CK2 activity is crucial for proper glucagon expression

Materials

Collagenase NB 4G was purchased from SERVA Electrophoresis (Heidelberg, Germany). Neutral red solution, Tween20 and Hoechst 33342 were purchased from Sigma-Aldrich (Taufkirchen, Germany). Ketamine (Ursotamin) was purchased from Serumwerke Bernburg (Bernburg, Germany) and xylazine (Rompun) from Bayer (Leverkusen, Germany). BSA and FCS were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Cell lysis reagent QIAzol and QuantiNova Reverse Transcription Kit were purchased from Qiagen (Hilden, Germany). HepatoQuick, as well as water-soluble tetrazolium (WST) and lactate dehydrogenase (LDH) assays, were purchased from Roche (Basel, Switzerland). The qScriber cDNA Synthesis Kit and ORA SEE qPCR Green ROX L Mix were purchased from HighQu (Kraichtal, Germany). The CK2 inhibitors CX-4945 and SGC-CK2-1, and linsitinib (OSI-906) were purchased from SelleckChem (Munich, Germany) and Sigma-Aldrich (Taufkirchen, Germany), respectively. Protein assay dye reagent and luminol-enhanced chemiluminescence (ECL) western blotting substrate were purchased from Bio-Rad Laboratories (Feldkirchen, Germany). Lipofectamine 3000, DMEM (1 g glucose/l [5.5 mmol/l] or 4.5 g glucose/l [25 mmol/l]) and RPMI medium 1640 (2 g glucose/l [11 mmol/l]) were purchased from Fisher Scientific (Schwerte, Germany). The μMacs Streptavidin Kit was purchased from Miltenyi Biotec (Gladbach, Germany). Biotinylated primers were obtained from Life Technologies (Darmstadt, Germany).

Antibodies

The anti-GCG antibody (catalogue no. ab92517), the anti-pAktS129 antibody (catalogue no. ab133458) and the anti-PAX6 antibody (catalogue no. ab109233) were from Abcam (Cambridge, UK). The anti-GAPDH antibody (catalogue no. 60004) and the insulin antibody (catalogue no. 15848-1-AP) were from Proteintech Germany (St Leon-Rot, Germany). The anti-Akt antibody (11E7, catalogue no. 4685) was from Cell Signaling Technology (Frankfurt am Main, Germany). The anti-CK2β antibody (E9, catalogue no. sc-46666) was from Santa Cruz Biotechnology (Heidelberg, Germany). Fructose 1,6-bisphosphatase (FBP1) was detected with a monoclonal rabbit antibody (D2T7F, catalogue no. 59172, Cell Signaling Technology). The anti-MafB (catalogue no. A700-046) antibody was from Bethyl Laboratories (Montgomery, TX, USA). Equal loading was verified using a mouse monoclonal α-tubulin antibody (clone DM1A, catalogue no. T9026, Sigma-Aldrich, München, Germany).The generation of the anti-CK2α antibody has been described previously [20]. PDX1 was identified with a polyclonal antiserum generated by immunising rabbits with recombinant mouse PDX1 [11]. The rabbit polyclonal serum no. 36 against nucleolin was generated by immunising rabbits with a C-terminal peptide of nucleolin (amino acids 696–707) [21]. The peroxidase-labelled anti-rabbit antibody (NIF 824) and the peroxidase-labelled anti-mouse antibody (NIF 825) were from GE Healthcare (Freiburg, Germany).

Cell culture

The murine pancreatic alpha cell line αTC1 clone 6 (ATCC: CRL-2934) was cultivated in DMEM (1 g/l glucose) supplemented with 10% (vol./vol.) FCS in a humidified atmosphere with 5% CO₂ at 37°C. The murine beta cell line MIN6 [22] was maintained in DMEM (4.5 g/l glucose) supplemented with 10% (vol./vol.) FBS in a humidified atmosphere with 5% CO₂ at 37°C. To study the effect of CK2 on pro-GCG expression, we knocked out the catalytic CK2α subunit in αTC1 cells (αTC1 KO) by the CRISPR/Cas9 technique using the plasmid pD1431-Apuro:441627 with a guide RNA for mouse CK2α designed by ATUM (Newark, USA). Transfection was carried out using Lipofectamine 3000 (Fisher Scientific) according to the manufacturer’s instructions. After 48 h, cells were exposed to puromycin (2 µg/ml), which was renewed every 3 days, to create a stable cell line (αTC1 KO cells). In addition, the αTC1 cells were exposed to CX-4945 (10 µmol/l), SGC-CK2-1 (10 µmol/l) or DMSO as control for 24 h. Transient transfection was done with the GCG promoter construct or different p3xFlagCMV7.1-PDX1-constructs [23] and Lipofectamine 3000 according to the manufacturer’s protocol. Used cell lines were free from mycoplasma contamination.

