Collagenase 5

β‐mercaptoethanol, aprotinin, ACh, Histopaque‐1077 and 1119 were obtained from Sigma‐Aldrich. Collagenase V was provided by Roche Diagnostics (Indianapolis, IN, USA). L‐Norepinephrine was purchased from J&K Scientific (Beijing, China). Lipofectamine 3000 was obtained from Invitrogen (California, USA). The antibodies to TH, NET and c‐Fos were from Cell Signaling Technology (Boston, USA). The antibodies to Mlxipl and Pdcd4 were purchased from Proteintech (Chicago, IL, USA). The antibodies to Synapsin, AChE, VAChT, Cadps, Sik2, TH (phospho S31), muscarinic receptors (M1‐M5) and adrenergic receptors (α1a‐α2c) were provided by Abcam (Cambridge, UK). Anti‐Cadps (phospho S259) monoclonal antibody was synthesized and produced by SGE Biotech (Suzhou, China). The phosphorylated antibodies including Sik2 (Phos S358), Mlxipl (Phos S196) and Pdcd4 (Phos S313) were synthesized by PTM BioLab (Hangzhou, China). The information on antibodies used in this study is listed in Table 1.

Islets were isolated from 8 to 12 weeks old C57BL/6 male mice as described before by our laboratory^{31 (link)–34 (link)}. Briefly, mouse islets were isolated using perfusion and digestion of pancreas with collagenase V (from Roche), density gradient purification with histopaque-1077 (Sigma), and then hand-picked. Isolated islets were cultured overnight in RPMI 1640 containing 10% FBS, 11 mM glucose, and then switched for 1 h to Krebs Ringer Bicarbonate buffer containing 2.6 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 4.9 mM KCl, 98.5 mM NaCl, and 25.9 mM NaHCO₃ (all from Sigma-Aldrich) supplemented with 20 mM Na-HEPES and 0.1% BSA. About 10 islets in each experimental condition were transferred to each well in 24-well plate containing 2.8 mM and 16.7 mM glucose concentration in Krebs Ringer Bicarbonate buffer for 1 h. The supernatants were collected for insulin measurements. The islets were lysed with 1% Triton to determine total protein content in the islets. Insulin levels were measured with an ELISA kit from ALPCO. About 200 isolated mouse islets from WT or sparc −/− mice were also collected for Western blot analysis of RGS4 and SPARC.