The ubiquitin–proteasome system regulates the stability and activity of the glucose sensor...

Biochem. J. (2013) 456, 173–184 (Printed in Great Britain) doi:10.1042/BJ20130262 173

The ubiquitin–proteasome system regulates the stability and activity of theglucose sensor glucokinase in pancreatic β-cellsAnke HOFMEISTER-BRIX*, Sigurd LENZEN* and Simone BALTRUSCH*†1

*Institute of Clinical Biochemistry, Hannover Medical School, 30625 Hannover, Germany, and †Institute of Medical Biochemistry and Molecular Biology, University of Rostock, 18057Rostock, Germany

The ubiquitin–proteasome system is important to maintainpancreatic β-cell function. Inhibition of the proteasomesignificantly reduced glucose-induced insulin secretion. Keyregulators of the stimulus/secretion cascade seem to be affectedby protein misfolding if the proteasome is down-regulatedas recently reported in humans with Type 2 diabetes. Itremains unknown, however, whether the glucose sensor enzymeglucokinase is involved in this process. A direct interactionbetween glucokinase and ubiquitin could be shown in vivo byFRET, suggesting regulation of glucokinase by the proteasome.After proteasome inhibition glucokinase activity was significantlyreduced in MIN6 cells, whereas the protein content was increased,indicating protein misfolding. Enhancing the availability ofchaperones by cyclohexamide could induce refolding and

restored glucokinase activity. Glucokinase aggregation due toproteasome blocking with MG132, bortezomib, epoxomicin orlactacystin could be detected in MIN6 cells, primary β-cellsand hepatocytes using fluorescence-based assays. Glucokinaseaggresome formation proceeded microtubule-assisted and wasavoided by cyclohexamide. Thus the results of the present studyprovide support for glucokinase misfolding and aggregation incase of a diminished capacity of the ubiquitin–proteasome systemin pancreatic β-cells. In the Type 2 diabetic situation this couldcontribute to reduced glucose-induced insulin secretion.

Key words: aggresome, glucose-induced insulin secretion,glucokinase, pancreatic β-cell, protein misfolding, ubiquitin–proteasome system.

INTRODUCTION

The cellular protein abundance and turnover rate is controlled bythe translation rate and the UPS (ubiquitin–proteasome system).Under physiological conditions, the proteasome degradesubiquitinated proteins once their specific lifetime has beenreached [1]. In cases of oxidative and ER (endoplasmic reticulum)stress, the UPS eliminates misfolded proteins [1,2]. In pancreaticβ-cells the balance between protein synthesis, folding anddegradation is important to maintain proper glucose-inducedinsulin secretion [2]. Incubation of human, mouse and ratpancreatic islets, as well as MIN6 β-cells, with a proteasomeinhibitor resulted in a significantly reduced glucose-inducedinsulin secretion, whereas the insulin content remained unchanged[3–5]. Thus, in addition to insulin biosynthesis [3], regulationof proteins involved in the pathway of insulin secretion suchas the ATP-dependent potassium and voltage-dependent calciumchannels by the UPS has been discussed [2,5,6]. However,the effect of the UPS on other key regulators of the insulinsecretion machinery is far less understood, but of growinginterest [2].

Recently, it has been reported that in pancreatic islets ofhumans with Type 2 diabetes, the activity of the UPS isreduced compared with healthy individuals [7,8]. Using qPCR(quantitative real-time PCR) it was demonstrated that genes of the26S proteasome were down-regulated [7]. This is in agreementwith another recently published study reporting accumulationof ubiquitinated proteins in β-cells of humans with Type 2diabetes [8]. Furthermore, low expression and activity of theubiquitin C-terminal hydrolase L1 has been shown, indicating

reduced access of ubiquitinated proteins to the proteasome inhumans with Type 2 diabetes [8]. Thus both studies underline thatalterations in the UPS can contribute to the pathogenesis of Type 2diabetes.

The glucose-phosphorylating enzyme glucokinase catalysesnot only the first step of glycolysis in β-cells, but has theimportant role of the glucose sensor [9–13]. Knockdown ofglucokinase resulted in a loss of glucose-induced insulin secretion[14]. In pancreatic β-cells glucokinase is mainly regulated atthe post-translational level [15–22]. The enzyme is activatedby glucose and the bifunctional enzyme phosphofructo-2-kinase/fructose-2,6-bisphosphatase [16,18,19,22]. In vitro it hasbeen described that recombinant glucokinase is ubiquitinated,suggesting quality control of glucokinase by the proteasome[23]. Using a multiplexing strategy the stability of ∼8000 humanproteins has been investigated. This cell-based approach revealedfor the long-living protein GAPDH (glyceraldehyde-3-phosphatedehydrogenase; gene ID 2597) a lifetime index of 6.24. Forglucokinase (gene ID 2645) a significantly lower lifetime indexof 4.34 has been observed, indicating medium half-life andregulation by the proteasome [24]. Thus the aim of the presentstudy was to investigate the impact of the UPS on glucokinaseactivity in pancreatic β-cells.

MATERIALS AND METHODS

Materials

MG132 (Z-Leu-Leu-Leu-al) and the ProteoStat® Aggre-some Detection kit were from EnzoLife Sciences. CHX

Abbreviations used: CHX, cyclohexamide; Cy2, carbocyanine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTOC, microtubule-organizingcentre; NA, numerical aperture; qPCR, quantitative real-time PCR; UPS, ubiquitin–proteasome system.

1 To whom correspondence should be addressed (email [email protected]).

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174 A. Hofmeister-Brix, S. Lenzen and S. Baltrusch

(cyclohexamide), camptothecin and nocodazole were fromSigma–Aldrich. Epoxomicin was from Merck. Bortezomib wasfrom New England Biolabs. All primers, including randomhexamer primers, and chemicals for TaqMan assays were obtainedfrom Life Technologies. The RevertAidTM H Minus M-MuLVreverse transcriptase was from Fermentas. The GoTaq® Taqpolymerase was purchased from Promega, and dNTPs were fromGenecraft.

