Correspondence: Per M. Thorsby, PhD, Hormone Laboratory, Oslo University Hospital, Aker, Oslo 0424, Norway. E-mail: p.m.thorsby@medisin.uio.no

(Received 21 August 2013 ; accepted 23 February 2014 )

ORIGINAL ARTICLE

Tmem27 is upregulated by vitamin D in INS-1 cells and its serum concentrations are low in patients with autoimmune diabetes

MILAIM PEPAJ , NINA GJERLAUGSEN , KARI JULIEN & PER M. THORSBY

Hormone Laboratory , Department of Medical Biochemistry , Oslo University Hospital , Oslo , Norway

Abstract Transmembrane protein 27 (Tmem27), which is expressed in pancreatic β -cells, plays an important role in insulin secretion and pancreatic β -cell proliferation. Analysis of the INS-1 cell proteome using stable isotope labeling by amino acids in cell culture (SILAC) in combination with LC-MS identifi ed Tmem27 as the one of most robustly (up to seven-fold) upregulated proteins after treatment with the active metabolite of vitamin D, 1,25-(OH) 2 D 3 . Furthermore, we report that Tmem27 which is cleaved and released from, i.e. pancreatic β -cells, is present in human serum and its levels are signifi cantly lower in subjects with autoimmune diabetes as compared to healthy individuals (13% of the levels). Additionally, Tmem27 cor-related positively (0.70) with C-peptide serum levels in healthy subjects. Our data indicate that Tmem27 could be of potential value as a serum marker for the pathogenesis of diabetes and as such may warrant the development of measure-ment methods with lower limit of detection for its further validation.

Key Words: Tmem27 , Diabetes , pancreatic β -cell , serum , vitamin D

Introduction

Experimental evidence indicates that vitamin D may play a benefi cial role in pancreatic β -cell function. Several studies have suggested that vitamin D may have a direct protective effect on β -cell function mediated by binding of the circulating active form, 1,25-Dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ), to the vitamin D receptor, which is highly expressed in beta-cells [1 – 3]. Alternatively, activation of the circulating metabolite 25-Hydroxyvitamin D 3 (25-(OH) 2 D 3 ), may occur within the β -cell by the 1 α -hydroxylase enzyme, which is expressed in β -cells [4]. According to recent studies, gene expression [5] and protein synthesis in β -cells [6] appear to be signifi cantly altered by vitamin D defi ciency and sub-sequent 1,25-(OH) 2 D 3 treatment. However, the use of low resolution techniques in the latter precluded identifi cation of any of these proteins.

Transmembrane protein 27 (Tmem27), a 46 kDa glycoprotein, is expressed in pancreatic β -cells [7 – 9] and in kidney [10] Tmem27 in pancreatic β -cells is identifi ed as a target of hepatocyte nuclear factor- α [7,8,11]. Studies with in vitro cell culture and transgenic mice revealed that Tmem27 plays an important role in insulin secretion and pancreatic

β -cell proliferation [7 – 9]. Overexpression of Tmem27 in INS-1 cells and transgenic mice resulted in signifi cant increase of insulin secretion and led to the promotion of SNARE (soluble N-ethyl-maleimide-sensitive factor attachment receptor) complex forma-tion [7,11]. The mechanism of how Tmem27 is involved in insulin secretion was derived from the discovery of the binding of Tmem27 to SNARE complex via interaction with the protein snapin.

Moreover, Akpinar et al. [8] showed that the abundance of Tmem27 protein in β -cells is regulated by ectodomain cleavage products, a 25 kDa N-terminal part that is released into the extracellular space, and a 22 kDa C-terminal fragment remaining in the membrane that is rapidly degraded. The same study also showed that the 25 kDa N-terminal extra-cellular domain is cleaved and shed from the plasma membrane of pancreatic β -cells in a constitutive manner.

