NMR and Mass Spectroscopy Based Comparison of Blood Metabolites from Dantrolene and SR59230A Treated Arctic Ground Squirrels

Abstract

Arctic ground squirrels (AGS) hibernate during winter, undergoing cycles of torpor, where body temperatures plummet as low as −3°C followed by rapid rewarming to 37°C during interbout arousals (IBA). While IBAs only occur in certain species that experience torpor, they offer a unique model into which one can observe the specific mechanisms responsible for thermogenesis. Rapid rewarming at IBA onset increases AGS body temperature above 15°C through the activation of brown adipose tissue (BAT). Above 15°C, shivering thermogenesis is able to contribute to warming back up to original body temperature. However, the relative contributions of these two thermogenic processes during IBA is not well understood. AGS were treated across periods of coupled torpor cycles, whereby during the second cycle AGS were treated with either carrier (control) or a selected inhibitor. BAT inhibition using SR59230A, a β3‐adrenergic receptor inhibitor, would be expected to strain the rapid heat production. Shivering thermogenesis utilizes calcium cycling and the Ryanodine receptor in skeletal muscles; the use of dantrolene reduces the thermogenic mechanism needed to reach 37°, effectively inhibiting the Ryanodine receptor in muscle. Relative concentrations of blood plasma metabolites were quantified using nuclear magnetic resonance (NMR) to compare whole and polar extracted plasma. The NMR method was compared against full metabolomic analysis performed using liquid chromatography tandem mass spectroscopy (LC MS‐MS). NMR analysis resulted in higher peak resolution quality and increased metabolite identification in polar and lipid extraction samples compared to whole samples, whereas LC MS‐MS provided a greater abundance of metabolites. Overall, total metabolites were significantly reduced during hibernation. Dantrolene treated AGS had alterations in metabolites indicative of increased energy demand. Furthermore, SR treatment resulted in profiles describing decreased beta‐oxidation and generally lower total metabolites compared to Dantrolene. These trends indicate that changes in calcium cycling and BAT stimulation during hibernation can have profound effects on the animals ability to efficiently balance energy usage.Support or Funding InformationResearch reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103395. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the NIH. BLaST is supported by the NIH with the linked awards; TL4GM118992, RL5GM118990, UL1GM118991.