Western blot analysis

For the generation of whole cell extracts, αTC1 and αTC1 KO cells, as well as αTC1 cells exposed to CX-4945, SGC-CK2-1 or DMSO as control for 24 h, were harvested and lysed for 30 min at 4°C with lysis buffer (10 mmol/l Tris-HCl, pH 7.5, 10 mmol/l NaCl, 0.1 mmol/l EDTA, 0.5% [vol./vol.] Triton X-100, 0.02% [wt/vol.] NaN₃) supplemented with 0.5 mmol/l phenylmethylsulfonyl fluoride (PMSF) and a protease and phosphatase inhibitor cocktail (1:75 vol./vol., Sigma-Aldrich). The cytoplasmic and nuclear extracts were generated and analysed as described previously in detail [24]. After electrophoresis and blotting onto PVDF membranes by a semi-dry blot procedure using a BioRad Trans-Blot-Turbo transfer system (BioRad, Munich Germany), the membranes were blocked using TBS (20 mmol/l Tris-HCl, pH 7.5, 150 mmol/l NaCl) supplemented with 0.1% (vol./vol.) Tween 20 (TBS-T) and 5% BSA (wt/vol.) for 1 h at room temperature. For the detection of proteins, primary antibodies were diluted 1:1000 with TBS-T and 1% BSA (wt/vol.) and incubated for 1 h at room temperature or overnight at 4°C. Subsequently, membranes were washed twice with TBS-T, and then incubated with the horseradish peroxidase-conjugated secondary antibodies (anti-rabbit antibody [NIF 824] or anti-mouse antibody [NIF 825]) at a dilution of 1:10,000 in TBS-T 1% BSA (wt/vol.) for 1 h at room temperature. After two further washing steps, the expression of the corresponding proteins was visualised by enhanced chemoluminescence using the ECL western blotting substrate from Bio-Rad Laboratories.

Real-time quantitative RT-PCR

For real-time quantitative RT-PCR (qRT-PCR), total RNA from αTC1 cells as well as isolated murine islets exposed to CX-4945, SGC-CK2-1 or DMSO for 24 h were extracted using QIAzol lysis reagent. The corresponding cDNA was synthesised from the total RNA by QuantiNova Reverse Transcription Kit and the qRT-PCR analysis was performed by means of ORA SEE qPCR Green ROX L Mix (highQu, Kraichtal, Germany). Primer sequences for qRT-PCR were coded as follows: Ins1 forward 5′-AACAACTGGAGCTGGGAGGAAG-3′ and reverse 5′-GGTGCAGCACTGATCCACAATG-3′; Gapdh forward 5′-CGGTGCTGAGTATGTC-3′ and reverse 5′-TTTGGCTCCACCCTTC-3′; and Gcg forward 5′-TGGACTCCCGCCGTGCTCAAG-3′ and reverse 5′-CCTTTGCTGCCTGGCCCTCC-3′.

Growth curves

αTC1 and αTC1 KO cells were seeded in a 24-well plate at a density of 1×10⁵/well. After 24 h, 48 h and 72 h, cells were detached, stained with Trypan Blue solution (0.4%, wt/vol.) and counted by a LUNA automated Cell Counter (Logos Biosystems, Villeneuve D’Ascq, France) according to the manufacturer’s protocol.

WST-1 assay

A WST-1 assay was used to analyse the effect of CK2 inhibition/loss on αTC1 and αTC1 KO cell viability by determining the activity of mitochondrial dehydrogenases. Cells were seeded in a 96-well culture plate at a density of 2×10³/well. After 24 h, 10 μl of WST-1 reagent (Roche) was added into each well and the absorbance was measured at 450 nm in a Tecan Infinite 200 Pro microplate reader (Tecan, Crailsheim, Germany).