Plasmids

The cDNA of human β-cell glucokinase was subcloned in-frame(ApaI and BamHI restriction sites) in the pDendra2-C vector(Evrogen). Generation of pECFP–glucokinase has been describedpreviously [25]. ECFP was replaced by mCherry (Clontech)using AgeI and BspEI to generate mCherry–glucokinase. Tubulinfrom the pEYFP–Tub vector (Clontech) was subcloned into theDendra2-C vector using XhoI and BamHI to generate Dendra2–α-tubulin. pEGFP–C1-Ub (addgene plasmid 11928) and pEGFP–C1-UbKO.G76V (addgene plasmid 11932) were generated anddeposited by Dantuma et al. [26]. EGFP was replaced byEYFP using AgeI and BsrGI restriction sites to produce EYFP–ubiquitin and EYFP–ubiquitinK0,G76V. The pcDNA3.3-d2eGFPvector (addgene plasmid 26821) was deposited by Rossi and co-workers [27,28].

Cell culture, isolation of primary cells and treatment

MIN6 and COS cells were grown in DMEM (Dulbecco’s modifiedEagle’s medium; Biochrom) supplemented with 25 mmol/lglucose, 10% (v/v) FBS, 10 units/ml penicillin, 10 μg/mlstreptomycin and 2 mmol/l glutamine in a humidified atmosphereat 37 ◦C and 5% CO2. MIN6 cells were transfected with the vectorDNA by the use of jetPEI (Qbiogene). Stable clones were selectedthrough resistance against G418 (1200 μg/ml) and characterizedfurther for Dendra2–glucokinase by fluorescence microscopy.Cells were seeded and grown 48 h before experiments. NMRI andC57/BL6 mice used in the present study were housed at the centralanimal care facility of the faculties, and islet and hepatocyteisolation were approved by the state’s Animal Care Committee.Pancreatic islets were isolated from 11-week-old female NMRImice by collagenase digestion in bicarbonate-buffered Krebs–Ringer solution. β-Cells were obtained by dispersion in calcium-free Krebs–Ringer solution and kept in RPMI 1640 mediumsupplemented with 5 mmol/l glucose for 24 h. Primary hepato-cytes were isolated from C57/BL6 mice [25]. Isolated cells weresuspended in Williams’ medium E supplemented with 10 mmol/lglucose, 5% (v/v) FBS, 1×10− 4 mmol/l dexamethasone and1×10− 5 mmol/l insulin. Hepatocytes were seeded at a densityof 4×104 cells on glass coverslips and incubated for 24 h in a hu-midified atmosphere at 37 ◦C and 5% CO2. MG132 (10 μmol/l),epoxomicin (1 μmol/l), CHX (10 μg/ml) and bortezomib (10, 50and 100 nmol/l) were added to the culture medium for 3 or 12 h,and camptothecin (2.5, 5 and 10 μmol/l) for 36 h.

Glucokinase enzyme activity

MIN6 cells were seeded in 10-cm dishes at a density of 3×106

cells and treated as indicated. Glucokinase enzyme activity wasmeasured in an enzyme-coupled photometric assay [22]. Cellswere homogenized in PBS (pH 7.4) and the protein concentra-tion was quantified using a Bio-Rad protein assay. Enzymeactivity was measured spectrophotometrically at 1 mmol/l glucoseand was subtracted from the value obtained at 10 mmol/lglucose to compensate for cellular hexokinase activity.

Measurement of insulin secretion

MIN6 cells were seeded in six-well plates at a density of 3×105

cells and treated as indicated. Finally, cells were incubatedfor 1 h in bicarbonate-buffered Krebs–Ringer solution withoutglucose, supplemented with 0.1% BSA and thereafter stimulatedfor 1 h with 3 or 25 mmol/l glucose. Thereafter, 1 ml of theincubation buffer from each well was carefully harvested andgently centrifuged to remove detached cells (720 g for 5 min at22 ◦C). In these supernatants the secreted insulin was determinedby RIA against a rat insulin standard. The protein concentrationwas quantified using the Bradford protein assay.

MTT cell viability and caspase 3 activity assays

MIN6 cells were seeded in 96-well plates at a density of 20000cells and treated as indicated. Cell viability was then determinedusing a microplate-based MTT assay [29]. MIN6 cells wereseeded at a density of 50000 cells per well and caspase 3-positive cells were detected using the NucViewTM 488 Caspase-3assay kit (Biotium). The total number of cells was determinedby nuclear staining with 1 μmol/l Hoechst 33342 for 15 minand corresponding fluorescence images were taken automaticallyusing a HC 387/11-433(50)-517(40)-613(60) filter set (AHFAnalysentechnik) and quantified with a scanR/IX81 microscopesystem (Olympus) as described previously [30].

