Excessive lipolysis from adipose tissue (AT) is considered a major risk factor for postpartum metabolic disease in dairy cows. Among those, clinical ketosis occurs when dysregulated lipolysis provides abundant fatty acids for ketone body synthesis. Since excessive and protracted lipolysis increases the risk for disease, limiting lipolysis becomes crucial to mitigate excessive fat mobilization and consequently disease risk. However, the mechanisms behind AT dysregulated lipolysis and how to inhibit it are not fully understood. Additionally, periparturient dairy cows often present inflammatory conditions such as coliform mastitis, metritis, pneumonia, and leaky gut, which result in circulating free endotoxin. Endotoxins, which are the outer component of gram-negative bacteria, can activate TLR4 in cells. Once TLR4 is activated, it leads to various effects including cytokine production, increased metabolic stress, and altered cellular metabolism. However, there is a lack of comprehensive understanding regarding the effects of endotoxins on AT in dairy cows. This work proposes that endotoxin plays a determinant role during the development of metabolic and inflammatory/infectious comorbidities around parturition through the exacerbation of lipolytic responses in AT and reduction of insulin sensitivity. Although some studies have examined the impact of endotoxemia on the metabolism of lipids, the molecular mechanisms underlying the interactions between endotoxins and AT remain poorly characterized. To address this gap, we first studied clinical ketosis, the most representative disease during AT lipolysis dysregulation. We found that cows with clinical ketosis had elevated plasma endotoxin and inflammatory markers. A randomized clinical trial was conducted to investigate the effectiveness of niacin and flunixin as inhibitors of lipolysis during clinical ketosis. Furthermore, given that the physiological role of AT is not fully understood during ketosis, we evaluated the AT function, and we characterize the AT macrophage phenotype involved in CK. We identified that endotoxins and inflammation were playing a determinant role during clinical ketosis and its recovery. Therefore, we developed an ex vivo model of AT inflammation to determine how endotoxins influence lipolytic and inflammatory markers. We found that endotoxin LPS triggers lipolysis and insulin resistance in AT from non-lactating and postpartum dairy cows. Among the pathways involved ERK1/2 was observed as a mediator of activation of lipases HLS and ATGL. To further characterize endotoxin-induced lipolysis, an in vitro cell culture model was developed to assess the mechanisms behind lipolysis in bovine adipocytes exposed to LPS. In our bovine adipocyte inflammation model, endotoxin depends upon the presence of TLR4 to activate lipolysis and inflammation. Adipocytes were also treated with lipolysis inhibitors niacin and flunixin meglumine and then exposed to endotoxin. Flunixin meglumine inhibits lipolysis in adipocytes and improves insulin sensitivity. This work suggests that inflammation is involved in lipolysis dysregulation in dairy cows with clinical ketosis. Our data also provide evidence for the novel therapy for inhibiting lipolysis with faster hyperketonemia recovery, lower lipolytic rates and reduced inflammation. Hence, treating cows with flunixin meglumine reduces inflammation and improves AT function during clinical ketosis. Additionally, our data suggest that TLR4 is mediating lipolysis and inflammation in bovine adipocytes. Therefore, if endotoxins are involved in lipolysis dysregulation, it is important to acknowledge the potential impact of endotoxin on metabolic diseases in dairy cows. Establishing the relationship between low-grade endotoxemia and AT effects, both in the short and long term, should be the focus of future studies. Moreover, future work should be based on further characterizing the mechanisms by which TLR4 activation impacts other adipose tissue functions, such as lipid accumulation.
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