Lupenone attenuates thapsigargin-induced endoplasmic reticulum stress and apoptosis in pancreatic beta cells possibly through inhibition of protein tyrosine kinase 2 activity

Abstract

Aims: Prolonged high levels of cytokines, glucose, or free fatty acids are associated with diabetes, elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_C), and depletion of Ca²⁺ concentration in the endoplasmic reticulum (ER) of pancreatic beta cells. This Ca²⁺ imbalance induces ER stress and apoptosis. Lupenone, a lupan-type triterpenoid, is beneficial in diabetes; however, its mechanism of action is yet to be clarified. This study evaluated the protective mechanism of lupenone against thapsigargin-induced ER stress and apoptosis in pancreatic beta cells.

Materials and methods: MIN6, INS-1, and native mouse islet cells were used. Western blot for protein expressions, measurement of [Ca²⁺]_C, and in vivo glucose tolerance test were mainly performed.

Key findings: Thapsigargin increased the protein levels of cleaved caspase 3, cleaved PARP, and the phosphorylated form of JNK, ATF4, and CHOP. Thapsigargin increased the interaction between stromal interaction molecule1 (Stim1) and Orai1, enhancing store-operated calcium entry (SOCE). SOCE is further activated by protein tyrosine kinase 2 (Pyk2), which is Ca²⁺-dependent and phosphorylates the tyrosine residue at Y³⁶¹ in Stim1. Lupenone inhibited thapsigargin-mediated Pyk2 activation, suppressed [Ca²⁺]_C, ER stress, and apoptosis. Lupenone restored impaired glucose-stimulated insulin secretion effectuated by thapsigargin and glucose intolerance in a low-dose streptozotocin-induced diabetic mouse model.

Significance: These results suggested that lupenone attenuated thapsigargin-induced ER stress and apoptosis by inhibiting SOCE; this may be due to the hindrance of Pyk2-mediated Stim1 tyrosine phosphorylation. In beta cells that are inevitably exposed to frequent [Ca²⁺]_C elevation, the attenuation of abnormally high SOCE would be beneficial for their survival.

Keywords: 2-Aminoethoxydiphenyl borate (PubChem CID: 1598); 4-Phenylbutyric acid (PubChem CID: 4775); Apoptosis; Beta-cell; Cyclopiazonic acid (PubChem CID: 54695722); ER stress; KN-93 (PubChem CID: 5312122); Lupenone; Lupenone (PubChem CID: 92158); Protein tyrosine kinase 2; SP600125 (PubChem CID: 8515); Sodium orthovanadate (PubChem CID: 61671); Store-operated calcium entry; Streptozotocin (PubChem CID: 29327); Thapsigargin (PubChem CID: 446378); W-7 (PubChem CID: 124887).