Discovery of a Novel Target for the Dysglycemic Chromogranin A Fragment Pancreastatin: Interaction with the Chaperone GRP78 to Influence Metabolism

Abstract

RationaleThe chromogranin A-derived peptide pancreastatin (PST) is a dysglycemic, counter-regulatory peptide for insulin action, especially in liver. Although previous evidence for a PST binding protein has been reported, such a receptor has not been identified or sequenced.Methods and ResultsWe used ligand affinity to purify the PST target, with biotinylated human PST (hCHGA273–301-amide) as “bait” and mouse liver homogenate as “prey”, and identified GRP78 (a.k.a. “78 kDa Glucose Regulated Protein”, HSPA5, BIP) as a major interacting partner of PST. GRP78 belongs to the family of heat shock proteins (chaperones), involved in several cellular processes including protein folding and glucose metabolism. We analyzed expression of GRP78 in the absence of PST in a mouse knockout model lacking its precursor CHGA: hepatic transcriptome data revealed global over-expression of not only GRP78 but also other heat shock transcripts (of the “adaptive UPR”) in CHGA(−/−) mice compared to wild-type (+/+). By contrast, we found a global decline in expression of hepatic pro-apoptotic transcripts in CHGA(−/−) mice. GRP78's ATPase enzymatic activity was dose-dependently inhibited by PST (IC50∼5.2 µM). PST also inhibited the up-regulation of GRP78 expression during UPR activation (by tunicamycin) in hepatocytes. PST inhibited insulin-stimulated glucose uptake in adipocytes, and increased hepatic expression of G6Pase (the final step in gluconeogenesis/glycogenolysis). In hepatocytes not only PST but also other GRP78-ATPase inhibitors (VER-155008 or ADP) increased G6Pase expression. GRP78 over-expression inhibited G6Pase expression in hepatocytes, with partial restoration by GRP78-ATPase inhibitors PST, VER-155008, or ADP.ConclusionsOur results indicate that an unexpected major hepatic target of PST is the adaptive UPR chaperone GRP78. PST not only binds to GRP78 (in pH-dependent fashion), but also inhibits GRP78's ATPase enzymatic activity, and impairs its biosynthetic response to UPR activation. PST decreases insulin-stimulated cellular glucose uptake, and PST as well as other chaperone ATPase activity inhibitors augment expression of G6Pase; GRP78 over-expression antagonizes this PST action. Analysis of the novel PST/GRP78 interaction may provide a new avenue of investigation into cellular glycemic control as well as dysglycemia.

Heat shock protein 70 dampens the inflammatory response of human PDL cells to mechanical loading in vitro

“…We recently demonstrated the effectiveness of VER15008 as an inhibitor of HSP70 activity that interacts with its ATPase binding domain, although its specificity does not seem to exist exclusively for HSP70, but also for glucose‐related protein 78, another chaperone of the HSP family involved in the process of protein folding and glucose metabolism . Thus, the effects reported here might result from the inhibition of targets other than HSP70.…”

Pancreastatin-Dependent Inflammatory Signaling Mediates Obesity-Induced Insulin Resistance

“…A guanosine triphosphate (GTP)-binding protein-coupled receptor-mediated signaling pathway leading to activation of conventional DAG/Ca 2+ -dependent PKC and downregulation of mature Srebp-1c (p68) is thought to play a role in the anti-insulin effects of PST (11). Recently, we observed that PST could also modulate endoplasmic reticulum (ER) stress by interacting with binding immunoglobin protein (BiP)/78 kDa glucoseregulated protein (GRP78) (45). Feeding an HFD creates obesity, leading to hyperinsulinemia and inflammation (18)(19)(20)(21)(22).…”

Pancreastatin Predicts Survival in Neuroendocrine Tumors

“…37,38 Although high pancreastatin has been recognized as a feature of neuroendocrine tumors for some time, pancreastatin’s role in normal physiology remains poorly understood. 36,39 Pancreastatin seems to exert its effects through activity at membrane-associated G-proteins and phospholipase C, but a specific membrane-bound pancreastatin receptor (PSTR) has not been identified. 37,39 Attempts to identify the PSTR have focused on affinity purification from rodent liver.…”

“…36,39 Pancreastatin seems to exert its effects through activity at membrane-associated G-proteins and phospholipase C, but a specific membrane-bound pancreastatin receptor (PSTR) has not been identified. 37,39 Attempts to identify the PSTR have focused on affinity purification from rodent liver. 37,39 NETs overexpress many hormone receptors such as those for somatostatin and gastric-inhibitory polypeptide, 40-42 making it tempting to speculate that the putative PSTR might be more abundant in, and more readily isolated from, NET tissue specimens.…”

“…37,39 Attempts to identify the PSTR have focused on affinity purification from rodent liver. 37,39 NETs overexpress many hormone receptors such as those for somatostatin and gastric-inhibitory polypeptide, 40-42 making it tempting to speculate that the putative PSTR might be more abundant in, and more readily isolated from, NET tissue specimens. Pancreastatin causes Ras-independent activation of the mitogen-activated protein kinase (MAPK) pathway, and also activates the phosphatidyl-inositol-3-kinase/Akt pathway.…”