This work was undertaken to determine the effects of the two vitamin D metabolites, 1,25-(OH) 2 D 3 and 25-(OH) 2 D 3 , on the proteome of INS-1 cells. To this aim, stable isotope labeling by amino acids in cell culture (SILAC) [12] in combination with two-dimensional liquid chromatography-tandem mass

Scandinavian Journal of Clinical & Laboratory Investigation, 2014; 74: 358–365

ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa HealthcareDOI: 10.3109/00365513.2014.898322

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Tmem27 as biomarker 359

spectrometry (2D LC-MS/MS) was used to identify proteins differentially regulated by the two metabo-lites. Over 5500 proteins were detected in these anal-yses and up to 200 proteins were found differentially abundant. Of these, Tmem27, whose expression was modulated by 1,25-(OH) 2 D 3 , was detected in whole cell lysates and in conditioned media. Based on our fi ndings from conditioned media, in combination with its reported release into extracellular space by pancreatic β -cells [9], we next aimed to detect circu-lating Tmem27 in human serum. Finally, we sought to determine whether Tmem27 serum levels were different in subjects with autoimmune diabetes com-pared to healthy individuals.

Materials and methods

Chemicals and materials

All chemicals, solvents and columns were purchased from Sigma (St Louis, MO, USA) unless noted otherwise. Sequencing grade trypsin was purchased from Promega (Madison, WI, USA) and Amicon Ultra centrifugal fi lters with a 3 kDa molecular weight cut-off were purchased from Millipore (Bedford, MA, USA).

Subjects

The autoimmune diabetes group consisted of 21 anonymized subjects from our routine lab (Hormone Laboratory, Oslo University Hospital, Aker, Nor-way). The inclusion criteria were; positive for at least one islet autoantibody against GAD65, IA-2 or ZnT8 and C-peptide values below 350 pmol/L (median � 162 pmol/L and IQR 71 – 216 pmol/L). Healthy controls were anonymous samples from the laboratory staff with C-peptide values (median � 612 pmol/L, IQR 542 – 807 pmol/L). The National ethics committee has approved the use of anonymous samples from the routine in the Hormone Laboratory in evaluation of laboratory methods.

Assays

Analyses of C-peptide and auto antibodies were per-formed with commercially available standardized methods at the Hormone Laboratory. Serum levels of C-peptide were determined using DELFIA kits from PerkinElmer Life Sciences, Wallac Oy, Turku, Finland.

Anti-GAD, anti-IA2 and anti ZnT8 were ana-lyzed using an in-house immunoprecipitation radio-ligand assay. Serum levels of Tmem27 were analyzed by ELISA in duplicate (Cusabio, Wuhan, P. R. China). This assay employs the quantitative sand-wich enzyme immunoassay technique with a detec-tion range of 2.4 – 32 pmol/L (60 – 800 pg/mL) and a

Lower Limit of Detection (LOD) at 0.64 pmol/L (16 pg/mL). Sample preparation and analysis were performed according to the manufacturer ’ s assay procedure. The Anthos 2010 micro plate reader (ASYS Hitech GmbH, Cambridge, UK) was used to measure the absorbance at 450 nm.

Cell culture and SILAC

Rat insulinoma INS-1 cells were grown in a RPMI1640 medium supplemented with 10% fetal bovine serum at 37 ° C, under 5% CO 2 and humidi-fi ed atmosphere. For SILAC experiments, the RPMI 1640 Flex medium (Invitrogen), which is formulated without glucose, lysine, arginine and glutamine, was supplemented with 10% dialyzed fetal bovine serum (FBS), 11.1 mM glucose and 2 mM Glutamine. Next, the medium was supplemented with 1.15 mM arginine and 0.27 mM Lysine (Invitrogen) to pre-pare the ‘ light ’ medium, and with 13 C6 arginine and 13 C6 Lysine (Invitrogen) in the same concentrations for ‘ heavy ’ medium. Cells were grown for 3 weeks in either ‘ light ’ or ‘ heavy ’ SILAC media for complete amino acid incorporation. For the vitamin D metab-olite treatment experiments, the INS-1 cells were pre-treated with either 10 nM 1,25-(OH) 2 D 3 , 25-(OH) 2 D 3 , or vehicle (0.001% ethanol) for 48 h in SILAC media containing FBS. Cells were then washed twice with PBS before incubation with serum-free SILAC media, containing either 10 nM 1,25-(OH) 2 D 3 , 10 nM 25-(OH) 2 D 3 or vehicle alone, for 24 and 48 h.

