Constitutive P2Y₂ receptor activity suppresses lipolysis in human adipocytes

S. Ali¹, J. Turner², S. Fountain¹. ¹Biological Sciences, University of East Anglia, Norwich, United Kingdom, ²Diabetes and Endocrinology, Norfolk and Norwich University Hospital NHS Foundation Trust, Norwich, United Kingdom.

Introduction: Obesity is a global epidemic that represents a significant health and economical issue. Adipocytes in obese individuals display decreased lipolytic (fat-breakdown) activity¹. P1 purinergic receptors have an established role in suppressing lipolysis^2,3, but little is known about P2 purinoceptors in adipocytes. This study aims to characterise the functional P2 receptors present in human adipocytes and identify their role in lipolysis.

Methods: Adipose-derived mesenchymal cells were isolated from healthy human subcutaneous adipose tissue and differentiated to adipocytes in vitro. Cells were loaded with Fura-2 and incubated with selective P2 receptor antagonists to evaluate effects on basal calcium (Ca²⁺) and nucleotide-evoked Ca²⁺ responses using a Flexstation-III. Lipolysis was measured by quantification of glycerol liberation in conditioned supernatants. cAMP concentrations were measured by competitive ELISA. Lentiviral transduction of adipocytes was used for shRNA-mediated knockdown P2Y₂ receptor.

Results: A robust response to ATP, ADP and UTP was observed (N=6 donors). Antagonists, MRS2500 (P2Y₁) and PSB0739 (P2Y₁₂), inhibited the ADP-evoked Ca²⁺ responses by 45.1±5% (IC₅₀ 77.1±38nM) and 39.7±5% (IC₅₀64.0±7nM) (N=6 donors) respectively. These antagonists also blocked the ATP-evoked responses (N=6 donors). Although AR-C118925XX (P2Y₂ antagonist) completely inhibited the UTP-evoked Ca²⁺ responses, IC₅₀ 318±399nM (N=6 donors), it only reduced the magnitude of response to ATP by 20.4±3%, IC₅₀ 683±116nM (N=6 donors). P2Y₁ and P2Y₁₂ antagonists and exogenous nucleotide application had no effect on lipolysis, but inhibition of P2Y₂, using AR-C118925XX and P2Y₂ shRNA, caused an increase in basal glycerol production by 32.2±7.8% and 31.6±8.6% respectively (N=3 donors). Inhibiting P2Y₂ decreased intracellular Ca²⁺ (11.3±3.6%, N=6 donors) and elevated cAMP (23.3±5.8%, N=4 donors) in adipocytes. Decreased Ca²⁺ can activate adenylate cyclase, thus increasing cAMP which can subsequently activate the protein kinase A-hormone sensitive lipase lipolytic pathway. P2Y₂ knockdown also caused a phenotypic difference in the lipid droplet size in adipocytes, which we hypothesise is due to increased lipolytic activity.

Conclusion: P2Y₁, P2Y₂ and P2Y₁₂ are involved in the nucleotide-evoked Ca²⁺ responses in adipocytes. P2Y₂ appears to be tonically active and plays a role in constitutively suppressing basal lipolysis by maintaining intracellular Ca²⁺ to inhibit cAMP production and subsequent downstream lipolysis induction. These findings suggest that P2Y₂ may be a novel drug target for controlling lipolysis in human adipocytes.

Reference:

1. Arner et al. (2011). Nature 478: 110-113.

2. Sollevi et al. (1981). Naunyn-Schmiedeberg’s Arch Pharmacol 316: 112-119.

3. Osisalo, JJ (1981). J Clin Endocrinol Metab 52: 359-363.