Background

Metabolic syndrome clusters the main risk factors for cardiovascular diseases and endocrine dysfunction. Novel studies show that the main underlying mechanism is a long term inflammation of adipose tissue as an endocrine organ with a gradual shift of the dominant macrophage subtype from the anti-inflammatory M2 (normal) towards the pro-inflammatory M1 macrophages (pathognomonic). Massive hypertrophy of fatty cells induces cell death, which releases DAMPs including nucleotides such as ADP and thus utilizing purinergic receptors to maintain the ongoing inflammatory process. Interestingly, we found out that the only ADP receptor expressed in the M1 Mϕ is the G_i coupled P2Y₁₃R, whereas in the M0 (unstimulated) and the M2 Mϕ it was not detectable.

Nevertheless, the role of P2Y₁₃R in the immune system and especially macrophages is currently unknown. Given its pivotal role in central metabolic processes (P2Y₁₃R has been described in insulin secretory signalling) together with its unique expression in its pathognomonic inflammatory Mϕ subtype makes it an interesting candidate to investigate its role in metabolic syndrome.

Purpose

Due to the unique expression of P2Y₁₃R on M1 Mϕ we hypothesise an improved outcome in a high-fat diet induced metabolic syndrome by interfering with the P2Y₁₃ signalling cascade.

Methods

BMDM isolation & differentiation to Mϕ using M-CSF and subsequent stimulation with medium (M0), LPS and IFNγ (M1) or IL4 (M2); Expression of P2Rs quantified using Taqman qPCR.

Male C57Bl6/J wild-type (WT) and P2Y₁₃-deficient (KO) mice were fed with a high-fat diet (HFD) for 20 weeks; weights of both the mice and their consumed food were recorded once a week. On week 15 we performed the ITT, on week 16 the GTT. Then the mice were put at different time points in a metabolic cage for 2 days. After 20 weeks several tissues were harvested from the mice and further analyzed.

Results

We observed a unique expression of P2Y₁₃R on M1 Mϕ (contrary to M0 & M2). Also, adult P2Y₁₃-deficient mice showed a higher O₂ consumption compared to adult wild-type mice (AUC of O₂ consumption during the 2^nd day= KO: 30299.3 ± 3153.1mL/kg vs. WT: 24266.5 ± 1992.6mL/kg, p<0.05). Although P2Y₁₃^-/- mice consumed slightly more food compared to WT littermates, they showed significantly decelerated weight gain (e.g. on week 15 → KO: 142 ± 7.63% vs. WT: 197.9 ± 17.2%, p<0.0001). After 6h fasting P2Y₁₃^-/- mice revealed decreased blood glucose levels compared to WT littermates (KO: 138.9 ± 21.1mg/dL vs. WT: 203.15 ± 24.11 mg/dL, p<0.05); Furthermore obese P2Y₁₃^-/- animals outperformed obese WT littermates in a peritoneal GTT (2h after glucose injection → KO: 176.9 ± 35.543mg/dL vs. WT: 372.7 ± 62.51mg/dL, p=0.0001). Interestingly, we observed a reduction in pathognomonic “crown-like-structures” in eWAT of P2Y₁₃^-/- mice (KO: 29.27 ± 17.1/100mm² vs. WT: 171.8 ± 99.22/100mm², p<0.001). Additionally, there was an increase in the amount of SVF cells (KO: 987.3 ± 412.6/mg fat vs. 2800 ± 615.9/mg fat, p<0.0001).

Conclusion

Global P2Y₁₃ deficiency leads to an improved outcome in metabolic syndrome with an increased protection against developing an insulin resistance as shown through an improved glucose tolerance and basal glucose levels, a decelerated weight gain and a better metabolic turnover. Observing these beneficial metabolic improvements, we hypothesise that antagonization of P2Y₁₃R could be a promising therapeutic tool in the field of metabolic syndrome.