Sample preparation and protein digestion

Secretome. After the fi nal incubation, conditioned media from each of the treated and control cultures were pooled (1:1 v/v ratio) and centrifuged at 20,000 rpm to remove cell debris. Paired samples were con-centrated using Amicon 3 kDa centrifugal fi lter units (Millipore Corporation, Billerica, MA, USA) fol-lowed by a buffer exchange against 0.1 M ammo-nium bicarbonate (pH 7.8) in the same fi lter unit. Proteins were then reduced using a denaturing buffer (8 M urea in 0.1 M ammonium bicarbonate and 5 mM DTT) for 1 h at 37 ° C, followed by alkylation of free sulfhydryl groups with 15 mM iodoacetamide at 37 ° C for 45 min in the dark. Reduced and alky-lated samples were then diluted with 50 mM ammo-nium bicarbonate (pH 7.8) before digestion with trypsin at 37 ° C overnight.

Cell lysate. Cells from each of the treated and control cultures were pooled and washed twice in PBS. Paired samples were then resuspended in 500 μ L lysis buffer (7 M urea, 2 M thiourea and a mixture of protease inhibitors [HALT EDTA-free protease inhibitor cocktail, Pierce, Thermo]) and lysed on ice

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360 M. Pepaj et al.

for 15 min. Cell debris was spun down by centrifuga-tion at 14,000 rpm for 15 min and the supernatant was transferred into new tubes. Samples were reduced and alkylated as outlined above (see secretome) before digestion. LysC (Pierce Thermo) was added and the samples were incubated for 6 h at 37 ° C. The sample was then diluted four times with 50 mM ammonium bicarbonate (pH 7.8) and trypsin was added. Incubation was carried out overnight at 37 ° C.

High pH reversed phase liquid chromatography fractionation

The tryptic digests derived from cell lysate- and secretome samples were fractionated using high pH reversed phase chromatography as previously described [13]. In short, high pH fractionation was performed on 4.6 � 250 mm C18 ACE column (Teknolab, Oslo, Norway). The fractionation was performed at a fl ow rate of 0.8 mL/min using a Dionex Ultimate 3000 LC analytical system con-nected to an UV detector (Dionex, Thermo Scien-tifi c) and a Gilson FC 203B fraction collector (Nerliens Mesanzky, Oslo, Norway). The mobile phases consisted of 10 mM ammonium formate (pH 10) as solvent (A) and 10 mM ammonium formate and 90% acetonitrile as solvent (B). Sample separa-tion was accomplished using the following linear gra-dient: From 0 – 5% B in 10 min, from 5 – 35% B in 60 min, from 35 – 70% B in 15 min, and held at 70% B for an additional 10 min. Sixty fractions were col-lected along with the LC separation and were con-catenated into 15 fractions [13]. The samples were then dried in vacuo , reconstituted in 1% formic acid and stored at � 20 ° C until LC-MS/MS analysis.

LC-MS/MS

The reconstituted peptide fractions were separated on a Discovery Bio Wide Pore C18 (1.0 � 250 mm) using a Dionex Ultimate 3000 micro LC system con-nected to a LTQ-Orbitrap XL hybrid mass spec-trometer (ThermoElectron, Bremen, Germany). The analytical separation was run for 180 min using a gradient of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The gra-dient was as follows: 3 – 25% eluent B in 165 min and 25 – 60% B in 15 min followed by 60% B in 10 min. All separations were performed at a fl ow rate of 40 μ L/min and a column temperature at 45 ° C. The mass spectrometer was operated in positive mode with a spray voltage set at 4.0 kV and the heated capillary temperature was kept at 195 ° C. The LTQ-Orbitrap XL was operated using Xcalibur 2.0.7 (Thermo Scientifi c) in data-dependent mode in which one cycle of experiments consisted of one full-MS survey scan using the Orbitrap mass ana-lyzer and subsequently fi ve sequential MS/MS events

of the most intense peaks using collision-induced dissociation (CID) in the LTQ. The MS survey scans were performed on the high resolution Orbitrap (R � 30 000) with an m/z range of 350 – 2000, and a dynamic exclusion of 60 s was applied to avoid repeated analyses. Precursor ions with charge 1 or unassigned charge were rejected and the precursor ion isolation window was set to 3 m/z units.