LDH assay

An LDH assay was used to evaluate the cytotoxic effects of CK2 downregulation. αTC1 and αTC1 KO cells were seeded in a 96-well culture plate at a density of 2×10³/well. After 24 h, an LDH assay was performed according to the manufacturer’s protocol (Roche). LDH-reaction mix (100 μl per 100 μl medium) was added to each well. After 10 min incubation, 50 μl stop solution was added and absorbance was measured at 492 nm in a Tecan Infinite 200 Pro microplate reader (Tecan, Crailsheim, Germany).

Reporter luciferase assay

The sequence of the mouse Gcg promoter was amplified using murine genomic DNA (primers: forward 5′-GTACCTGAGCTCGCTAGCCGACCCTCAAATGAGACTAGG-3′ and reverse 5′-CAACAGTACCGGATTGCCAAGCTGCCCTTCTGCACCAGGGTG-3′). The resulting 379 bp construct was cloned into the XhoI restriction site of the luciferase reporter vector pGL4.10 (Promega, Mannheim, Germany). The identity of pGL4.10-Gcg was verified by sequencing. The transcriptional activity of the Gcg promoter was assessed by reporter gene assays according to the manufacturer’s instructions (Promega). Briefly, αTC1 or αTC1 KO cells were seeded in a 24-well plate. The culture medium (DMEM, 1 g glucose/l) was renewed on the day of transfection and the inhibitor (10 µmol/l CX-4945 or 10 µmol/l SGC-CK2-1) was added. Subsequently, cells were transfected with pGL4 or pGL4-Gcg reporter vector by using Lipofectamine 3000 for 24 h. In addition, pGL4-Gcg-transfected αTC1 cells were exposed to CX-4945, SGC-CK2-1 or DMSO for 24 h. Then, cells were lysed and the luciferase activity was detected by a luminescence plate reader.

DNA pull-down assay

To study the transcription factor binding capacity to the Gcg promoter, we used the μMacs Streptavidin Kit and biotinylated primers (forward 5′-biotin-GACCCTCAAATGAGACTAGG-3′ and reverse 5′-biotin-GCCCTTCTGCACCAG-3′) for the generation of a biotinylated DNA probe ranging from −329 to +8 of the murine Gcg gene. For the pull-down assay, 1 mg of nuclear extracts were preincubated with salmon sperm DNA (100 µg/ml) for 20 min at 4°C. Thereafter, 1 µg of the biotinylated DNA probe was added and incubated for 30 min at 4°C. Fifty microlitres of streptavidin–agarose beads were added and incubated for 12 min at room temperature. The DNA–protein complex was passed through an equilibrated μMacs column, the column was washed and proteins were eluted. The eluates were then analysed by the western blot method.

Pseudoislet formation

Pseudoislets (PIs) were generated by the liquid-overlay technique in 96-well plates covered with 1% agarose as described previously in detail [25]. Briefly, 3500 MIN6 cells and 1500 αTC1 or αTC1 KO cells were seeded per well and incubated for 5 days. The culture medium (DMEM 4.5 g glucose/l) was changed every second day. After 5 days of incubation at 37°C and 5% CO₂, the spheroids were harvested and stored for immunohistochemical analyses or ELISA.

Immunohistochemical analyses

PIs from MIN6 cells and either αTC1 or αTC1 KO cells were incubated for 45 min at 37°C in 100 µl HepatoQuick, 50 µl human citrate plasma and 10 µl 10% CaCl₂ solution. The resulting clot was also fixed for 24 h in 4% (wt/vol.) paraformaldehyde at 4°C. After dehydration, the paraffin-embedded samples were cut into sections (3 μm thick). Antigens in samples were demasked by citrate buffer and the unspecific binding sites were blocked by goat serum. Cells were stained with specific primary antibodies (1:300), which were detected by the corresponding fluorescence-coupled secondary antibodies (1:1000). Cell nuclei were stained with Hoechst 33342. The sections were analysed using a BX60F fluorescence microscope (Olympus, Hamburg, Germany).

Animals

All animal care and experimental procedures were performed according to the German legislation on protection of animals and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, Washington DC, USA). The experiments were approved by the local governmental animal protection committee (Landesamt für Verbraucherschutz LAV, Saarland, permit number 18/2017 and 46/2018). Mice were maintained on a standard 12 h light–dark cycle. Standard pellet chow (Altromin, Lage, Germany) and water were provided ad libitum. C57BL/6J mice (RRID:IMSR_JAX:000664, The Jackson laboratory, USA; https://www.jax.org/strain/000664) with an age of 3–12 months were used as donors for islet isolation, as recipients for transplantation and for treatment with the CK2 inhibitor CX-4945. Details of the mice used are found in electronic supplementary material (ESM) Table 1.

