Different downstream signalling of CCK₁ receptors regulates distinct functions of CCK in pancreatic beta cells

Abstract

BACKGROUND AND PURPOSECholecystokinin (CCK) is secreted by intestinal I cells and regulates important metabolic functions. In pancreatic islets, CCK controls beta cell functions primarily through CCK 1 receptors, but the signalling pathways downstream of these receptors in pancreatic beta cells are not well defined. EXPERIMENTAL APPROACHApoptosis in pancreatic beta cell apoptosis was evaluated using Hoechst-33342 staining, TUNEL assays and Annexin-V-FITC/PI staining. Insulin secretion and second messenger production were monitored using ELISAs. Protein and phospho-protein levels were determined by Western blotting. A glucose tolerance test was carried out to examine the functions of CCK-8s in streptozotocin-induced diabetic mice. KEY RESULTSThe sulfated carboxy-terminal octapeptide CCK 26-33 amide (CCK-8s) activated CCK 1 receptors and induced accumulation of both IP 3 and cAMP. Whereas G q -PLC-IP 3 signalling was required for the CCK-8s-induced insulin secretion under low-glucose conditions, G s -PKA/Epac signalling contributed more strongly to the CCK-8s-mediated insulin secretion in high-glucose conditions. CCK-8s also promoted formation of the CCK 1 receptor/β-arrestin-1 complex in pancreatic beta cells. Using β-arrestin-1 knockout mice, we demonstrated that β-arrestin-1 is a key mediator of both CCK-8s-mediated insulin secretion and of its the protective effect against apoptosis in pancreatic beta cells. The anti-apoptotic effects of β-arrestin-1 occurred through cytoplasmic latephase ERK activation, which activates the 90-kDa ribosomal S6 kinase-phospho-Bcl-2-family protein pathway. CONCLUSIONS AND IMPLICATIONSKnowledge of different CCK 1 receptor-activated downstream signalling pathways in the regulation of distinct functions of pancreatic beta cells could be used to identify biased CCK 1 receptor ligands for the development of new anti-diabetic drugs.

A cullin 4B-RING E3 ligase complex fine-tunes pancreatic δ cell paracrine interactions

“…However, although the insulin secretion of islets isolated from Sst-Cre +/-Cul4b fl/Y mice was still much lower than that of their WT littermates after SNX482 treatment, this difference was eliminated after nicardipine treatment ( Figure 3D). These data are in agreement with the observed somatostatin secretion patterns and reinforce the conclusion that L-type calcium channels in pancreatic δ cells are key components mediating the increased somatostatin secretion and impaired glucose metabolism in Sst In addition to intracellular calcium, the second messenger cAMP, the G q -PLC pathway, and kinase signaling are actively involved in hormone secretion from pancreatic islets (5,13,(39)(40)(41). Therefore, we preincubated the islets with several inhibitors, including cAMP-PKA signaling inhibitors Rp-CAMPs and H89, G q -PLC inhibitor U73122, MEK inhibitor U0126, and adenyl cyclase (AC) inhibitor 2′,5′-dideoxyadenosine (DDA), and we examined glucose-induced somatostatin secretion patterns ciency in the pancreatic β cells of Ins2-Cre +/-Cul4b fl/Y mice did not significantly affect insulin and somatostatin secretion levels in response to high glucose relative to trends found for Ins2-Cre +/-mice (Supplemental Figure 3, A and B).…”

“…L-type calcium channels are responsible for the influx of extracellular calcium during hormone secretion. We therefore measured high glucose-and high potassium-induced calcium signals in iso- In addition to intracellular calcium, the second messenger cAMP, the G q -PLC pathway, and kinase signaling are actively involved in hormone secretion from pancreatic islets (5,13,(39)(40)(41). Therefore, we preincubated the islets with several inhibitors, including cAMP-PKA signaling inhibitors Rp-CAMPs and H89, G q -PLC inhibitor U73122, MEK inhibitor U0126, and adenyl cyclase (AC) inhibitor 2′,5′-dideoxyadenosine (DDA), and we examined glucose-induced somatostatin secretion patterns ciency in the pancreatic β cells of Ins2-Cre +/-Cul4b fl/Y mice did not significantly affect insulin and somatostatin secretion levels in response to high glucose relative to trends found for Ins2-Cre +/-mice (Supplemental Figure 3, A and B).…”

Cholecystokinin expression in the β-cell leads to increased β-cell area in aged mice and protects from streptozotocin-induced diabetes and apoptosis

“…We have clearly demonstrated CCK1(A)R mRNA expression in pancreatic islets [ Fig. 5 and (37)], and blockade of the CCK1(A)R prevents downstream signaling and CCK-stimulated insulin secretion in mouse islets (44). Therefore, the CCK1(A)R is very likely to be the mediator of CCK signaling in the ␤-cell.…”

“…In previous studies, CCK concentrations of 100 pM to 100 nM were used to document enhanced insulin secretion (13,15,21,44). Although we cannot estimate the concentration of CCK within the islet microenvironment in vivo in MIP-CCK mice, the amount of CCK in medium from isolated MIP-CCK islets was only ϳ20 pM after 24 h (Fig.…”

“…The downstream signaling of the GLP-1, GIP, and CCK1(A) receptors converge on the pro-survival signaling of protein kinase A (11,31). A recent paper found CCK1(A)R signaling via ␤-arrestin, extracellular signal-regulated protein kinase (ERK), the 90-kDa ribosomal S6 kinase (p90RSK), and Bad regulates the ␤-cell antiapoptotic effects of CCK in high-glucose conditions (44). The expression of CCK receptors in the pancreatic islet has been controversial, with several studies finding disparate results in different species and using different antibodies (5,11,23,25,28,43,48).…”