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Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro
Published on: March 17, 2023
The P2Y2 receptor mediates terminal adipocyte differentiation and insulin resistance: Evidence for a dual G-protein
Shenqi Qian1, Yi Shi2, Jared Senfeld2
1Department of Drug Discovery and Development, Harrison College of Pharmacy, Auburn University, Auburn, Alabama, USA; Department of Pharmacy, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Abstract:
Several P2Y nucleotide receptors have been shown to be involved in the early stage of adipocyte differentiation in vitro and insulin resistance in obese mice; however, the exact receptor subtype(s) and its underlying molecular mechanism in relevant human cells are unclear. Here, using human primary visceral preadipocytes as a model, we found that during preadipocyte-to-mature adipocyte differentiation, the P2Y2 nucleotide receptor (P2Y2R) was the most upregulated subtype among the eight known P2Y receptors and the only one further dramatically upregulated after inflammatory TNFα treatment. Functional studies indicated that the P2Y2R induced intracellular Ca2+, ERK1/2, and JNK signaling but not the p38 pathway. In addition, stimulation of the P2Y2R suppressed basal and insulin-induced phosphorylation of AKT, accompanied by decreased GLUT4 membrane translocation and glucose uptake in mature adipocytes, suggesting a role of P2Y2R in insulin resistance. Mechanistically, we found that activation of P2Y2R did not increase lipolysis but suppressed PIP3 generation. Interestingly, activation of P2Y2R triggered Gi-protein coupling, and pertussis toxin pretreatment largely inhibited P2Y2R-mediated ERK1/2 signaling and cAMP suppression. Further, treatment of the cells with AR-C 118925XX, a selective P2Y2R antagonist, significantly inhibited adipogenesis, and P2Y2R knockout decreased mouse body weight gain with smaller eWAT mass infiltrated with fewer macrophages as compared to WT mice in response to a Western diet. Thus, we revealed that terminal adipocyte differentiation and inflammation selectively upregulate P2Y2R expression and that P2Y2R mediates insulin resistance by suppressing the AKT signaling pathway, highlighting P2Y2R as a potential new drug target to combat obesity and type-2 diabetes.
Insights
The P2Y2 receptor (P2Y2R) is upregulated during fat cell development and inflammation. P2Y2R activation promotes insulin resistance by inhibiting AKT signaling, suggesting it as a target for obesity and type-2 diabetes treatments.
Area of Science:
- Molecular Endocrinology
- Metabolic Signaling
- Adipocyte Biology
Background:
- P2Y nucleotide receptors are implicated in adipocyte differentiation and insulin resistance.
- The specific P2Y receptor subtypes and mechanisms involved in human cells remain unclear.
- Obesity and inflammation are linked to altered metabolic processes.
Purpose of the Study:
- To identify the P2Y receptor subtype(s) involved in human adipocyte differentiation and insulin resistance.
- To elucidate the molecular mechanisms by which P2Y receptors influence these processes.
- To evaluate the therapeutic potential of targeting specific P2Y receptors for metabolic disorders.
Main Methods:
- Utilized human primary visceral preadipocytes and mature adipocytes.
- Investigated P2Y receptor expression during adipogenesis and after TNFα treatment.
- Assessed intracellular signaling pathways (Ca2+, ERK1/2, JNK, AKT, cAMP) and glucose uptake.
- Employed P2Y2 receptor antagonist (AR-C 118925XX) and knockout mouse models.
- Analyzed body weight, adipose tissue mass, and macrophage infiltration.
Main Results:
- P2Y2 nucleotide receptor (P2Y2R) was the most upregulated P2Y subtype during adipogenesis and further induced by TNFα.
- P2Y2R activation stimulated Ca2+, ERK1/2, and JNK signaling, suppressed AKT phosphorylation, GLUT4 translocation, and glucose uptake.
- P2Y2R activation inhibited PIP3 generation and triggered Gαi-protein coupling, impacting cAMP levels.
- P2Y2R antagonist treatment inhibited adipogenesis; P2Y2R knockout reduced diet-induced weight gain and adipose tissue inflammation in mice.
Conclusions:
- Terminal adipocyte differentiation and inflammation selectively upregulate P2Y2R expression in human cells.
- P2Y2R mediates insulin resistance by suppressing the AKT signaling pathway and glucose uptake.
- P2Y2R represents a potential therapeutic target for combating obesity and type-2 diabetes.
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