The influence of equine body weight gain on inflammatory cytokine expressions of adipose tissue in response to endotoxin challenge
Authors: Blaue Dominique , Schedlbauer Carola , Starzonek Janine , Gittel Claudia , Brehm Walter , Blüher Matthias , Pfeffer Martin , Vervuert Ingrid 
Published: April 2020

Abstract
Background
Human obesity is linked with systemic inflammation. However, it is still controversial if equines produce more inflammatory cytokines with increasing body weight and if the production of those show breed type specific patterns. The main objective of this study was to determine if diet induced obesity is associated with increased inflammatory signatures in adipose tissue of equines and if a breed predisposition exists between ponies and horses. Additionally, we aimed to identify adipose tissue depot differences in inflammatory cytokine expression. Nineteen healthy, non-overweight and metabolically healthy equines received a hypercaloric diet for 2 years. Body weight, body condition score and cresty neck score were assessed weekly throughout the study. At three time points, insulin sensitivity was determined by a combined glucose-insulin test. Adipose tissue samples were collected from two intra-abdominal and two subcutaneous depots under general anesthesia at each time point after an endotoxin trigger. In the adipose tissue samples levels of CD68 mRNA (a marker of macrophage infiltration) and pro-inflammatory cytokine mRNA (IL-1β, IL-6 and TNFα) were analyzed with RT-qPCR. As markers of lipid metabolism mRNA levels of lipoprotein lipase (LPL) and fatty acid binding protein 4 (FABP4) were determined with RT-qPCR.

Results
CD68 mRNA levels increased with body weight gain in several adipose tissue (AT) depots (Wilcoxon signed rank test with Bonferroni correction; retroperitoneal AT horses: P = 0.023, mesocolonial AT horses: P = 0.023, subcutaneous tail head AT ponies: P = 0.015). In both abdominal depots CD68 mRNA levels were higher than in subcutaneous adipose tissue depots (Kruskal–Wallis-ANOVA with Bonferroni correction: P < 0.05). No breed related differences were found. Pro-inflammatory cytokine mRNA IL-1β, IL-6 and TNFα levels were higher in subcutaneous depots compared to abdominal depots after body weight gain. IL-1β, IL-6 and TNFα mRNA levels of mesocolon adipose tissue were higher in obese horses compared to obese ponies (Mann–Whitney-U test; IL-1β: P = 0.006; IL-6: P = 0.003; TNFα: P = 0.049). In general, horses had higher FABP4 and LPL mRNA levels compared to ponies in neck AT and tail AT at all time points.

Conclusion
Our findings suggest an increased invasion of macrophages in intra-abdominal adipose tissue with increasing body weight gain in equines in combination with a low dose endotoxin stimulus. This might predispose equines to obesity related comorbidities. In obese horses mesocolon adipose tissue showed higher inflammatory cytokine expression compared to obese ponies. Additionally, subcutaneous adipose tissue expressed more pro-inflammatory cytokines compared to intra-abdominal adipose tissue. Horses had higher FABP4 and LPL mRNA levels in selected AT depots which may indicate a higher fat storage capacity than in ponies. The differences in lipid storage might be associated with a higher susceptibility to obesity-related comorbidities in ponies in comparison to horses.