Isolation of pancreatic islets

Murine pancreatic islets were isolated by collagenase-induced enzymatic digestion and purified by hand picking as described previously in detail [25]. Isolated islets were cultivated in RPMI 1640 supplemented with 10% (vol./vol.) FCS, 100 U/ml penicillin and 0.1 mg/ml streptomycin for 24 h at 37°C and 5% CO₂. For the determination of insulin secretion, we used ten islets per well of a 24 well plate and for the determination of GCG secretion 20 islets per well of a 24 well plate.

Human islets were isolated at the Alberta Diabetes Institute IsletCore [26] and incubated overnight in DMEM supplemented with l-glutamine, 110 mg/l sodium pyruvate, 10% (vol./vol.) FCS and 100 U/ml penicillin/streptomycin (15140, Gibco) for 24 h at 37°C and 5% CO₂. All human islet studies were approved by the Human Research Ethics Board (Pro00013094; Pro00001754) at the University of Alberta and all families of organ donors provided written informed consent. Details about the donors are shown in the human islet checklist (ESM).

Kidney capsule model

The kidney capsule model was performed as previously described in detail [27]. Sham-transplanted mice, which were subjected to the operation procedure but did not receive any transplants, served as control. Briefly, normoglycaemic C57BL/6J mice were anaesthetised using ketamine and xylazine and 5×10⁶ αTC1 or αTC1 KO cells were injected under the left kidney capsule. Body weights and fasting blood glucose levels (7 h fasting period) of the mice were measured twice a week during the entire observation period of 28 days. Blood samples were taken from the tail vein and analysed by a portable blood glucose monitoring system (GL50; Breuer, Ulm, Germany).

IPGTT

An IPGTT was performed on day 28 after cell transplantation under the kidney capsule of mice [25, 28]. After 14 h of fasting, the mice were intraperitoneally injected with a 10% glucose (wt/vol.) solution (10 µl/g body weight). Tail-vein blood glucose levels were determined after 0, 15, 30, 45, 60, 120 and 180 min using a portable blood glucose monitoring system (GL50; Breuer).

Mouse islet and cell line GCG and insulin secretion measurements

For GCG secretion, αTC1 and αTC1 KO cells, murine islets or PIs from MIN6 cells and either αTC1 or αTC1 KO cells were incubated in KRB buffer (135 mmol/l NaCl, 3.6 mmol/l KCl, 5 mmol/l NaHCO₃, 0.5 mmol/l NaH₂PO₄, 0.5 mmol/l MgCl₂, 1.5 mmol/l CaCl₂, 10 mmol/l HEPES, pH 7.4, BSA [0.1%] [wt/vol.]) with 25 mmol/l glucose for 1 h [29]. Subsequently, the buffer was removed and the cells, islets or PIs were incubated in KRB buffer containing 0.5 mmol/l glucose for 2 h.

For insulin secretion, cells, islets or PIs were incubated in KRB buffer for 1 h. Subsequently, the buffer was removed and the cells, islets or PIs were incubated in KRB buffer containing 25 mmol/l glucose for 1 h.

The incubation buffer was collected and any residual cells, islets or PIs were removed from the incubation buffer by centrifugation at 250 g for 10 min at 4°C. The supernatant fractions were collected and GCG and insulin secretion were determined by specific ELISA kits according to the manufacturer’s protocol (Invitrogen by Fisher Scientific, Schwerte, Germany).

Analysis of plasma GCG and insulin levels and FBP1 expression in mice

C57BL/6J mice were given a daily i.p. injection of CX-4945 (1.5 mg/kg dissolved in DMSO/PBS) for 3 days. Mice were killed and the blood, kidney and liver samples were collected. GCG secretion was analysed by a GCG or an insulin ELISA as described above. For extraction of protein from mouse liver and kidney, the tissue sample was frozen in liquid nitrogen and crushed in a mortar. The broken tissue was resuspended in three volumes of RIPA buffer (50 mmol/l Tris/HCl, pH 8.0, 150 mmol/l NaCl, 0.5% sodium desoxycholate [wt/vol.], 1% Triton X-100 [vol./vol.], 0.1% sodium dodecylsulphate [wt/vol.]) with the protease and phosphatase inhibitor cocktail Complete (1:25) and the phosphatase inhibitor PhosSTOP (1:10) (both from Roche Diagnostics, Mannheim, Germany). The suspension was incubated on ice for 30 min. After lysis, the debris was removed by centrifugation (30 min, 4°C, 12,500 g). The protein content was determined according to a modified Bradford method (Bio-Rad, Munich, Germany).