SILAC protein identifi cation and quantifi cation

Proteome Discoverer computational proteomics platform (version 1.3, Thermo Scientifi c) with default settings was used to identify and quantify proteins. In brief, SILAC raw data fi les were searched against the rat Swiss-Prot database using the SEQUEST search engine with the precursor and fragment mass tolerances set to 10 ppm and 0.8 Da, respectively. Parameters for modifi cation included a static cysteine modifi cation of � 57.02 Da and the following vari-able modifi cations: � 15.99 Da for oxidized Met, and � 6.02 Da for stable isotope labeled Arg and Lys. For all experiments only unique peptides were con-sidered for protein quantifi cation. To determine the average change in protein abundance after treatment (SILAC ratios), relative peak intensities of multiple isotopically distinct peptides from each protein were used. The SILAC ratios were calculated as ratios of the areas of the monoisotopic peaks of the unlabelled (treated) versus labelled (control) peptides. Peptide and protein false discovery rate was set to 1% using default fi lters.

Statistical analysis

Statistical analysis was performed using MedCalc statistical software (MedCalc, Ostend, Belgium). Data are presented as median and interquartile range (IQR). For comparisons of Tmem27 concentrations in autoimmune diabetes and controls, we used a two-sided Mann-Whitney rank sum test. The Spear-man correlation test was used for correlation analysis. Differences and correlations were considered to be signifi cant at p � 0.05.

Results

The effect of 1,25-(OH) 2 D 3 on the Tmem27 expression

The INS-1 cell proteome was characterized for each culture condition, and SILAC was used to identify proteins differentially regulated by the two vitamin D metabolites in cell lysates and in conditioned media. Over 5500 proteins were detected in these analyses and up to 200 proteins were found differ-entially abundant (data not shown). Of these, Tmem27 was identifi ed as the one of most robustly up-regulated proteins after treatment with 1,25-(OH) 2 D 3

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Tmem27 as biomarker 361

Table I. Peptides used for identifi cation and quantifi cation of Tmem27 in cell lysates and conditioned media.

Lysate Conditioned media

1,25-(OH) 2 D 3 (24 h) GGHINDGFLTEDER AYVWDTDEEYLFRAYVWDTDEEYLFRVSFWFVVTDPLKINSAFFLDDHTLEFLKKGPPGVEDAEDKGPPGVEDAEDK

1,25-(OH) 2 D 3 (48 h) GGHINDGFLTEDER AYVWDTDEEYLFRCENIITIENGIPCDPLDMKVSFWFVVTDPLKINSAFFLDDHTLEFLKKGPPGVEDAEDKGPPGVEDAEDK

25-(OH) 2 D 3 (24 h) ND ND25-(OH) 2 D 3 (48 h) INSAFFLDDHTLEFLK AYVWDTDEEYLFR

AYVWDTDEEYLFR

ND: Tmem27 was not detected.

Figure 1. Quantitative analysis of Tmem27 in INS-1 cells after treatment with either 1,25-(OH) 2 D 3 or 25-(OH) 2 D 3 . LC-MS/MS analyses of Tmem27 were conducted on INS-1 cell lysates and conditioned media incubated for 24 and 48 h with either 10 nM 1,25-(OH) 2 D 3 or 10 nM 25-(OH) 2 D 3 . The SILAC ratio for Tmem27 represents the relative expression difference between treated and control cells. ND, Tmem27 was not detected; CM, conditioned media.

for 24 and 48 h. The peptides used to identify and quantify Tmem27 for the different experiments are summarized in Table I. For the 1,25-(OH) 2 D 3 treatment, Tmem27 extracted from cell lysates was identifi ed with fi ve peptides (quantifi ed with 3 pep-tides) derived from the 46 kDa full length protein (Table I). Next, Tmem27 secreted into conditioned media was identifi ed and quantifi ed with one peptide derived solely from the 25 kDa shed N-terminal extracellular fragment (Table I). The fold changes of Tmem27 in cell lysates and conditioned media shows that 25-(OH) 2 D 3 had no impact on the regulation of the Tmem27, SILAC ratio 1:1 at 48 h (Figures 1 and 2B). Treatment of INS-1 cells with 1,25-(OH) 2 D 3 , on the other hand, resulted in up to 7-fold

