Diabetes
Title page
“Function of isolated pancreatic islets from patients at onset of type 1 diabetes; Insulin secretion can be restored after some days in a non-diabetogenic environment in vitro. Results from the DiViD study”
Short running title: “In vitro studies of islets from type 1 diabetes patients”
List of authors Lars Krogvold,1,2,* Oskar Skog,3 Görel Sundström,4 Bjørn Edwin,2,5 Trond Buanes,2,6 Kristian F Hanssen,2,7 Johnny Ludvigsson,8 Manfred Grabherr,4 Olle Korsgren,3 and Knut Dahl-Jørgensen1,2
1Paediatric Department, Oslo University Hospital 2Faculty of Medicine, University of Oslo, Norway 3Department of Immunology, Genetics and Pathology, Uppsala University, Sweden 4Department of Medical Biochemistry and Microbiology, Uppsala University, Sweden 5The Intervention Centre and Department of Surgery, Oslo University Hospital 6Division of Cancer, Surgery and Transplantation, Oslo University Hospital 7Department of Endocrinology, Oslo University Hospital 8Division of Paediatrics, Department of Clinical Experimental Medicine, Linköping University, Sweden
*Corresponding author: Lars Krogvold, Paediatric Department, Oslo University Hospital HF, P. O. Box 4950 Nydalen, N-0424 Oslo, Norway
Phone: +4797522277. Fax number: +4723015790. E-mail: [email protected]
LK, OS and GS contributed equally to the manuscript and are listed in alphabetical order. KDJ and OK contributed equally to the manuscript. KDJ is Principal Investigator of DiViD-study.
Word count, manuscript: 2024
Number of tables: 0 (1 online supplemental table) Number of figures: 3 (1 online supplemental figure)
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Diabetes Publish Ahead of Print, published online February 12, 2015 Diabetes Page 2 of 24
Abstract:
The understanding of the etiology of type 1 diabetes (T1D) remains limited. One objective of the Diabetes Virus Detection study (DiViD) was to collect pancreatic tissue from living subjects shortly after the diagnosis of T1D. Here we report the insulin secretion ability by in vitro glucose perifusion and explore the expression of insulin pathway genes in isolated islets of Langerhans from these patients. Whole genome RNA sequencing was performed on islets from six DiViD patients and two organ donors that died at the onset of T1D and compared with findings in three non diabetic organ donors. All human transcripts involved in the insulin pathway were present in the islets at onset of T1D. Glucose induced insulin secretion was present in some patients at the onset of T1D, and a perfectly normalized biphasic insulin release was obtained after some days in a non diabetogenic environment in vitro. This indicates that the potential for endogenous insulin production is good, which could be taken advantage of if the disease process was reversed at diagnosis.
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Our understanding of the etiology of type 1 diabetes remains limited (1). Animal
models show only partial similarities with the human disease (2), and there has been
lack of well preserved human tissue samples (3). The functionality of islets of
Langerhans at onset of type 1 diabetes in humans remains poorly characterized.
Previous in vitro studies (4;5) have shown function of islet cells obtained several
months after diagnosis but so far there has been lack of in vitro access to isolated
islets obtained from subjects at onset of type 1 diabetes required to obtain in depth
understanding. Studies demonstrating the gene expression profiles for the human
pancreas and purified islets in type 1 diabetes have been published, providing
interesting data supporting that type 1 diabetes is caused by a chronic inflammatory
process with participation of innate immunity (6;7). The beta cells express and release
cytokines and chemokines, providing a link between the beta cell and the immune
system in early type 1 diabetes (8). However, these studies are based on few diseased
cases, mainly including tissue from subjects with long standing type 1 diabetes (6;7).
Insulin secretion from islets obtained from non diabetic human subjects exhibits a
typical bi phasic pattern when stimulated with glucose (9). At diagnosis most patients
with type 1 diabetes have significant, but insufficient insulin secretion, even though
40 50% of the β cells may be present (10) suggesting beta cell dysfunction (11;12).
Endoplasmic reticulum (ER) stress causes beta cell dysfunction, suggestively by
entrapment of the protein Wolfram syndrome 1 (WFS1), inhibiting the synthesis of
cAMP and thereby the secretion of insulin (13).
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Here we report the ability of insulin secretion and explore the expression of insulin pathway genes in isolated islets of Langerhans obtained from subjects with recent onset type 1 diabetes and non diabetic controls.