Human islet GCG and insulin secretion measurements

Isolated human islets were perfused using a BioRep perifusion system (BioRep, Miami, FL, USA). Batches of 35 islets were preincubated in perifused KRB buffer containing 140 mmol/l NaCl, 3.6 mmol/l KCl, 2.6 mmol/l CaCl₂, 0.5 mmol/l NaH₂PO₄, 0.5 mmol/l MgSO₄, 5 mmol/l HEPES, 2 mmol/l NaHCO₃ and 0.5 mg/ml essentially fatty acid free BSA (Sigma A6003) for 30 min at 5.5 mmol/l glucose, and then perfused in KRB with changes in glucose, 100 nmol/l glucose-dependent insulinotropic polypeptide (GIP) (Anaspec, Fremont, USA), 10 mmol/l alanine (Sigma, Oakville, ON, Canada) and KCl as indicated. Samples were collected at intervals of 120–300 s and stored at −20°C until assay of GCG (U-PLEX Mouse Glucagon Assay, no. K1525YK, Meso Scale Diagnostics, Rockville, MD, USA). The insulin concentration in the same samples was determined using the Stellux Insulin Chemiluminescence ELISA (80-INSHU-CH01, Alpco, Salem, New Hampshire, USA).

Statistical and database analysis

All in vitro experiments were reproduced at least three times. The in vivo experiments were performed with at least six animals per group and no mice were excluded from the statistical analysis. After testing the data for normal distribution and equal variance, differences between two groups were assessed by the unpaired Student’s t test; one-way ANOVA was applied when comparing multiple groups. This was followed by the Tukey post hoc test by means of Prism software 8 (GraphPad, USA). The results were expressed as mean ± SD. Statistical significance indicated as *p<0.05, **p<0.01 and ***p<0.001.

We utilised the PhosphoSitePlus (www.phosphosite.org, accessed 12 January 2023) and Scansite 4.0 (https://scansite4.mit.edu, accessed 12 January 2023) tools to identify the CK2 phosphorylation sites in PAX6 and transcription factor MafB.

Effect of CK2 downregulation on proliferation, viability and GCG expression of αTC1 cells

To check whether the murine pancreatic alpha cell line αTC1 is an appropriate model to study the impact of CK2 on GCG expression and secretion, we first analysed the expression of GCG and CK2 in αTC1 cells. These cells express high levels of pro-GCG when compared with the insulin-expressing beta cell line MIN6, which served as negative control (Fig. 1a–c). Moreover, we detected both CK2 subunits, the catalytic CK2α and the regulatory CK2β (Fig. 1c). Accordingly, the αTC1 cell line was deemed suitable to study the effect of CK2 inhibition on GCG expression. The pharmacological inhibitors CX-4945 [30] and SGC-CK2-1 [31] were used to specifically repress CK2 kinase activity. In addition, we knocked out Csnk2a1 in αTC1 cells by means of the CRISPR/Cas9 technique (αTC1 KO cells). We found that the two inhibitors had only minor effects on CK2 protein expression, whereas the deletion of the catalytic subunit abolished CK2α expression and markedly reduced CK2β protein levels (Fig. 1d–g). We further determined the effect of CK2 inhibition or loss on its catalytic activity by analysing phosphorylation of Akt on serine 129, a specific CK2 phosphorylation site [32]. As expected, we did not detect Akt phosphorylation in αTC1 cells exposed to CX-4945 and SGC-CK2-1 or in αTC1 KO cells when compared with control DMSO-treated cells (Fig. 1d,h).