up-regulation of this protein in both cell lysates and conditioned media after 48 h (Figures 1 and 2A). In Figure 2, we show a mass spectrum obtained for a peptide unique to Tmem27 (INSAFFLDDHTLE-FLK) extracted from cell lysates upon 48 h of 1,25-(OH) 2 D 3 and 25-(OH) 2 D 3 treatment, respectively. The mass spectrum shows a SILAC peptide doublet with peaks at m/z 637.33 and 639.33 corresponding to unlabelled (treated) and labelled (control) pep-tides, respectively. The unlabelled and labelled pep-tides are in a ratio of 7:1 for 1,25-(OH) 2 D 3 treatment (spectrum A) and 1:1 for 25-(OH) 2 D 3 treatment (spectrum B). Consistently, the ratio of peak intensi-ties of unlabelled versus labelled peptides refl ects differences in protein abundance.

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637,0 637,5 638,0 638,5 639,0 639,5 640,0 640,5 641,0m/z

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638,3358639,6739639,3392

640,0077638,6699 640,3412

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638,3349 640,6763

641,0104638,6712 639,0032

(A)

(B)

Figure 2. LC-MS/MS quantifi cation of Tmem27 using INSAFFLDDHTLEFLK peptide fragment. (A) MS spectrum showing a SILAC peptide pair from Tmem27 after treatment with 1,25-(OH) 2 D 3 for 48 h. The peaks at m/z 637.33 and 639.33 correspond to unlabelled (treated) and labelled (control) peptides, exhibiting a SILAC ratio of 7:1. (B) MS spectrum showing the same peptide pair as shown in (A) after treatment with 25-(OH) 2 D 3 for 48 h in a SILAC ratio of 1:1. SILAC ratio refers to fold changes in protein expression, as described in SILAC protein identifi cation and quantifi cation .

ELISA of Tmem27 in serum samples

Based on the in vitro results from conditioned media and in combination with its reported high degree of pancreatic β -cell selectivity, we wondered whether shed circulating Tmem27 (25 kDa N-ter-minal fragment) could be detected in human serum samples. Human serum samples from 21 healthy individuals were analyzed using a commercially available ELISA kit and Tmem27 was detected in all individuals with levels ranging from 1.76 – 18.4 pmol/L (Figure 3). We next sought to determine whether Tmem27 serum levels were affected in

subjects with autoimmune diabetes (21 subjects not matched for age or gender with controls), and found that Tmem27 levels were signifi cantly lower in this group compared with controls (median � 0.64 pmol/L versus median � 4.84 pmol/L, p � 0.0001, Figure 3). Notably, 14 out of 21 serum samples from the autoimmune diabetes group showed Tmem27 levels below 0.64 pmol/L , which is the LOD of the assay used. Furthermore, because Tmem27 has been impli-cated in insulin exocytosis process, we next explored whether there is a correlation between Tmem27 and C-peptide serum levels in the control group.

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Tmem27 as biomarker 363

Figure 3. Serum levels of Tmem27 in autoimmune diabetes versus healthy controls. Box and-whisker plot showing median and interquartile range (IQR). The median in n � 21 autoimmune diabetes subjects is 0.64 pmol/L with IQR of 0.64 – 1,16 pmol/L and the median in n � 21 control subjects is 4.84 pmol/L and IQR is 2.71 – 7.18 pmol/L. A Mann-Whitney U-test of the groups resulted in * p � 0.0001 for differences between autoimmune diabetes and controls.

Figure 4. Correlation of Tmem27 in healthy controls. Tmem27 correlation with C-peptide (Spearman rho � 0.70, p � 0.0004, n � 21).

We found a signifi cant positive correlation between Tmem27 and C-peptide levels (Spearman rho � 0.70, p � 0.0004, Figure 4).