Western blot analyses

MIN6 cells and hepatocytes were seeded in 6-cm dishes ata density of 2.5×105 cells and treated as indicated. Finally,cells were homogenized by sonication in lysis buffer containing5 mmol/l Tris (pH 7.5), 5 mmol/l EDTA, 1 mmol/l PMSF, 1 %Triton X-100 and protease inhibitors and insoluble materialwas pelleted by centrifugation (7000 g for 10 min at 4 ◦C).Total cellular protein was fractioned by reducing SDS/PAGE(10% gel) and electroblotted on to PVDF membranes. Non-specific-binding sites of the membranes were blocked withOdyssey Blocking Buffer (Li-Cor Biosciences) for 30 min atroom temperature (22 ◦C). Blots were incubated with glucokinaseantibody [Santa Cruz Biotechnology; catalogue number sc-7908(diluted 1:200) or catalogue number sc-1980 (diluted 1:500)] forβ-cell and liver glucokinase respectively and GAPDH [Santa CruzBiotechnology; catalogue number sc-137179 (diluted 1:2000)]at 4 ◦C overnight, followed by incubation with the appropriateIRDye secondary antibodies (Li-Cor Biosciences) for 30 minat room temperature. Specific protein bands were visualizedin the Li-Cor Infrared Imaging System (Li-Cor Biosciences).Quantification of specific protein bands was performed usingOdyssey application software (Li-Cor Biosciences).

qPCR analysis

Total RNA was isolated from MIN6 or MIN6 Dendra-GKcells using the Qiagen RNeasy kit and glucokinase (GCK)gene expression was measured with TaqMan assays (humanGCK, Hs01564555_m1; mouse Gck, Mm00439129_m1). Thereactions were performed using the ViiA 7 real-time PCR system(Life Technologies) [31,32]. The housekeeping genes Actb (β-actin), G6pdx (glucose-6-phosphate dehydrogenase X-linked),Gapdh and Ppia (peptidylprolyl isomerase A) were used fornormalization. Data analysis was performed with qBasePLUS(Biogazelle) [31,32].


Ubiquitination of glucokinase 175

Immunocytochemistry

MIN6 cells and hepatocytes at a density of 80000 cells, andprimary β-cells from 20 islets were seeded on to glass coverslipsand treated as indicated. Thereafter cells were washed twicewith PBS and fixed overnight with 4% paraformaldehyde. Cellswere permeabilized for 30 min with 0.5 % Triton X-100 and3 mM EDTA in ProteoStat® aggresome assay buffer (pH 8) onice. Non-specific-binding sites were blocked with 1% BSAand 1% Triton X-100 in PBS for 20 min at room temperature.Cells were immunostained as described previously [30] witha goat anti-glucokinase antibody [Santa Cruz Biotechnology;catalogue number sc-1979 (diluted 1:100)] and the appropriateCy2 (carbocyanine)-labelled secondary antibody (diluted 1:200,Dianova) for 1 h at room temperature. Thereafter cells werestained with the aggresome detection reagent containing nuclearstaining (Hoechst 33342) and Proteostat® red dye. Cells weremounted with Mowiol/DABCO anti-photobleaching mountingmedia (Sigma). Finally, samples were analysed with an OlympusFluoview1000 confocal microscope system. For sequentialscanning the multi-line argon laser (488 nm) to excite Cy2,the diode laser (405 nm) to excite Hoechst 33342, and theDPSS (diode-pumped solid state) laser (559 nm) to excite theProteoStat® dye and an UPLSAPO 60×1.35 NA (numericalaperture) oil-immersion objective were used. Hoechst 33342 wasdetected at a 425–475 nm wavelength range together with Cy2at 500–545 nm. Light emitted by ProteoStat® dye was detected at575–675 nm. Image processing was done using FV10-ASWsoftware (Olympus).

Live-cell imaging

Analyses of MIN6 Dendra2–glucokinase cells and FRETexperiments were performed with a cellR/Olympus IX81 invertedmicroscope system (Olympus) equipped with a Cellcubator(Olympus) using 60% humidity, 37 ◦C and 5% CO2. Glass-bottomed dishes were fixed on the microscope stage and imageswere taken with an UPLSAPO 60×1.35 NA oil-immersionobjective (Olympus). ET 470/40 and 556/20 filter sets (AHF)were used to excite green and red Dendra2 respectively. Dendra2green and red emission were detected using a 510/30-630/100dual-band filter (AHF). An S360/40 filter (AHF) was usedfor photoconversion of Dendra2. Image processing was doneusing xcellence software (Olympus). The FRET set-up hasbeen described previously [19]. Sensitized emission-based FRETefficiency (FRETN) was calculated from the ECFP emission withexcitation at 436 nm, EYFP emission with excitation at 436 nmand EYFP emission with excitation at 500 nm, on the basis of thecalculation of Vanderklish et al. [33].

Data analyses

Data are expressed as means+−S.E.M. Statistical analyses wereperformed by ANOVA followed by Bonferroni’s test for multiplecomparisons or by Student’s t test using the Prism analysesprogram (Graphpad).

RESULTS

Effect of MG132 and CHX on glucose-induced insulin secretion andcell viability

MIN6 β-cells were responsive to a glucose stimulus, whereastreatment with the reversible proteasome inhibitor MG132, thetranslation inhibitor CHX or a combination of both for 12 h led

to a loss of insulin secretion at basal (Figure 1A) and stimulating(Figure 1B) glucose concentrations. Treatment with bortezomib,a proteasome inhibitor specific for the β5 proteasome subunit,significantly reduced both basal and glucose-induced insulinsecretion in a concentration-dependent manner. Cell viabilitydetermined using the MTT test was reduced only by 10 and 30%for bortezomib (50 nmol/l) and MG132 and/or CHX respectively(Figure 1C), and the number of caspase 3-positive MIN6 cellswas only increased 2-fold (Figure 1D). In contrast, camptothecin,with a comparable effect on insulin secretion (Figures 1A and1B), decreased cell viability by 50% (Figure 1C) and resulted ina 7-fold higher number of caspase 3-positive cells (Figure 1D).Therefore loss of cell viability could not be the major reason forthe loss of insulin secretion observed by proteasome inhibition.