Materials and Methods
Patients and pancreas donors
Six patients (case 1 6), age 24 35 years who gave their written informed consent were recruited to the DiViD study (14). In addition, the pancreases from two organ donors
(case 7 8) who died at the onset of type 1 diabetes and from three organ donors
(controls 1 3) without pancreatic disease were included in the study. Both donors with type 1 diabetes died of brain edema and total brain infarction described previously
(15). Clinical data regarding cases and controls are shown in eTable 1. The
Government’s Regional Ethics Committee in Norway and the Regional Ethics
Committee in Uppsala approved the study. For details regarding the methods, see supplementary material.
Results
Islet function
The mean glucose stimulated insulin secretion (GSIS) was reduced in islets from type
1 diabetes subjects (Fig. 1A). GSIS was lowest when islets were examined day 1 after isolation but seemed to increase after 3 and 6 days of in vitro culture (Fig. 1A). Islets from individual subjects with type 1 diabetes had varying levels of GSIS (Fig. 1B).
When examined day 1 after isolation, islets from all subjects except case 6 had a very low or undetectable GSIS (Fig. 1B). After 3 and 6 days of culture, islets from two of the subjects (Case 1 and 2) secreted slightly increased amount of insulin upon glucose
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stimulation, but did not display biphasic insulin secretion, whereas islets from case 4
that displayed a poor GSIS one day after isolation, recovered to a normal biphasic
secretion after 3 and 6 days of culture. Case 6 displayed a close to normal GSIS
already one day after isolation, and the insulin secretion levels increased further after
3 and 6 days of culture (Fig. 1B). For the rest of the cases (3, 5, 7 and 8) the insulin
secretion remained low or undetectable after culturing. Islets from non diabetic
subjects responded with a biphasic insulin release already on day one and were not
further stimulated.
Whole transcriptome sequencing
We generated a total of 362 million reads and mapped those to the human genome.
Both cases and controls generated approximately the same amount of mapable reads.
The full data set (reads) is openly available on doi: 10.17044/BILS/g000002.
When comparing expression similarities, using RPKM values, across all genes, islets
from 5 of the 6 live subjects (Case 1 5, but not case 6) are grouped together and
separate from the brain dead donors (case 7 and 8 and control 1 3), regardless of
whether the islets are from type 1 diabetes or non diabetic subjects (Fig. 2A),
reflecting the major impact of brain death on the results obtained from the whole
transcriptome analysis. Similarly, genes that are part of the complement system
pathway also reflect the differences between islets from live and brain dead subjects
(Fig. 2B). By contrast, the insulin secretion pathway groups islet samples by type 1
diabetes or not, notably resulting in longer branch lengths compared to the
complement system pathway (Fig. 2C).
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In all cases, except case 6, the genes that (according to KEGG database) is involved in the secretion of insulin, the insulin pathway, were less expressed when compared to the controls (Fig. 3). That includes all the genes in the insulin pathway, both the insulin gene INS itself, and the genes involved in production and release of insulin. In case 6, all the genes in the pathway were up regulated compared to the non diabetic controls. INS is about ten fold lower expressed in all other diabetic samples, together with lowered expression of two upstream regulators, the pancreatic and duodenal homeobox 1 (PDX1), and MAFA, a transcription factor that binds RIPE3b, a conserved enhancer element that regulates pancreatic beta cell specific expression of
INS (16). Additional genes that are consistently lower in expression include (a) the adenylate cyclase activating polypeptide (PACAP) and its receptor PACAPR; (b)
FFAR1, a member of the GPR40 family of G protein coupled receptors and may be involved in the metabolic regulation of insulin secretion; (c) G protein controlled integral membrane protein and inward rectifier type potassium channel (KCNJ11); (d)
ATP binding cassette transporter of the MRP subfamily (ABCC8), which is involved in multi drug resistance but also functions as a modulator of ATP sensitive potassium channels and insulin release; (e) calcium activated nonselective ion channel that mediates transport of monovalent cations across membranes (TRPM4); and (f) three major glucose transporters in the mammalian blood brain barrier, which are found primarily in the cell membrane and on the cell surface, where they also function as receptors for human T cell leukemia virus I and II (GLUT1/2/3). By contrast, the muscarinic cholinergic receptor CHRM3, and the G protein coupled receptor CCKAR, which binds non sulfated members of the cholecystokinin family of peptide hormones and acts as a major physiologic mediator of pancreatic enzyme secretion, are unchanged in expression, or slightly more highly expressed.