Next, we assessed the effect of CK2 on alpha cell viability and proliferation by a panel of different assays. Our results showed that CX-4945 and SGC-CK2-1 decrease the activity of mitochondrial dehydrogenases and that this activity was not affected by the loss of CK2α (Fig. 1i). Furthermore, inhibition or loss of CK2 did not increase LDH release in αTC1 cells, except for when SGC-CK2-1 was used for inhibition (Fig. 1j). The analyses of growth curves and Trypan Blue dye exclusion revealed that both pharmacological inhibition and loss of CK2α slightly reduced αTC1 cell proliferation and viability (Fig. 1k,l). It has already been shown that CK2 inhibition promotes insulin expression [33]. Therefore, we assumed that CK2 inhibition may suppress pro-GCG expression. Indeed, the exposure of αTC1 cells to CX-4945 and SGC-CK2-1, as well as the loss of CK2α in αTC1 cells, markedly decreased pro-GCG expression (Fig. 2a,b). This, in turn, resulted in a markedly reduced GCG secretion (Fig. 2c). Whereas the median secretion of untreated cells was 1094±124 ng/l, it was reduced to 545± 82 ng/l by the use of CX-4945, 459±110 ng/l by the use of SGC-CK2-1, and 582± 160 ng/l in αTC1 KO cells. Additional analyses demonstrated that the inhibition or loss of CK2 reduced Gcg mRNA levels (Fig. 2d). This indicates that CK2 most probably regulates Gcg expression at the transcriptional level.

Effect of CK2 downregulation on Gcg gene expression in αTC1 cells

To gain further insight into the mechanisms underlying CK2-regulated GCG expression, we generated a luciferase reporter construct containing the enhancer region (boxes G3, G2 and G5 in Fig. 3a) and the minimal promoter (boxes G4 and G1 in Fig. 3a) of the murine Gcg promoter. This construct was highly active in αTC1 cells (Fig. 3b) and its activity was dramatically reduced after inhibition or downregulation of CK2 (Fig. 3c). The G1 box of the promoter is crucial for alpha cell specificity and Gcg gene expression [34]. It has been reported that the transcription factors PAX6 and MafB bind to this box, resulting in an enhanced Gcg transcription (Fig. 3d) [35]. Hence, we analysed the nuclear localisation of both transcription factors, depending on CK2 activity. We detected an increased cytoplasmic and decreased nuclear localisation of MafB in αTC1 KO cells when compared with αTC1 wild-type (WT) cells (Fig. 3e–g). In contrast, the nuclear localisation of PAX6 was not affected by CK2 downregulation (Fig. 3e,h). We next performed a DNA pull-down assay to study the binding capacity of the two transcription factors in WT αTC1 cells and αTC1 KO cells. As expected, MafB and PAX6 bound to the promoter in WT αTC1 cells; however, this binding was markedly reduced after CK2α loss (Fig. 3i,j).

Effect of CK2 downregulation on PDX1-mediated Gcg gene expression in αTC1 cells

We have already shown that CK2 phosphorylates murine PDX1 at residues T231 and S232 and that the loss of the CK2-dependent phosphorylation stabilises and increases the transcriptional activity of PDX1 in beta cells [10, 11, 23]. Pancreatic alpha cells exhibit very low levels of PDX1 and we herein found that downregulation of CK2 increases the nuclear fraction of PDX1 (Fig. 4a,b). It has been shown that PDX1 is capable of suppressing Gcg gene expression in beta cells by binding to the G1 box and, thus, displacing MafB and PAX6 [34]. To address the possibility that CK2 reduces Gcg expression via PDX1 in αTC1 cells (Fig. 4c), we performed a DNA pull-down assay. The loss of CK2α in αTC1 KO cells significantly increased the binding of PDX1 to the Gcg promoter (Fig. 4d). To further analyse the importance of the CK2-specific phosphorylation of PDX1 for pro-GCG expression, we overexpressed PDX1 WT or PDX1 T231A/S232A (Mut) in αTC1 cells and determined GCG expression. In fact, we detected lower promoter activity and protein expression of pro-GCG in PDX1 Mut-overexpressing cells (Fig. 4e,f and ESM Fig. 1). On the other hand, overexpression of PDX1 WT in αTC1 KO cells did not result in higher PDX1 levels when compared with PDX1 Mut-overexpressing αTC1 KO cells (Fig. 4g,h). Taken together, these results indicate that the loss of CK2 activity reduces GCG expression in a PDX1-dependent manner.