Effect of MG132, epoxomicin, lactacystin and CHX on glucokinaseenzyme activity and protein content

Treatment of MIN6 cells with MG132 significantly decreasedthe activity of the glucose sensor enzyme glucokinase byapproximately 50% (Figure 2A). Although CHX had noeffect on glucokinase activity compared with the control, co-treatment with MG132 could counteract the reduction caused byMG132. In agreement with previous studies [17], glucokinase-overexpressing MIN6 Dendra2–glucokinase cells showed ahigher glucokinase activity compared with MIN6 cells alone(results not shown). Treatment with MG132 and/or CHX resultedin a significantly reduced activity compared with the control(Figure 2B). To test whether the reduction in glucokinase activitywas due to a reduced protein content, Western blot analyses wereperformed. Quantification of the typical line at ∼53 kDa showedthat MG132 treatment of MIN6 cells resulted in a significantincrease in the glucokinase protein content (Figure 2C). Treatmentof MIN6 cells for 12 h with more specific proteasome inhibitors,namely epoxomicin (1 μmol/l) or lactacystin (10 μmol/l) alsoresulted in a higher glucokinase protein content (results notshown). Glucokinase protein expression was reduced after 12 htreatment with the translation inhibitor CHX (Figure 2C).Simultaneous inhibition of the proteasome and translation resultedin a protein expression comparable with the control situation.To test whether long-living proteins are also influenced bytreatment with MG132 and/or CHX, the immunoreactivity ofGAPDH was quantified (Figure 2D). In contrast with the effecton glucokinase, a 12 h treatment with the inhibitors alone or incombination had no significant effect on GAPDH expressionin MIN6 cells. qPCR analyses revealed a down-regulation inendogenous GCK gene expression after treatment with MG132and/or CHX after 3 and 12 h by ∼40% and 60% respectively(Supplementary Figure S1A at http://www.biochemj.org/bj/456/bj4560173add.htm). MIN6 Dendra2–glucokinase cellsshowed a comparable response to MG132 and CHX with respectto Gck expression (Supplementary Figure S1B), whereas humanGCK mRNA under the control of the CMV (cytomegalovirus)promoter was solely significantly influenced by 12 h treatmentwith CHX (Supplementary Figure S1C), indicating that inhibitionof the translation has only an effect on transgene expression.

Aggregation of endogenous glucokinase after proteasomeinhibition

Glucokinase aggregation after inhibition of the proteasome wasfurther analysed by fluorescence microscopy. MIN6 cells weretreated with MG132 and/or CHX for 12 h and thereafter stainedfor glucokinase and the ProteoStat® dye specific for aggresomes


http://www.biochemj.org/bj/456/bj4560173add.htm



Figure 1 Treatment with MG132, bortezomib and/or CHX results in loss of insulin secretion in MIN6 cells

MIN6 cells (white bars) were treated with MG132 (10 μmol/l, black bars), CHX (10 μg/ml, grey bars), MG132 + CHX (grey striped bars) or bortezomib (10, 50 or 100 nmol/l, white cross-stripedbars) for 12 h, or camptothecin (2.5, 5 or 10 μmol/l, white lengthwise-striped bars) for 36 h. For insulin secretion cells were starved for 1 h (A and B) and thereafter stimulated with 3 mmol/l glucose(A) or 25 mmol/l glucose (B). Insulin secretion is expressed per insulin content per protein content. Cell viability was measured with the MTT assay (C) and caspase 3-positive cells were quantifiedusing an automated fluorescence microscopy approach (D). Results are means+−S.E.M. for three to ten experiments. **P < 0.01 and ***P < 0.001 compared with the control, ◦◦◦P < 0.001compared with MG132 (ANOVA/Bonferroni’s multiple comparison test).

[34]. In MIN6 cells, glucokinase was homogenously distributedin the cytoplasm (Figure 3A). Inhibition of degradation byMG132 resulted in small aggregates which co-localize with theaggresome detection marker (Figure 3B). The aggregates weredistributed in the cytoplasm with a somewhat higher densityin the perinuclear region. Treatment with CHX had nearly noeffect on glucokinase distribution (Figure 3C). Simultaneousinhibition of translation and degradation reduced cytoplasmicglucokinase aggregation (Figure 3D). Likewise, treatment withthe β5 proteasome-subunit-specific inhibitor bortezomib evokedformation of glucokinase aggregates in the cell cytoplasmwhich co-localized with the aggresome detection marker. Forquantification (Figure 3H), regions of interest were determined inthe cytoplasm. Proteasome inhibition increased the range of pixelintensity significantly representing aggregation of glucokinase,whereas co-treatment with CHX could significantly diminishthis aggregation. Aggregation of endogenous glucokinase couldbe confirmed in primary mouse β-cells (Figure 3F). Treatmentfor 12 h with MG132 resulted in glucokinase aggregates in thecytoplasm, which co-localized with the aggresome detectionmarker (Figure 3G).

Effect of MG132 and CHX on glucokinase in hepatocytes

Glucokinase expression in hepatocytes is significantly highercompared with pancreatic β-cells [35]. After incubation at highglucose, glucokinase showed a homogenous distribution in mouse

hepatocytes (Figure 4A). Inhibition of the proteasome evokeda strong aggregation of glucokinase in the cytoplasm withsignificant co-localization with the ProteoStat® dye (Figure 4B).Simultaneous inhibition of the translation could counteract thisaggregation (Figure 4C). Proteasome inhibition resulted in anincrease in glucokinase in relation to GAPDH expression inhepatocytes (Figure 4D), which could be partly diminished byco-treatment with CHX.

