1H-NMR analysis reveals cold acclimation induces homeoviscous adaptation in membranes from liver, but not in blood cells or skeletal muscle in the freeze tolerant Cope's gray treefrog
Authors: Yokum Elizabeth , Erb Jeremy , Goldstein David , Krane Carissa 
Published: May 2023

Cope’s gray treefrog Dryophytes chrysoscelis is a seasonally freeze tolerant anuran capable of repeatedly freezing and thawing as much as two-thirds of body water by accumulating and mobilizing cryoprotectants. In cold-acclimated D. chrysoscelis, RBCs are inherently freeze tolerant in vitro, suggesting that membrane composition may contribute to cellular freeze tolerance. The objective of this study is to compare the effects of cold acclimation on lipid profiles of membranes from liver, muscle, and blood cells, to assess the role of membrane composition in cellular and organismal freeze tolerance. It is hypothesized that membrane biochemical composition is tissue-specific and is sensitive to environmental fluctuation. Membranes were prepared from liver (N=7), skeletal muscle (N=7), and blood cells (WBCs, RBCs, and platelets) (N=5) collected from wild-caught male treefrogs that were either cold-acclimated (5°C, 8 hrs daylight) or maintained in warm conditions (22°C, 12 hrs daylight). Fatty acid characterization and cholesterol and triacylglycerol (TAG) content were evaluated using 1H-NMR spectra collected in a 400 MHz Bruker Avance spectrometer. Principal component analysis demonstrated that membrane lipid profiles were tissue-specific, but distinct warm and cold profiles were detected only in liver. Fatty acids in liver membranes did not vary with acclimation state in chain length ( P=0.46), but total unsaturation was nearly 2-fold greater in cold compared with warm frogs ( P<0.05). Monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids were elevated by 1.7-fold in cold frogs ( P<0.05). Of the PUFA lipid species assessed, linoleic acid (LA) was more than doubled in cold frogs ( P<0.05), while other Ω-3 and Ω-6 fatty acids did not vary between groups ( P>0.5). TAG, free cholesterol, and esterified cholesterol were elevated by 2.5-fold, 2-fold, and 1.4-fold, respectively, in cold frogs (P<0.05), while free cholesterol did not vary between groups ( P=0.38). In membranes from muscle and blood cells, neither fatty acid characterization nor TAG and cholesterol varied between warm and cold frogs, but trends suggested that MUFA ( P=0.10) and LA ( P=0.06) content in blood cells may be elevated in warm frogs. Overall, liver membranes are remodeled during cold acclimation, supporting the hypothesis that membrane composition is adapted to maintain cell function and integrity (i.e. homeoviscous adaptation). Specifically, elevated fatty acid unsaturation and cholesterol in liver likely contribute to cellular freeze tolerance and may support the central role of liver in mobilizing metabolites and cryoprotectants during cold acclimation. However, the absence of such changes (or even changes in the opposite direction) in muscle and blood cells indicates that additional environmental cues, such as exposure to freezing conditions, may be required to induce remodeling and/or that membrane lipid composition does not contribute to freeze tolerance in some tissues. This study was supported by a UD STEM Catalyst Grant awarded to CMK and JE, a Graduate Student Summer Fellowship and Dittrich-Spotila Research Fellowship awarded to EEY, and an NSF-MRI Grant awarded to JE and CMK that was supported by the Ohio Action Fund. This is the full abstract presented at the American Physiology Summit 2023 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.