Characterising fat tissue structure and function in response to hypoxia in grey seals : an animal model of extreme fat deposition and mobilisation, using novel in vivo and in vitro approaches

Phocid seals go through dramatic seasonal changes in body mass and composition because of the spatial and temporal separation of foraging, reproduction and moulting. The regulation of fat mobilisation during these processes is a key factor for their survival. Unlike in humans, reproductive fitness and first-year survival are positively linked to adiposity in seals. Therefore, the rapid fluctuations in body mass and metabolic profile suggest that seals could be a good study model for understanding how fat expansion and loss can be achieved in a mammalian system without significant comorbidities. In humans, excess fat accumulation leads to tissue remodelling, potential hypoxia, chronic inflammation and oxidative stress. Although these processes are strongly interrelated, evidence suggest hypoxia, as result of limited vascularity or impaired diffusion, is the underlying trigger. How seals avoid the potential detrimental effects of large, fluctuating adiposity is unknown. Given that grey seals (Halichoerus grypus) have a similar fat percentage to obese humans, I hypothesised that oxygen availability in blubber will be affected by increasing tissue depth and adiposity. In chapter 2, I measured partial pressure of oxygen (pO₂) and temperature in vivo at different blubber depths in healthy juvenile grey seals. Adipose tissue pO₂ was similar to other species. Blubber pO₂ dropped significantly with increased fatness, but not with blubber depth, which is consistent with other animal models of rapid fat deposition and most human studies. Grey seal blubber therefore not only undergoes oxygen restriction during diving as previously reported, but also during fattening. In chapter 3 I investigated if fatness changes were reflected in tissue morphologic modifications and if adipocyte area and vascularity could impair oxygen diffusion in adult females and their pups during the breeding season, during which mothers reduced their mass by 23% while pups mass increased by 72%. Adipocyte area did not significantly shrink during the mothers' fast v but adipocytes did significantly enlarge during suckling in pups. Moreover, the rate of adipocyte growth was faster in inner than in outer blubber. Adipocyte diameter in mothers and weaned pups was close to the maximum O₂ diffusion distance, suggesting that the bigger cells could be susceptible to hypoxia. Vascularity did not reduce in mothers during mass loss, but angiogenesis was associated with adipocyte enlargement in inner blubber in pups. Whether vascularity growth is quick enough during adipogenesis to provide sufficient oxygen is unknown. Collagen area per adipocyte was associated with adipocyte area in females, but not in pups, suggesting pups' adipocytes are not mechanically constrained while expanding. In chapter 4, I investigated how hypoxia related molecular pathways were associated with adipocyte area alterations. Despite the expected reduced oxygenation described in chapter 2 and 3, no significant changes in hypoxia inducible factor (HIF-1α) were detected in mothers, which is consistent with the limited changes in adipocyte area. However, Hif-1α did significantly increase in pups at late suckling, and was reduced at the post-weaned fast. These changes were paralleled by alterations in adipogenic markers and thus the role of HIF during blubber development warrants further attention. Blubber collagen showed a unique distribution pattern of parallel bundles. This repeated orientation was not observed in human adipose tissue. Moreover, collagen area was similar between both species despite adipocyte area being significantly bigger in seals. Collagen content and distribution might allow blubber to expand further than subcutaneous adipose tissue in humans, which could help prevent ectopic fat accumulation that is a key trigger for comorbidities. Downstream effects of HIF-1α accumulation were investigated in chapter 6, with a novel explant in vitro approach. The HIF-1α downstream metabolic pathways seem to be regulated conversely to what is reported in the literature. We suggest that the metabolic pathways are more susceptible to nutritional state than oxygen availability. This thesis contributes to the understanding of oxygen management during blubber development and contraction in seals, the molecular shifts induced by hypoxia and key insights into the comparative anatomy and physiology of adipose tissue structure and function.