Discussion

Emerging evidence indicates that 1,25-(OH) 2 D 3 may protect β -cells against various types of stress by affecting the expression of a number of different genes [1 – 3,5]. Accordingly, protein synthesis appears to be signifi cantly altered by 1,25-(OH) 2 D 3 treat-ment [6]. However, the use of low resolution tech-niques have hereto precluded identifi cation of any of these proteins. In the present study, using a combina-tion of SILAC and 2D LC-MS/MS, we were able to quantify proteins specifi cally regulated by the two vitamin D metabolites. SILAC is a high-throughput, quantitative proteomic technique which can be used

to examine proteome changes and is ideal for comparing relative protein levels between different cell states [12]. The cell states are differentiated by growing each cell population in media containing different stable isotope labelled amino acids that become incorporated into newly synthesized pro-teins. In this study we used 13 C6 Lys and 13 C6 Arg to ensure that all tryptic peptides contain isotopically labelled amino acids that can be distinguished from their sister peptides derived from a different cell state. Careful examinations of the differentially regu-lated proteins identifi ed Tmem27 as the one the most profoundly increased in expression in the presence of 1,25-(OH) 2 D 3 , but not 25-(OH) 2 D 3 . Tmem27, a cell surface N-glycoprotein, is expressed in pancreas and kidney. In pancreas, Tmem27 is specifi cally expressed in β -cells [7 – 9]. It has been shown that the 46 kDa full length (FL) protein is constitutively cleaved by the Bace2 protease to a 25 kDa N-terminal shed fragment that is released into the extracellular space, and a 22 kDa C-terminal membrane bound fragment that is further processed by γ -secretase [14].

In the present study, Tmem27 was detected in whole cell lysates and in conditioned media of INS-1 cells. Tmem27 extracted from whole cell lysates was identifi ed with fi ve peptides (quantifi ed with three peptides) derived from the FL protein, whereas Tmem27 secreted into conditioned media was iden-tifi ed and quantifi ed with one peptide derived solely from the shed extracellular fragment. These fi ndings are in accordance with a previous study [15], where only a fi fth of the total lysate Tmem27 was found at the cell surface, a critical compartment for Tmem27 and Bace2 interaction leading to shed Tmem27 in the supernatant. Nevertheless, increase of Tmem27 protein levels by 1,25-(OH) 2 D 3 leads to proportional enrichment of the shed fragment in conditioned media as shown by SILAC fold changes (Figure 1).

While SILAC data shows that the active metabo-lite, 1,25-(OH) 2 D 3 , signifi cantly increased Tmem27 protein expression levels in a time course manner, stimulation with 25-(OH) 2 D 3 had no effect on Tmem27 levels. Recently Saisho et al. [16] showed that glucose enhanced Tmem27 protein expression in insulin-producing MIN6 β -cells; however, to the authors ’ best knowledge this is the fi rst study show-ing that Tmem27 protein levels are also regulated by 1,25-(OH) 2 D 3 in insulin producing cells. Although, the molecular mechanism(s) on how 1,25-(OH) 2 D 3 is involved in Tmem27 expression are currently unknown, it is tempting to hypothesize that Tmem27 may be one of many potential mediators through which 1,25-(OH) 2 D 3 exerts its reported protective effects in pancreatic β -cells. Of note, in vitro cell cul-ture and transgenic mice studies have showed that Tmem27 plays a positive role in insulin exocytosis and β -cell proliferation [7 – 9]. Finally, our fi ndings are in line with a recent study showing an up-regulation of the Tmem27 gene in a time-course manner in mice

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supported by the low serum levels of Tmem27 in autoimmune diabetes group, presumably having low β -cell mass. Second, future validation of Tmem27 as a potential serum marker for diabetes will require development of a high throughput assay that can operate at low pg/mL limit of detection.

In summary, the present study demonstrates that Tmem27 protein expression in INS-1 cells is modu-lated by 1,25-(OH) 2 D 3 . Our fi ndings that Tmem27 serum levels in subjects with autoimmune diabetes are only 13% of those in healthy individuals call for development of assays with lower limits of detection for its further development and validation as a poten-tial serum marker for the pathogenesis of diabetes.

Acknowledgements

The authors thank Sandra Dahl, Hormone Laboratory, Oslo University Hospital, for help with preparing the manuscript.

Declaration of interest: The authors report no confl ict of interest. The authors alone are responsible for the content and writing of the paper.