Effect of CK2 downregulation on insulin and GCG secretion ex vivo

Next, we examined whether CK2 inhibition also decreases GCG secretion ex vivo. For this, islets were isolated from C57BL/6J mice and exposed to CX-4945 or SGC-CK2-1 to determine the expression and secretion of insulin and GCG (Fig. 5a). In line with our in vitro findings, we found a markedly reduced expression of Gcg after CK2 inhibition (Fig. 5b). It is known that insulin affects GCG secretion in a paracrine manner [36]. Therefore, CK2-induced insulin secretion in islets may attenuate the secretory capacity of alpha cells. In line with our previous studies [33], we here demonstrated that the pharmacological inhibition of CK2 promoted Ins1 expression as well as insulin secretion (Fig. 5c,d). However, results from isolated islets exposed to either of the two inhibitors, with or without the insulin receptor blocker linsitinib, clearly showed that most of the decreased GCG secretion is due to CK2 inhibition in alpha cells and only a minor effect is due to the paracrine action of insulin (Fig. 5e). We additionally strengthened our findings by the generation of PI build from MIN6 (3500 cells) and αTC1 cells (1500 cells) (PI WT) or build from MIN6 (3500 cells) and αTC1 KO cells (1500 cells) (PI KO) (Fig. 5f,g). CK2 knockout did not affect the cellular composition of the PIs (Fig. 5h). As expected, in line with the results from treatment with inhibitors, PI KO exhibited a significantly lower GCG secretion when compared with PI WT (Fig. 5i).

Effects of CK2 downregulation in mice

To assess the influence of CK2 on GCG expression in vivo, we transplanted αTC1 WT or KO cells under the kidney capsule of C57BL/6J mice. Moreover, we used sham-transplanted mice as an additional control group. Fasting blood glucose levels and body weights were measured over 28 days twice a week (Fig. 6a). We did not observe any differences in the body weights between the groups (Fig. 6b,c). We measured significantly higher blood glucose levels after fasting in mice receiving αTC1 WT cells when compared with sham-transplanted mice or mice transplanted with αTC1 KO cells over the entire observation period (Fig. 6d). Accordingly, the AUC for glucose in the WT group was significantly increased when compared with that in the KO and sham groups (Fig. 6e). Based on the fasting-induced hyperglycaemia observed in the mice transplanted with αTC1 WT cells, we evaluated the effect of CK2-mediated altered GCG expression on glucose tolerance and clearance. The results of the IPGTT demonstrated higher blood glucose levels in mice receiving αTC1 WT cells (Fig. 6f,g). This is not surprising given the fact that these mice received additional alpha cells that are not under paracrine control of pancreatic islet cells, and, thus, gluconeogenesis is enhanced. Indeed, we measured elevated plasma GCG levels and lower insulin levels in mice receiving WT αTC1 cells when compared with mice receiving KO cells and the sham-transplanted group (Fig. 6h,i).

To study the effect of the reduced GCG expression after CK2 inhibition under physiological conditions, mice were treated with CX-4945 or DMSO (as control) for 3 days and plasma GCG and insulin levels were examined. As a key enzyme of gluconeogenesis, FBP1 expression in liver and kidney was analysed (Fig. 6j). As expected, we measured markedly lower plasma GCG levels and higher plasma insulin levels after CK2 inhibition (Fig. 6k,l). Moreover, we detected a diminished expression of FBP1 in liver and kidney samples of CX-4945-treated mice (Fig. 6m,n) and reduced plasma glucose levels of CX-4945-treated mice when compared with controls (Fig. 6o).

Rodent and human islets have differences in their cellular composition and intra-islet cell interactions [37]. To exclude that the observed effects of CK2 on GCG expression are specific for mice, we finally determined GCG secretion in islets from healthy human donors. For this, human isolated islets were treated with CX-4945 and SGC-CK2-1 for 24 h and subsequently GCG secretion was induced by low-glucose buffer (3 mmol/l glucose) complemented with GIP and alanine (Fig. 7a). In line with our results from αTC1 cell lines and murine islets, CK2 inhibition significantly reduced GCG secretion from human islets (Fig. 7b). Accordingly, the AUC of GCG secretion in CX-4945- and SGC-CK2-1-exposed human islets was significantly reduced compared with control islets (Fig. 7c). We additionally analysed insulin release from these islets. As expected, we detected only low levels of released insulin within all groups, and the release was not affected by 3 mmol/l of glucose (Fig. 7d). These findings indicate that CK2 also represses human GCG gene expression, resulting in a reduced GCG secretion.