Interaction of glucokinase with ubiquitin

In vitro ubiquitination of glucokinase has been reported previouslyusing a rabbit reticulocyte lysate system [23]. In additionto wild-type conjugation-efficient EYFP–ubiquitin, a mutantconjugation-deficient EYFP–ubiquitinK0,G76V lacking all internallysine residues and the C-terminal glycine residue [26] wasused to investigate ubiquitination of glucokinase in vivo. EYFP–ubiquitin (Figure 5A) and EYFP–ubiquitinK0,G76V (Figure 5B) werevisible in the cytoplasm and the nucleus in MIN6 cells. EYFP–ubiquitin fluorescence was highest in the nucleus. In the cytoplasma punctate pattern was observed, partly co-localized withglucokinase. In contrast, the conjugation-deficient mutant showeda homogeneous expression pattern throughout the cytoplasm andthe nucleus. For FRET analyses, ECFP–glucokinase and EYFPas a control, EYFP–ubiquitin wild-type or mutant were co-overexpressed in COS cells. In ECFP–glucokinase and EYFP–ubiquitin co-overexpressing cells FRETN was 4.5-fold higher



Figure 2 Treatment with MG132 reduces glucokinase enzyme activity but increases glucokinase protein content in MIN6 cells

MIN6 cells (white bars) were treated with MG132 (10 μmol/l, black bars), CHX (10 μg/ml, grey bars) or MG132 + CHX (grey striped bars) for 12 h. Cell extracts were prepared from MIN6 cells (A,C and D) or MIN6 Dendra2–glucokinase cells (B) stably overexpressing glucokinase. Glucokinase enzyme activity (A and B) measured at 1 mmol/l glucose was subtracted from the value obtainedat 10 mmol/l glucose to exclude the cellular hexokinase activity. Cell extracts (C and D) were analysed by SDS/PAGE and immunoblotted using antibodies against glucokinase and GAPDH. Theglucokinase line (∼53 kDa) (C) and GAPDH line (∼30 kDa) (D) were quantified. Results are means+−S.E.M. for three to ten individual experiments. *P < 0.05, **P < 0.01 and ***P < 0.001(ANOVA/Bonferroni’s multiple comparison test).

than in the negative control (Figure 5C). In contrast, no interactionwas detected with the EYFP–ubiquitinK0,G76V mutant (Figure 5C).

Effect of proteasome inhibition on mCherry–glucokinase incomparison with d2EGFP in MIN6 cells

The EGFP has a long half-life and high protein stability,and showed homogenous distribution in mammalian cells aftertransfection [27,28]. Peptide sequences which are rich in proline,glutamic acid, serine and threonine residues, also called PESTsequences, are known to serve as a signal for protein degradation.Subcloning of a PEST sequence to the C-terminus of EGFPresulted in d2EGFP, an EGFP variant with a short half-life[27,28]. MIN6 cells were transiently transfected with mCherry–glucokinase, a red fluorescence glucokinase fusion protein andd2EGFP and analysed over 9 h (Figure 6A). The horizontalintensity profile demonstrated distribution of both fluorescenceproteins at 0, 3, 6 and 9 h (Figure 6B). MG132 inducedaggregation of mCherry–glucokinase (Figure 6C), as observed forendogenous glucokinase in MIN6 cells (Figure 3). AggregatedmCherry–glucokinase near the cell nucleus accumulated overtime, reflected by the strong increase in fluorescence at theaggregation point in the profile (Figure 6D). In contrast, despitethe short half-life, d2EGFP showed a homogenous distributionwithout aggregation (Figure 6C). The intensity profile wascomparable at 0, 3, 6 and 9 h (Figure 6D). Treatment ofMIN6 cells with epoxomicin (Supplementary Figures S2A andS2B at http://www.biochemj.org/bj/456/bj4560173add.htm) andbortezomib (Supplementary Figures S2C and S2D) resulted in thesame effect.

Aggregation of Dendra2–glucokinase in MIN6 cells

MIN6 cells, transiently transfected (Figures 7A–7C) or stablyoverexpressing a Dendra2 fusion protein with glucokinase (Fig-ures 7D–7L) were incubated with MG132 alone or in combinationwith CHX. No differences were detected in the distribution ofDendra2–glucokinase compared with endogenous glucokinase(Figure 3), mCherry–glucokinase (Figure 6), and betweentransiently transfected or stably overexpressing cells (Figures 7Aand 7D). The proteasome inhibitors MG132 (Figures 7B and7E), lactacystin (Figure 7G), epoxomicin (Figures 7H–7L) andbortezomib (Supplementary Figures S3B and S3C at http://www.biochemj.org/bj/456/bj4560173add.htm) evoked aggrega-tion of Dendra2–glucokinase in the cytoplasm with a higherlocalization around the nucleus, whereas co-treatment with CHXcould counteract this aggregation (Figures 7C and 7F). In the 3Dslice view the compactness of Dendra2–glucokinase aggregationis clearly visible (Figure 7I). Smaller aggregates arranged arounda dense core, resulting in a growing globular aggregate. Thehomogeneous distribution of Dendra2–glucokinase (Figure 7J)changed to small (Figure 7K) and globular (Figure 7L) aggregates6 and 8 h after treatment with epoxomicin respectively. Thefluorescent protein Dendra2 was homogeneously distributed inMIN6 cells (Supplementary Figure S3D) and did not aggregateafter 12 h treatment with MG132 (Supplementary Figure S3E)or bortezomib (Supplementary Figure S3F). Susceptibility ofthe tubulin network to proteasome inhibitors, because ofhyperacetylation of α-tubulin, has been reported previously [36].Dendra2–α-tubulin showed a comparable filamentous structure inMIN6 cells (Supplementary Figure 3G) as the endogenous protein[17]. After 12 h treatment with MG132 or bortezomib tubulin






Figure 3 Proteasome inhibition evokes cytoplasmic aggregation of glucokinase in β-cells, partly avoidable by co-treatment with CHX