References

Riachy R , Vandewalle B , Belaich S , Kerr-Conte J , Gmyr V , [1] Zerimech F , d ’ Herbomez M , Lefebvre J , Pattou F . Benefi cal effect of 1,25 dihydroxyvitamin D3 on cytokine-treated human pancreatic islets . J Endocrinol 2001 ; 169 : 161 – 8 . Riachy R , Vandewalle B , Moerman E , Belaich S , Lukowiak [2] B , Gmyr V , Muharram G , Kerr-Conte J , Pattou F . 1,25 Dihydroxyvitamin D3 protects human pancreatic islets against cytokine-induced apoptosis via down-regulation of the Fas receptor . Apoptosis 2006 ; 11 : 151 – 9 . Gysemans CA , Cardozo AK , Callewaert H , Giulietti A , [3] Hulshagen L , Bouillon R , Eizirik DL , Mathieu C . 1,25 Dihydroxyvitamin D3 modulates expression of chemokines and cytokines in pancreatic islets: implications for preven-tion of diabetes in nonobese diabetic mice . Endocrinology 2005 ; 146 : 1956 – 64 . Bland R , Markovic D , Hills CE , Hughes SV , Chan SL , [4] Squires PE , Hewison M . Expression of 25-hydroxyvitamin D3-1 alpha-hydroxylase in pancreatic islets . J Steroid Bio-chem Mol Biol 2004 ; 89-90 : 121 – 5 . Wolden-Kirk H , Overbergh L , Gysemans C , Brusgaard K , [5] Naamane N , Van Lommel L , Schuit F , Eizirik DL , Christesen H , Mathieu C . Unraveling the effects of 1,25(OH)2D3 on global gene expression in pancreatic islets . J Steroid Biochem Mol Biol 2013 ; 136 : 68 – 79 . Bourlon PM , Faure-Dussert A , Billaudel B . The de novo [6] synthesis of numerous proteins is decreased during vitamin D3 defi ciency and is gradually restored by 1,25-dihydroxyvi-tamin D3 repletion in the islets of Langerhans of rats . J Endocrinol 1999 ; 162 : 101 – 9 . Fukui K , Yang Q , Cao Y , Takahashi N , Hatakeyama H , [7] Wang H , Wada J , Zhang Y , Marselli L , Nammo T , Yoneda K , Onishi M , Higashiyama S , Matsuzawa Y , Gonzalez FJ , Weir GC , Kasai H , Shimomura I , Miyagawa J-I , Wolheim CB , Yamagata K . The HNF-1 target Collectrin controls insulin exocytosis by SNARE complex formation . Cell Metab 2005 ; 2 : 373 – 84 .

pancreatic islets after stimulation with 1,25-(OH) 2 D 3 [5]. Taken together, our fi ndings should pave the way for future studies providing insights into molecular mechanisms by which 1,25-(OH) 2 D 3 regulates Tmem27 protein expression in pancreatic β -cells.

Esterhazy et al. [14] found reduced Tmem27 protein levels in pancreatic islets from type 2 diabetic patients. Another study showed that Tmem27 mRNA levels were signifi cantly reduced in islets from patients with type 2 diabetes [9]. These fi ndings are in line with a recent report showing that Tmem27 gene expression is 10-fold lower in islets from recent diagnosed type 1 diabetic pancreatic donors as com-pared with healthy individuals [17]. Against this background and based on our own fi ndings from conditioned media, we wondered whether shed cir-culating Tmem27 could be detected in human serum samples. Importantly, in the present study, we report that Tmem27 is detectable in serum and its levels are signifi cantly lower in subjects with autoimmune diabetes compared to healthy individuals. It should be noted that Tmem27 levels were below the LOD of the assay used in 14 out of 21 serum samples from the autoimmune diabetes group.

As demonstrated in healthy subjects, a signifi cant positive correlation was found between Tmem27 serum levels and C-peptide levels. This fi nding is in accordance with a recent report [9] showing a sig-nifi cant positive correlation between Tmem27 and insulin in human pancreatic islets. Thus, our study support the notion that Tmem27 is implicated in insulin exocytosis process, most likely as a compo-nent of a multiprotein submembrane complex that regulates the plasma membrane docking and exocy-tosis of insulin granules [7,11].

