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. 2015 Dec 8;112(49):E6818-24.
doi: 10.1073/pnas.1519430112. Epub 2015 Nov 23.

CK2 acts as a potent negative regulator of receptor-mediated insulin release in vitro and in vivo

Mario Rossi  1 , Inigo Ruiz de Azua  1 , Luiz F Barella  1 , Wataru Sakamoto  1 , Lu Zhu  1 , Yinghong Cui  1 , Huiyan Lu  2 , Heike Rebholz  3 , Franz M Matschinsky  4 , Nicolai M Doliba  4 , Adrian J Butcher  5 , Andrew B Tobin  5 , Jürgen Wess  6
Affiliations

CK2 acts as a potent negative regulator of receptor-mediated insulin release in vitro and in vivo

Mario Rossi et al. Proc Natl Acad Sci U S A. .

Abstract

G protein-coupled receptors (GPCRs) regulate virtually all physiological functions including the release of insulin from pancreatic β-cells. β-Cell M3 muscarinic receptors (M3Rs) are known to play an essential role in facilitating insulin release and maintaining proper whole-body glucose homeostasis. As is the case with other GPCRs, M3R activity is regulated by phosphorylation by various kinases, including GPCR kinases and casein kinase 2 (CK2). At present, it remains unknown which of these various kinases are physiologically relevant for the regulation of β-cell activity. In the present study, we demonstrate that inhibition of CK2 in pancreatic β-cells, knockdown of CK2α expression, or genetic deletion of CK2α in β-cells of mutant mice selectively augmented M3R-stimulated insulin release in vitro and in vivo. In vitro studies showed that this effect was associated with an M3R-mediated increase in intracellular calcium levels. Treatment of mouse pancreatic islets with CX4945, a highly selective CK2 inhibitor, greatly reduced agonist-induced phosphorylation of β-cell M3Rs, indicative of CK2-mediated M3R phosphorylation. We also showed that inhibition of CK2 greatly enhanced M3R-stimulated insulin secretion in human islets. Finally, CX4945 treatment protected mice against diet-induced hyperglycemia and glucose intolerance in an M3R-dependent fashion. Our data demonstrate, for the first time to our knowledge, the physiological relevance of CK2 phosphorylation of a GPCR and suggest the novel concept that kinases acting on β-cell GPCRs may represent novel therapeutic targets.

