CRFR1 is expressed on pancreatic beta cells, promotes beta cell proliferation, and potentiates insulin secretion in a glucose-dependent manner - PubMed (original) (raw)

CRFR1 is expressed on pancreatic beta cells, promotes beta cell proliferation, and potentiates insulin secretion in a glucose-dependent manner

Mark O Huising et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Corticotropin-releasing factor (CRF), originally characterized as the principal neuroregulator of the hypothalamus-pituitary-adrenal axis, has broad central and peripheral distribution and actions. We demonstrate the presence of CRF receptor type 1 (CRFR1) on primary beta cells and show that activation of pancreatic CRFR1 promotes insulin secretion, thus contributing to the restoration of normoglycemic equilibrium. Stimulation of pancreatic CRFR1 initiates a cAMP response that promotes insulin secretion in vitro and in vivo and leads to the phosphorylation of cAMP response element binding and the induction of the expression of several immediate-early genes. Thus, the insulinotropic actions of pancreatic CRFR1 oppose the activation of CRFR1 on anterior pituitary corticotropes, leading to the release of glucocorticoids that functionally antagonize the actions of insulin. Stimulation of the MIN6 insulinoma line and primary rat islets with CRF also activates the MAPK signaling cascade leading to rapid phosphorylation of Erk1/2 in response to CRFR1-selective ligands, which induce proliferation in primary rat neonatal beta cells. Importantly, CRFR1 stimulates insulin secretion only during conditions of intermediate to high ambient glucose, and the CRFR1-dependent phosphorylation of Erk1/2 is greater with elevated glucose concentrations. This response is reminiscent of the actions of the incretins, which potentiate insulin secretion only during elevated glucose conditions. The presence of CRFR1 on beta cells adds another layer of complexity to the intricate network of paracrine and autocrine factors and their cognate receptors whose coordinated efforts can dictate islet hormone output and regulate beta cell proliferation.

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Figures

Fig. 1.

Fig. 1.

CRFR1 is expressed on insulinoma cells and on primary mouse β cells. CRFR1 expression levels are relatively abundant in the mouse insulinoma cell line MIN6 compared with the α cell line αTC-1 or the δ cell line TU6 (A). Similarly, GLP-1R (B) and GIPR (C) are expressed abundantly in the MIN6 insulinoma cells. Using dissociated primary islets from an mIP-GFP transgenic reporter mouse (D), we obtained primary β cells by FACS separation of GFP-positive and GFP-negative cells (E). The expression profile of the GFP-positive population demonstrates the retention of expression of β cell markers such as insulin, GLP-1R, and GIPR, whereas glucagon and Sst are not expressed (F). CRFR1 expression in GFP-positive cells demonstrates β cell localization of CRFR1. GFP and insulin are stained green and red, respectively, in (D); DAPI (blue) is applied as a nuclear counterstain.

Fig. 2.

Fig. 2.

CRFR1 activation induces a cAMP response that augments GSIS in MIN6 insulinoma cells and primary rodent and human islets. CRF dose-dependently increases intracellular cAMP levels in MIN6 cells; this increase is blocked by the simultaneous administration of the CRFR1-selective antagonist antalarmin (A). Stimulation of MIN6 cells with the CRFR1-selective agonist oCRF rapidly induces robust phosphorylation of CREB (B) and leads to the stimulation of the expression of immediate-early genes including cFos, nuclear receptor subfamily 4 group A member 2 (NR4A2), regulator of G protein signaling 2 (RGS2), and dual-specificity phosphatase 1 (DUSP1) (C). These transcriptional changes are enhanced by high ambient glucose concentrations and can be blocked by the CRFR1 antagonist antalarmin. Stimulation of MIN6 cells with a relatively low dose of oCRF (1 nM) suffices to augment insulin secretion in the presence of intermediate (11 mM) or high (16.8 mM) glucose concentrations (D). The augmentation of GSIS induced by selective or preferential CRFR1 agonists (oCRF, r/hCRF, stressin1-A) is completely inhibited by general (astressin) or CRFR1-selective (antalarmin) antagonists but not by the CRFR2-selective antagonist astressin2-B (E). Stimulation with oCRF potentiates GSIS in mouse (F) and human (G) primary islets in a CRFR1-dependent manner, because coadministration of antalarmin complete blocks the effects of oCRF.

Fig. 3.

Fig. 3.

Activation of CRFR1 activates the MAPK signaling cascade and induces proliferation in rat neonatal β cells. Stimulation of CRFR1 dose-dependently increases phosphorylation of Erk1/2, an increase that is inhibited by coadministration of antalarmin (A). The induction of pErk1/2 is rapid (5 min) and persists for at least 3 h (B). oCRF and exendin-4 synergize with glucose (C). Isolated primary rat islets respond to the CRFR1-selective agonist oCRF with increased levels of pErk1/2 (D). Stimulation of rat neonatal islet cells with r/hCRF increased the nuclear incorporation of EdU into insulin-positive cells; this incorporation was blocked by antalarmin (E_–_G). Proliferation was quantified as the fraction of EdU insulin-positive cells (H). Numbers indicate the total number of insulin-positive cells; the number of insulin-positive EdU cells is given in parentheses. The total number of islets isolated from CRFR1-null pancreata was markedly lower than in control animals (I).

Fig. 4.

Fig. 4.

Acute stimulation of CRFR1 in vivo augments GSIS leading to enhanced glucose tolerance. Fasted plasma insulin levels in CRFR1-null males receiving a replacement dose of glucocorticoids to mitigate HPA deficiency were significantly lower than in age-matched wild-type males (A). Despite reduced fasting and glucose-induced insulin levels, glucose tolerance paradoxically was superior in CRFR1-null mice than in wild-type controls (B). CRFR1-null mice have reduced relative eWAT weight (C). Circulating insulin levels in fasted adrenalectomized wild-type animals were unaffected by prior stressin1-A administration (0.5 mg/kg i.p.) as compared with controls (D). Although glucose challenge (2 g/kg i.p.) significantly elevated circulating insulin in both groups, preadministration of stressin1-A significantly elevated GSIS (D). Stressin1-A (2.5 mg/kg, i.p.) administered 15 min before a glucose tolerance test significantly improved glucose tolerance as compared with vehicle-treated controls (E). *, P < 0.05; **, P < 0.01 vs. wild-type or vehicle-treated controls.

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