MIN6 cells (A and H, white bar) were treated with MG132 (10 μmol/l; B and H, black bar), CHX (10 μg/ml; C and H, grey bar), MG132 + CHX (D and H, grey striped bar) or bortezomib (50 nmol/l;E and H, white cross-striped bar) for 12 h. Finally, cells were fixed and stained for glucokinase (green), for aggresomes (red) and for DNA (blue). Representative images are shown (A–E). Scalebar, 10 μm. For quantification (H) regions of interest were determined and the range of pixel intensity was measured as a marker of cellular aggregation. Results are means+−S.E.M. for three orfour individual experiments. ***P < 0.001 compared with the control, and #P < 0.05 and ###P < 0.001 compared with MG132 treatment (ANOVA/Bonferroni’s multiple comparison test). Primarymouse β-cells (F) were treated with MG132 (G) for 12 h. Finally, cells were fixed and stained for glucokinase (green), aggresomes (red) and DNA (blue). Images shown were obtained from z-stacksafter deconvolution and are representative of three individual experiments. Scale bar, 5 μm.

bundles and focal aggresomes were detectable (SupplementaryFigures S3H and S3I).

Live-cell imaging of MIN6 Dendra2–glucokinase cells aftertreatment with MG132

Using the special feature of Dendra2, irreversible photoconversion[37], the effect of the proteasome inhibitor MG132 on folded(red Dendra2) and newly synthesized Dendra2–glucokinase(green Dendra2) in living cells can be analysed over time.MIN6 Dendra2–glucokinase cells were treated with MG132 andfluorescence images of living cells were taken every hour afterinitial irreversible photoconversion of green into red Dendra2(Figure 8A). After 5 h of treatment small aggregates were visiblein the cytoplasm with a somewhat higher concentration near

the nucleus. After 9 h the small aggregates accumulated in theperinuclear region forming a dense globular aggregate. After 24 ha high amount of aggregated Dendra2–glucokinase was detectablearound the nucleus. Nocodazole, an inhibitor of microtubulepolymerization, prevented accumulation of the aggregates nearthe nucleus and after 12 h small aggregates were distributed inthe whole cytoplasm (Figure 8B).

DISCUSSION

In pancreatic β-cells the rate of transcription and translationof the glucose sensor glucokinase appears to be very stable[38,39]. Regulation of glucokinase enzyme activity occursmainly at the post-translational level [21] by interaction withthe bifunctional enzyme phosphofructo-2-kinase/fructose-2,6-



Figure 4 MG132 causes not only an increase in glucokinase protein, but also cytoplasmic aggregation of glucokinase in hepatocytes

Primary hepatocytes (A and D, white bar) were treated with MG132 (10 μmol/l; B and D, black bar), CHX (10 μg/ml; D, grey bar) or MG132 + CHX (C and D, grey striped bar) for 12 h. Finally, cellswere fixed and stained for glucokinase (green), aggresomes (red) and DNA (blue). Representative images from two individual experiments are shown (A–C). Scale bar, 10 μm. Cell extracts wereanalysed by SDS/PAGE and immunoblotted using antibodies against glucokinase and GAPDH. Glucokinase expression is shown in relation to GAPDH expression (D). Results are means+−S.E.M.for three individual experiments.

bisphosphatase [16,18,19,22], cytoplasmic compartmentation viabinding to insulin granules mediated through S-nitrosylation[15,20], to mitochondria [12], and association with tubulinfilaments [17]. However, another aspect of post-translationalregulation, namely glucokinase protein stability, remains anunexploited field in vivo. Unstructured regions and aminoacids which are correlated with protein instability were mostlypostulated from in vitro experiments. However, a global proteinstability profiling, which has been performed in mammaliancells indicates the need for cellular approaches to investigateprotein half-life regulation [24]. In the present study we coulddemonstrate ubiquitination of glucokinase in vivo by FRETanalyses, as previously demonstrated in vitro [23].

Thermal instability, structural changes and self-association ofglucokinase mutations causing maturity-onset diabetes of theyoung have been shown [40–45]. We investigated aggregation ofthe wild-type glucokinase protein using the computer algorithmTANGO [46]. This program predicted regions between Val181

and Leu185, and between Val302 and Asp311 regions with high β-sheet aggregation propensities. Additionally taking into accountthat the glucokinase protein has a high flexibility and can bepresent in at least five different conformations by undergoingextensive conformational changes [10,47,48], it becomes obvious

that glucokinase has high susceptibility to protein misfolding and,thus, requires a sufficient control by the UPS.

Two decades ago the half-life of glucokinase in rat hepatocyteshas been estimated to be 12.7 h [49]. In the same study the half-lifeof lactate dehydrogenase was 17 h, and the authors concluded thatnon-autophagic mechanisms are involved in cytoplasmic enzymeturnover [49]. Interestingly, the recently performed cellularproteome-scale protein-turnover analyses using the proteasomeinhibitor MG132 suggested that both proteins are regulated bythe proteasome and provided, in agreement with the resultsobtained by Kopitz et al. [49], a higher lifetime index forlactate dehydrogenase (gene ID 3945) than for glucokinase (4.80compared with 4.34) [24]. Treatment of MIN6 cells with theproteasome inhibitor MG132 for 3 h resulted in an increase inthe glucokinase protein by 50% [40]. Extending the incubationtime to 12 h, which corresponds to the calculated half-life ofthe enzyme, we observed a comparable increase in glucokinaseprotein. As a control, GAPDH immunoreactivity was taken.The content of this long-living protein (lifetime index 6.24)remained unchanged. Glucokinase gene expression decreased,most probably as an adaptive process, after incubation withMG132, but notably did not increase and, thus, cannot accountfor causing the higher glucokinase protein content. However,



Figure 5 In vivo detection of co-localization and interaction between glucokinase and ubiquitin