Keywords: G protein-coupled receptors; GPCR regulation; glucose homeostasis; mouse models; β-cell function.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Knockdown of CK2α expression selectively augments M3R-mediated increases in [Ca2+]i in MIN6 cells. (A and B) MIN6 cells were electroporated with CK2α siRNA or scrambled control siRNA. The insert in A shows a representative Western blot indicating that the use of CK2α siRNA led to a very efficient knockdown of CK2α expression. Cells were then incubated with increasing concentrations of the muscarinic agonist, OXO-M (A), which acts on endogenous M3Rs or AVP (B), which stimulates endogenous V1B vasopressin receptors, respectively. Agonist-induced increases in [Ca2+]i were determined via FLIPR. Data are expressed as means ± SEM of three independent experiments, each carried out in quadruplicate. AU, arbitrary units. ***P < 0.001, compared with the corresponding control value.
Fig. 2.
Fig. 2.
Knockdown of CK2α specifically augments M3R-mediated insulin secretion in MIN6 cells. Insulin release assays were carried out with MIN6 cells that had been treated with CK2α siRNA or scrambled control siRNA. (A–D) Cells were incubated with increasing concentrations of OXO-M (acting on endogenous M3Rs) (A), AVP (acting on endogenous V1B receptors) (B), glucose (C), or glibenclamide (D), an inhibitor of ATP-sensitive K+ channels. CK2α knockdown greatly enhanced M3R-mediated insulin release but had little or no effect on AVP-, glucose-, or glibenclamide-induced insulin secretion. Note that basal insulin secretion was slightly increased (P < 0.05) in cells treated with CK2α siRNA (insulin in ng/mL; control siRNA vs. CK2α siRNA: (A) 23.2 ± 1.5 vs. 28.1 ± 1.2; (B) 23.2 ± 1.6 vs. 28.2 ± 0.8; (C) 16.6 ± 0.6 vs. 22.0 ± 0.8; (D) 27.3 ± 0.6 vs. 31.0 ± 1.0. Data are expressed as the percentage increase in insulin release above basal levels and represent means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the corresponding control value.
Fig. 3.
Fig. 3.
CK2 inhibition or CK2α deletion selectively increases M3R-mediated insulin secretion from pancreatic islets. (A and C–E) Isolated pancreatic islets from adult WT mice were incubated for 1 h in Krebs solution containing 16.7 mM glucose in either the absence or the presence of the selective CK2 inhibitor CX4945 (10 μM) and the muscarinic agonist OXO-M (100 μM) (A), AVP (100 nM) (C), palmitate (0.5 mM) (D), or exendin-4 (10 nM) (E). (B) Isolated pancreatic islets prepared from adult β-CK2α-KO mice and control littermates were incubated with OXO-M, as described above. Note that deletion of CK2α in mouse β-cells leads to a similar enhancement in M3R-stimulated insulin release as observed after CX4945 treatment of WT islets. (F) Glucose-induced insulin secretion in WT mouse islets in the absence or presence of CX4945 (10 μM). In A–F, the amount of insulin secreted into the medium during the 1-h incubation period was normalized to the total insulin content of each well (islets plus medium). Data are expressed as means ± SEM of three independent experiments, each carried out in triplicate. *P < 0.05 and **P < 0.01, compared with the corresponding control value. n.s., no statistically significant difference. (G) Insulin perifusion studies carried out with human islets. Perifused human islets were stimulated with increasing concentration of ACh in the presence of a stimulatory concentration of glucose (8 mM; the initial glucose concentration was 3.3 mM). Experiments were carried out in the absence (control) or the presence of CX4945 (10 μM). (Right) ACh-induced augmentation of insulin release at 8 mM glucose expressed as area under the curve (AUC). For experimental details, see SI Appendix, Methods. Each curve represents the mean ± SEM of four independent perfusion experiments (180 human islets per group and perifusion; ***P < 0.001).
Fig. 4.
Fig. 4.
Acute inhibition of CK2 selectively augments bethanechol-induced insulin secretion in vivo. WT mice (age: ∼8–12 wk) received a single dose of CX4945 (25 mg/kg, i.p.), followed by a 4-h fast. Mice were then injected with the muscarinic agonist bethanechol (2 mg/kg, s.c.) (A), glibenclamide (5 mg/kg i.p.) (B), arginine (1 g/kg i.p.) (C), or exendin-4 (12 nmol/kg i.p.) (D). Before CX4945 treatment, mice were either fed ad libitum (A, B, and D) or fasted for 5 h (C). Under these experimental conditions, bethanechol stimulates insulin release in WT mice via activation of β-cell M3Rs (17). Plasma insulin levels were measured at the indicated time points. Values are given as means ± SEM (n = 7 or 8 per group). *P < 0.05, compared with the corresponding control value.
Fig. 5.
Fig. 5.
Inhibition of CK2 prevents diet-induced hyperglycemia and glucose intolerance in an M3R-dependent fashion. (A) WT or (B) M3R-deficient (M3R KO) mice (males) that had been maintained on a HFD for 9–10 wk were injected twice a day for 6 d with CX4945 (25 mg/kg, i.p.) or vehicle (DMSO). At the end of the 6-d injection period, an IGTT was carried out as described in SI Appendix, Methods. Data are given as means ± SEM (A: vehicle, n = 7; CX4945, n = 6; B: vehicle, n = 4; CX4945, n = 6). *P < 0.05, **P < 0.01, compared with the corresponding control value.
Fig. 6.
Fig. 6.
CK2 phosphorylates the M3R in vitro in a CX4945-sensitive fashion. (A) CK2 phosphorylation assays. HEK-293 cells were transiently transfected with HA-tagged versions of the WT mouse M3R or a PD mutant M3R (see text for details). Phosphorylation assays were carried out by incubating receptor-expressing membranes with 500 units of CK2 in the absence or the presence of OXO-M (100 μM) and/or CX4945 (10 μM) (for experimental details, see SI Appendix, Methods). (A) A representative autoradiograph and a corresponding Western blot demonstrating that equal amounts of receptor protein were loaded. (B) A summary of three independent phosphorylation experiments (means ± SEM). **P < 0.01, compared with the corresponding WT M3R value.
Fig. 7.
Fig. 7.
CX4945-sensitive phosphorylation of mouse β-cell M3Rs. Lysates were prepared from pancreatic islets of WT or β-M3R Tg mice (note that the transgenic mice overexpress an HA-tagged version of the WT M3R selectively in β-cells). (A) Immunoblotting studies using Phos-tag technology. Proteins were separated via Zn-Phos-tag 5.5% SDS/PAGE (∼5 μg islet protein per lane; for details, see SI Appendix, Methods) and probed with a monoclonal anti-HA antibody to detect HA-tagged β-cell M3Rs. Note that two distinct HA-M3R bands can be detected in islet lysates from β-M3R Tg mice. Because the Zn-Phos-tag slows the migration of phosphorylated proteins, the upper band is predicted to represent a phosphorylated (or hyper-phosphorylated) form of the receptor. OXO-M (100 μM) treatment of transgenic islets enhanced the intensity of this upper band. This effect was significantly reduced in the presence of CX4945 (10 μM). (B) Quantification of the OXO-M data shown in A. In each individual experiment, the intensity of the higher molecular mass band observed with the OXO-M–treated transgenic islet was set equal to 100. The data shown are means ± SEM of three independent experiments. **P < 0.01, compared with control samples. (C) Aliquots of islet lysates corresponding to samples run in A were also subjected to regular 5.5% SDS/PAGE, and blots were probed with anti-HA and anti–β-actin antibodies. Note that only a single HA-M3R band was detectable under these conditions. The blots shown are representative of three independent experiments.
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