Though our data from serum samples are encour-aging with regard to its clinical relevance, two main issues have to be addressed before suggesting that Tmem27 serum levels may be useful as a biomarker in diabetes. First, its pancreatic β -cell specifi city have to be determined; In vitro data suggest that circulat-ing Tmem27 in serum can possibly have two differ-ent origins: pancreatic β -cells and kidney cells [9]. A possible release of Tmem27 from kidney cells could thus infl uence the quantitative results obtained in our study. A recent study, on the other hand, dem-onstrates the lack of endogenous Tmem27 cleavage from kidneys due to absence of Bace2 protease (the main protease cleaving Tmem27 in primary pancre-atic β -cells) in the kidney [14]. Obviously, further studies are needed to confi rm the origin and to what extent Tmem27 is released from other cell types than pancreatic β -cells. Indeed, in the present study we found a signifi cant positive correlation between Tmem27 levels and C-peptide in healthy individuals. These fi ndings suggest that circulating Tmem27 in serum may originate predominantly from pancre-atic β -cells, although secretion from the kidney can-not be entirely ruled out. This suggestion is further

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Klemke RL , Camp DG II , Smith RD . Reversed-phase chro-matography with multiple fraction concatenation strategy for proeteome profi ling of human MCF10A cells . Proteom-ics 2011 ; 11 : 2019 – 26 . Esterhazy D , Stutzer I , Wang H , Rechsteiner MP , [14] Beauchamp J , Dobeli H , Hilpert H , Matile H , Prummer M , Schmidt A , Lieske N , Boehm B , Marselli L , Bosco D , Kerr-Conte J , Aebersold R , Spinas GA , Moch H , Migliorini C , Stoffel M . Bace2 is a β cell-enriched protease that regu-lates pancreatic β cell function and mass . Cell Metab 2011 ; 14 : 365 – 77 . Esterhazy D , Akpinar P , Stoffel M . Tmem27 dimerization, [15] deglycosylation, plasma membrane depletion, and the extra-cellular Phe-Phe motif are negative regulators of cleavage by Bace2 . Bio Chem 2012 ; 393 : 473 – 84 . Saisho K , Fukuhara A , Yasuda T , Sato Y , Fukui K , [16] Iwahashi H , Imagawa A , Hatta M , Shimomura I , Yamagata K . Glucose enhances collectrin protein expression in insulin-producing MIN6 β cells . Biochem Biophys Res Commun 2009 ; 389 : 133 – 7 . Planas R , Carrillo J , Sanchez A , de Villa MC , Nunez F , [17] Verdaguer J , James RF , Pujol-Borrel R , Vives-Pi M . Gene expression profi les for the human pancreas and purifi ed islets in type 1 diabetes: new fi ndings at clinical onset and in long-standing diabetes . Clin Exp Immunol 2009 ; 159 : 23 – 44 .

Akpinar P , Kuwajima S , Krutzfeldt J , Stoffel M . Tmem27: [8] a cleaved and shed plasma membrane protein that stimulates pancreatic beta cell proliferation . Cell Metab 2005 ; 2 : 385 – 97 . Altirriba J , Gasa R , Casas S , Ramirez-Bajo MJ , Ros S , [9] Gutierrez-Dalmau A , Ruiz de Villa MC , Barbera A , Gomis R . The role of transmembrane protein 27 (TMEM27) in islet physiology and its potential use as a beta cell mass biomarker . Diabetologia 2010 ; 53 : 1406 – 14 . Zhang H , Wada J , Hida K , Tsuchiyama Y , Hiragushi K , [10] Shikata K , Wang H , Lin S , Kanwar YS , Makino H . Collec-trin, a collecting duct-specifi c transmembrane glycoprotein, is a novel homolog of ACE2 and is developmentally regu-lated in embryonic kidneys . J Biol Chem 2001 ; 276 : 17132 – 9 . Yamagata K , Nammo T , Sato Y , Saisho K , Shoda H , [11] Fukui K . The HNF-1 α -SNARE connection . Diabetes, Obes Metabolism 2007 ; 9 : 40 – 5 . Ong SE , Blagoev B , Kratchmarova I , Kristensen DB , [12] Steen H , Pandey A , Mann M . Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics . Mol Cell Proteom 2002 ; 1 : 376 – 86 . Wang Y , Yang F , Gritsenko MA , Wang Y , Clauss T , Liu T , [13] Shen Y , Monroe ME , Lopez-Ferrer D , Reno T , Moore RJ ,

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