Background

The prevalence of obesity in America is a major health concern leading to preventable diseases such as type 2 diabetes and cardiovascular disease. Adipose tissue inflammation is an emerging factor thought to play a role in obesity-linked complications. Crown-like structures (CLSs) serve as local indicators of inflammation that predict complications of excess weight.

Methods

We developed an in-vitro CLS model to interrogate metabolic and necrotic queues that drive CLS formation and resolution. This model consists of treatment of mature 3T3-L1 adipocytes with isoproterenol (ISO) or TNF-α, followed by coculture of J774 monocyte-like cells (J7). In collaboration, we showed that our in-vitro CLS system recapitulates monocyte-like cell aggregation around necrotic adipocytes, and patterns cytokine secretion observed in inflamed adipose tissue (increased TNF-α in IL-6 and decreased IL-10). Using reverse-phase LCMS, we quantitatively detect over 100 target metabolites secreted from the in-vitro CLS.

Results

TNF-α stimulation of mature 3T3-L1 adipocytes leads to significantly increased secretion of palmitate, linoleate, and oleate compared to ISO stimulation. In contrast, secretion of lipokine 12,13-diHOME is comparable between ISO and TNF-α stimulation. We also observe an increase in cyclic compound secretion including cholesterol derivatives and sphingomyelin secretion including ceramides with TNF-α stimulation. Following addition of J7s, we observe low levels of palmitate, linoleate, and oleate secretion with TNF-α stimulation, and an increase of residual palmitate secretion with ISO stimulation. Addition of J7s abrogate 12,13-diHOME production under both stimuli. To further interrogate lipid flux from adipocytes to adipose tissue macrophages, we use carbon-13 and deuterium tracing to reconstruct metabolic in-vitro CLS rewiring.

Conclusions

This work may result in novel pathways and targets to treat adipose tissue inflammation in addition to weight loss.