MIN6 cells were transiently transfected with ECFP–glucokinase and EYFP–ubiquitin wild-type (A) or the conjugation-deficient mutant EYFP–ubiquitinK0,G76V (B). Fluorescence images were taken inliving cells 2 days after transfection. In the merged images, ECFP is depicted in green, EYFP is depicted in red and co-localization is indicated in yellow. Sensitized emission-based FRET efficiencywas calculated in COS cells (C) transfected with ECFP–glucokinase and EYFP (white bar), ECFP–glucokinase and EYFP–ubiquitin wild-type (black bar), or EYFP–ubiquitinK0,G76V (white striped bar).Results are means+−S.E.M. for three individual experiments. ***P < 0.001 (ANOVA/Bonferroni’s multiple comparison test).

despite the high protein content we observed a 50% decreasein glucokinase activity, indicating formation of inactive misfoldedglucokinase in the absence of proteasome activity. In fact,incubation of MIN6 cells with MG132 and in addition with CHX,a compound inhibiting translation and increasing the availabilityof chaperones, restored glucokinase activity to a certain extent.

At the single-cell level we observed direct evidence for regula-tion of glucokinase by the UPS. Using a novel fluorescence-basedassay [34] it could be demonstrated in MIN6 cells, primary mouseβ-cells and hepatocytes that glucokinase is within aggresomesafter inhibition of the proteasome and that this aggregationcould be, at least in part, avoided by CHX through chaperone-mediated refolding. The same effect could be shown usingdifferent classes of proteasome inhibitors, namely the peptidealdehyde MG132, a reversible proteasome inhibitor, thepeptide epoxyketone epoxomicin and the naturally occurringlactacystin, both irreversible proteasome inhibitors interactingwith β subunits of the proteasome, and bortezomib, which has,with the β5 proteasome subunit, a specific target structure [50].Fluorescent fusion proteins of glucokinase were used in thepresent study to investigate the process of protein aggregation inliving MIN6 cells over time. We are aware that the glucokinaseprotein content in MIN6 cells after overexpression was somewhathigher than in primary β-cells and in the range of that of

hepatocytes. However, aggregation due to proteasome inhibitionresulted from transiently and stably overexpressed glucokinase inMIN6 cells in a manner comparable with that of the endogenousprotein.

The use of a fusion protein between glucokinase and thephotoconvertible protein Dendra2 [37] in a time series analysis,which allows discrimination between older and newly synthesizedprotein, argue in favour of microtubule-assisted aggresomeformation in β-cells. At the beginning of proteasome inhibition,small glucokinase-containing aggresomes were distributed in thecytoplasm, whereas later on they formed bigger aggregates andwere transported to a so-called MTOC (microtubule-organizingcentre) [51,52], forming a ‘ribbon-like’ huge aggresome near thenucleus [53] (Figure 8C). Consistently, blocking both microtubulepolymerization and proteasomal degradation prevented formationat the MTOC. Because unfolded polypeptides can attach tosuch MTOC-located aggresomes this is a dangerous knock-onprocess for the cell, which has been associated so far withneurodegenerative diseases, cancer and metabolic syndromes[53]. Impaired clearance of misfolded and ubiquitinated proteinsdue to reduced proteasome activity has been recently reportedin patients with Type 2 diabetes [7,8]. In agreement withprevious studies [3,4] we demonstrated that insulin secretionis significantly reduced after inhibition of the proteasome.


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Figure 6 MG132 evokes aggregation of mCherry–glucokinase, but not of the short half-life protein d2EGFP in MIN6 cells

MIN6 cells were transiently transfected with mCherry–glucokinase and d2EGFP. At 24 h after transfection medium without MG132 (A) or with MG132 (10 μmol/l, C) was added and fluorescence pictures were taken directly, and after 3, 6 and 9 h. The horizontalfluorescence intensity profiles (B and D) of mCherry–glucokinase (red) and d2EGFP (green) without (B) or with (D) MG132 illustrate the distribution at the red line in the corresponding images. Scale bar, 10 μm. Fluorescence images shown are representative ofthree individual experiments. FORTRAN values are used on the x-axis (e.g. 1.0e-002 = 0.01).

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Figure 7 Aggregation pattern of Dendra2–glucokinase after treatment with MG132, lactacystin or epoxomicin in MIN6 cells

MIN6 cells transiently transfected with Dendra2–glucokinase (A–C) or MIN6 Dendra2–glucokinase cells (D–H) were incubated with MG132 (10 μmol/l; B and E), MG132 + CHX (C and F),lactacystin (10 μmol/l; G) or epoxomicin (1 μmol/l; H–L) for 12 h. (I) 3D distribution pattern of cytoplasmic aggregated Dendra2–glucokinase after treatment with epoxomicin. (J–L) MIN6Dendra2–glucokinase cells were treated with epoxomicin and fluorescence images of the same cell were taken directly (J), and 6 (K) and 8 h (L) thereafter. Fluorescence images shown arerepresentative of two to four individual experiments.

Despite its protective effect against MG132-induced glucokinaseaggregation CHX could not restore insulin secretion, because itinterrupts translation, and thus, biosynthesis of insulin and otherkey proteins of β-cells. An effect of MG132 on cell viabilitywas detectable and has been described previously [3,4], but at theconcentration of 10 μmol/l used the number of apoptotic cells wasnegligible compared with that of the cytotoxic quinoline alkaloidcamptothecin, which had in the present study a comparable effecton insulin secretion. In addition, bortezomib, at a concentrationof 10 nmol/l, caused a significant aggregation of glucokinase anda reduction in glucose-induced insulin secretion by 80%, but hadvirtually no effect on cell viability. This clearly indicates that

beyond autophagy [2,54], the UPS is essential for regulating keyfactors of the stimulus/secretion cascade in pancreatic β-cells.

