Signs of life: Oxygen sensors confirm viability, measure oxygen consumption and provide rapid, effective contamination monitoring for field‐based tissue culture

Abstract

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Consequences of in vitro benzyl butyl phthalate exposure for blubber gene expression and insulin-induced Akt activation in juvenile grey seals

“…Total Akt content was higher in inner compared to outer tissue, which is consistent with tissue depth differences in metabolism (Robinson et al, 2018(Robinson et al, , 2021. Insulin treatment for 30 min increased Akt phosphorylation, regardless of tissue depth and moulting status.…”

“…Three 10 mm and one 6 mm blubber biopsies were taken from each animal. The 10 mm biopsies were used for the generation of explants as described previously (Bennett et al, 2017;Robinson et al, 2018Robinson et al, , 2021 and the 6 mm biopsy was used for measurement of lipid percentage (Robinson et al, 2019). Tissue was cut immediately, using sterile surgical scissors, into inner (blubber layer closest to the muscle) and outer (blubber layer closest to the skin) sections, and placed into separate 15 mL centrifuge tubes filled with sterile Krebs Ringer solution (37 • C, pH 7.4; NaCl; 7.89 g L − 1 ; KCl; 0.373 g L − 1 ; MgSO 4 ; 0.12 g L − 1 ; K 2 HPO 4 ; 0.07 g L − 1 ; glucose; 0.99 g L − 1 ; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); 4.77 g L − 1 ; CaCl 2 ; 0.11 g L − 1 ) with 1% Antibiotic Antimycotic Solution (all chemicals supplied by Sigma Aldrich, Gillingham, UK) for transport back to the laboratory.…”

“…Mass or moult-dependent differences in gene expression also differed between tissue depths. Blubber is not homogeneous throughout its depth in terms of lipid composition (Guerrero and Rogers, 2017;Tverin et al, 2019) and function (Strandberg et al, 2008;Robinson et al, 2018Robinson et al, , 2021. Inner blubber is characterised by more dynamic lipid metabolism compared to outer (Robinson et al, 2018(Robinson et al, , 2021, which is thought to be more important for insulation (Strandberg et al, 2008).…”

“…The impact on glucose handling by the tissue was not established here due to technical limitations. In previous experiments on pups we measured glucose in media as a measure of glucose uptake (Robinson et al, 2018(Robinson et al, , 2021, but the response to insulin was too small to detect over only a 30 min exposure (Data unpublished). Additional experiments to establish whether insulin induces glucose uptake in blubber explants using fluorescent tracers would help establish the functional consequences of insulin-induced activation of Akt, particularly in light of the apparent decoupling of GLUT4 regulation from insulin signalling in other seal species (Viscarra et al, 2011b).…”

“…Here we employed an explant approach (Bennett et al, 2017(Bennett et al, , 2021Robinson et al, 2018) using blubber from juvenile grey seals (Halichoerus grypus) during periods of mass loss (moulting/recently moulted) and mass gain (post moult) to examine the effect of an overnight ex vivo exposure to micromolar concentrations of BBzP on insulin sensitivity and the expression of genes involved in fat metabolism. We used BBzP because it has a higher K ow than low molecular weight phthalates, making it more lipophilic and thus likely to be more problematic for adipose tissue; because its recent high production volumes for PVC manufacture mean it continues to be identified in marine food webs (Beltifa et al, 2017), including in marine mammal blubber (Baini et al, 2017); and because it is typically reported as one of the most potent endocrine disrupting phthalates, with adipogenic and insulin signalling effects (Pereira-Fernandes et al, 2013Yin et al, 2016;Sakuma et al, 2017;Zhang and Choudhury, 2021).…”