3, LepR, Opn5, Tacr3) express multiple G-protein coupled receptors (GPCRs). The stimulatory Gαs subunit (encoded by Gnas) generates cAMP through adenylyl cyclase and may activate these neurons. The authors hypothesized that Gαs-signaling activates QPLOT neurons to decrease metabolism, and that deleting Gnas would prevent activation and increase metabolism.

**Methods:** Male Opn5-cre; Gnas-fl/fl (experimental, n=9) and Opn5-cre; Gnas+/+ (control, n=7) mice were analyzed using Promethion Core metabolic chambers. Mice were housed on a 12:12 light:dark cycle and studied at three ambient temperatures: 22°C (standard), 10°C (cold challenge), and 28°C (thermoneutrality). Each temperature was preceded by one day of acclimation, with 48 hours of recording. Parameters measured included oxygen consumption, carbon dioxide expiration, food intake, water intake, and locomotor activity. Energy expenditure (EE) and respiratory exchange ratio (RER) were calculated. Statistical analysis used two-way ANOVA with Sidak's multiple comparison tests and CircaCompare for circadian rhythm analysis (MESOR, amplitude, phase). M-FISH was used to confirm Gnas deletion efficiency in Opn5 neurons.

**Key Results:** M-FISH confirmed substantially reduced Gnas transcript in Opn5-cre; Gnas-fl/fl; Ai14 neurons compared to controls. Two-way ANOVA showed no significant differences in EE, RER, food intake, or water intake at any temperature. However, a marked decrease in nocturnal locomotion was observed in experimental mice at 28°C (interaction p<0.0001, genotype p=0.0385) and 22°C (interaction p=0.0023). CircaCompare analysis revealed: (1) EE MESOR was significantly higher in experimental mice at 22°C and 10°C, with a 0.64-hour phase delay at 28°C; (2) RER MESOR was higher in experimental mice at 28°C but lower at 22°C and 10°C, with a 1.16-hour phase delay at 28°C; (3) Food intake MESOR was higher at 28°C and 22°C, with increased amplitude at 22°C; (4) Water intake MESOR was higher only at 28°C; (5) Locomotion MESOR and amplitude were lower at all temperatures, with no phase differences. No differences in sleep duration were detected.

**Clinical Implications:** This study demonstrates that Gαs-signaling in QPLOT neurons plays a role in regulating daily metabolic rhythms, particularly at higher ambient temperatures. The findings suggest that QPLOT neurons integrate multiple GPCR inputs through Gαs to modulate energy expenditure, substrate utilization, and locomotor activity in a temperature-dependent manner. Understanding these mechanisms may inform approaches to metabolic disorders involving circadian disruption. The relatively mild phenotypes observed may reflect compensatory mechanisms through other signaling pathways, suggesting redundancy in QPLOT neuron regulation. Further research is needed to identify specific receptors responsible and the role of the suprachiasmatic nucleus in regulating circadian metabolic patterns through the POA.