In conclusion, we demonstrated that glucokinase is regulated bythe UPS to avoid misfolding and reduced activity of the enzyme.In addition, our fluorescence-based single-cell analyses suggest amicrotubule-assisted aggresome formation of misfolded proteinsin β-cells. This helps to understand the pathogenesis of Type 2diabetes, where a reduced proteasome activity has been reported[7,8]. Inclusion of glucokinase in aggresomes in β-cells of patientswith Type 2 diabetes could impair glucose recognition and, thuscontribute, at least in part, to a reduced glucose-induced insulinsecretion.



Figure 8 Time-resolved description of Dendra2–glucokinase aggresome formation after treatment with MG132

(A) MIN6 Dendra2–glucokinase cells were treated with MG132 (10 μmol/l). Directly after the addition of the proteasome inhibitor MG132, part of the green Dendra2–glucokinase was photoconvertedinto the stable red form. Representative fluorescence images are shown 5, 7, 9, 12 and 24 h after photoconversion. (B) MIN6 Dendra2–glucokinase cells were treated with MG132 (10 μmol/l) andnocodazole (10 μg/ml) for 12 h. Images shown are representative of two individual experiments. (C) The aggregation process of glucokinase starting in the cell periphery and resulting in aggresomesclose to the nucleus is depicted in the scheme.

AUTHOR CONTRIBUTION

Anke Hofmeister-Brix performed experiments, analysed and interpreted data, and wrote thepaper. Sigurd Lenzen discussed the data and revised the paper. Simon Baltrusch designedand supervised the study, performed experiments, analysed and interpreted data, andwrote the paper.

ACKNOWLEDGEMENTS

We thank Jasmin Kresse and Rica Waterstradt for technical assistance. We also thank DrOrtwin Naujok for support with real-time PCR analyses.

FUNDING

This work was supported by the Innovative Medicines Initiative Joint Undertaking [grantnumber 155005 (IMIDIA)], resources of which are composed of financial contributionfrom the European Union’s Seventh Framework Programme [grant number FP7/2007-2013] and EFPIA (European Federation of Pharmaceutical Industries and Associations)companies.

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Received 20 February 2013/22 August 2013; accepted 12 September 2013Published as BJ Immediate Publication 12 September 2013, doi:10.1042/BJ20130262


Biochem. J. (2013) 456, 173–184 (Printed in Great Britain) doi:10.1042/BJ20130262

SUPPLEMENTARY ONLINE DATAThe ubiquitin–proteasome system regulates the stability and activity of theglucose sensor glucokinase in pancreatic β-cellsAnke HOFMEISTER-BRIX*, Sigurd LENZEN* and Simone BALTRUSCH*†1

*Institute of Clinical Biochemistry, Hannover Medical School, 30625 Hannover, Germany, and †Institute of Medical Biochemistry and Molecular Biology, University of Rostock, 18057Rostock, Germany

See the following pages for Supplementary Figures S1–S3.

1 To whom correspondence should be addressed (email [email protected]).


A. Hofmeister-Brix, S. Lenzen and S. Baltrusch

Figure S1 Down-regulation of endogenous glucokinase gene expressionafter incubation with MG132 in MIN6 cells

(A) MIN6 cells (white bars) were treated with MG132 (10 μmol/l, black bars), CHX (10 μg/ml,grey bars) or MG132 + CHX (grey striped bars) for 3 or 12 h. Finally, total RNA was isolatedand expression of the mouse Gck gene (TaqMan assay Mm00439129_m1) was analysed.(B and C) MIN6 Dendra2–glucokinase cells (white bars) were treated with MG132 (blackbars), CHX (grey bars) or MG132 + CHX (grey striped bars) for 12 h. Finally, total RNA wasisolated and the expression of the mouse Gck gene (B) and the overexpressed human GCKgene (C) was analysed. Relative expression levels normalized to housekeeping genes Actb,G6pdx, Gapdh and Ppia are shown. Results are means+−S.E.M. for three to six experiments.*P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control, #P < 0.05 comparedwith 3 h (ANOVA/Bonferroni’s multiple comparison test).


UbiquitinationofglucokinaseFigure S2 Epoxomicin and bortezomib evoke aggregation of mCherry–glucokinase, but not of the short half-life protein d2EGFP in MIN6 cells

MIN6 cells were transiently transfected with mCherry–glucokinase and d2EGFP. At 24 h after transfection, medium with epoxomicin (1 μmol/l, A) or with bortezomib (50 nmol/l, C) was added and fluorescence pictures were taken directly, and after 3, 6 and 9 h.The horizontal fluorescence intensity profiles (B and D) of mCherry–glucokinase (red) and d2EGFP (green) with epoxomicin (B) or bortezomib (D) illustrate the distribution at the red line in the corresponding images. Scale bar, 10 μm. Fluorescence imagesshown are representative of two individual experiments. FORTRAN values are used on the x-axis (e.g. 1.0e − 002 = 0.01).

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A. Hofmeister-Brix, S. Lenzen and S. Baltrusch

Figure S3 MG132 and bortezomib evoke aggregation of Dendra2–glucokinase or Dendra2–tubulin but not of Dendra2 in MIN6 cells

MIN6 Dendra2–GK cells (A–C) and MIN6 cells transiently transfected with Dendra2 (D–F), Dendra2–tubulin (G–I) were incubated with bortezomib (10 nmol/l, B) or bortezomib (50 nmol/l, C, Fand I) or MG132 (10 μmol/l, E and H), for 12 h. Scale bar, 10 μm. Fluorescence images shown are representative of two individual experiments.

Received 20 February 2013/22 August 2013; accepted 12 September 2013Published as BJ Immediate Publication 12 September 2013, doi:10.1042/BJ20130262


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