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Discussion
The results show that the expression of key regulatory elements, the insulin pathway
and the GSIS were reduced in islets isolated from 7 of the 8 subjects with type 1
diabetes compared to islets from non diabetic organ donors. However, in 1 of the 8
cases, the insulin pathway expression and the GSIS were remarkably preserved
already at the day of isolation. The observed GSIS improvement after three and six
days of culture in 4 of the 8 cases suggests that when the islets are removed from the
‘diabetic milieu’, remaining beta cells can recover. Soluble factors released within the
vicinity of the islets could induce a functional impairment of the beta cells, which
could be reversed after islet isolation and in vitro culture. However, a screening of 41
cytokines and chemokines in isolated islets from the six type 1 diabetes patients
showed no difference from that in isolated islets from brain dead organ donors (data
not shown).
It is known that prolonged exposure of human islets to high glucose impairs beta cell
function (17) and since even short episodes of hyperglycemia dramatically affect
glucose tolerance (18) it is possible that glucotoxicity could play a role in the reduced
GSIS observed in islets from these patients. In 1940 Jackson described that intensive
insulin therapy at diagnosis of T1D lead to a long partial remission of the disease (19).
Later these findings were confirmed using C peptide determinations (20). However,
in an experimental transplantation model human islets were exposed 4 6 weeks in
vivo to hyperglycemia followed by normoglycemia for 2 weeks (21). The results
showed that even if the hyperglycemia induced impairment in glucose metabolism,
depletion of insulin mRNA, decreased (pro)insulin biosynthesis, increased glycogen
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accumulation and depletion of insulin content was reversed by the 2 week period of normoglycemia, the deleterious effects of the diabetic state on human islet insulin release remained.
Endoplasmatic reticulum (ER) stress has been proposed as an important contributor to beta cell dysfunction in type 1 diabetes (22). In this study, the gene expression of ER stress markers (BiP, CHOP, ATF4, and XBP1) was high in islets from all subjects with type 1 diabetes, but did not differ from the islets from the brain dead non diabetic controls. The process of brain death has been shown to induce ER stress (23) and the finding of a similar level of expression of these genes in islets isolated from subjects with recent onset type 1 diabetes imply that the ER stress in these islets is a feature of type 1 diabetes. The expression of WFS1 was lower in islets from subjects with T1D (both brain dead and live subjects) than in islets from the control donors, with the exception of case 6 from whom the islets with almost preserved GSIS were isolated. It may be speculated that ER stress and low expression of WFS1 contribute to the reduced beta cell function in T1D.
The level of transcription of the genes in the insulin pathway was reduced in most of the diabetic cases, compared to the non diabetic controls. However, all genes in the insulin pathway were expressed, also in islets from the subjects with no or very low
GSIS. This is in line with earlier studies, describing insulin synthesis and storage remaining in islets even many years after type 1 diabetes onset (6;24), and show that the destruction of the β cells is a slow ongoing process (24). A limitation in all studies of transcriptome profiling of an unfractioned tissue is that any changes observed can be due to differences in either modulation of gene expression or from changes in its
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cell composition. The current analysis was conducted on handpicked islets, thereby
avoiding to a large extent the problem with analyzing pancreatic biopsies in which the
islets constitute only about 1%. However, the insulin producing beta cells constitute
only about 60% of the total cell number in human islets. A reduction in the number of
insulin producing cells in the islets isolated from subjects with recent onset type 1
diabetes could be in agreement with the observation of a reduction in the insulin
pathway.
The most obvious limitation of the present study is the small number of cases
examined. The number of recruited patients was limited by the unexpectedly high
frequency of complications arising from the biopsy procedure (14). Therefore we
considered it unethical to continue to enrol patients. A further important consideration
is the degree of matching between the tissues obtained from the patients with T1D and
those used as controls. This study did not include pancreatic biopsies from healthy
individuals and we therefore chose to employ tissue harvested by alternative methods.
Non diabetic organ donors were chosen as their clinical characteristics are well
defined, all having normal HbA1c and being negative for 4 auto antibodies. The islets
from the controls were not cultured and stimulated with glucose on day 3 and 6, but it
has previously been shown that culturing of normal beta cells does not improve their
insulin secretion ability (25). Due to the limited number of islets available from each
of the cases, we chose to stimulate with glucose only, knowing that additional
secretagogues would potentially provide more complete information.
In summary, our findings illustrate the importance of β cell dysfunction, and not only
loss of number of insulin producing cells, at onset of type 1 diabetes. The restoration
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of specific function of the isolated islets removed from the diabetogenic milieu should encourage further characterization of the underlying mechanism(s) of this functional impairment. Hopefully, this would allow initiation of clinical intervention trials specifically aiming to restore beta cell function alone or combined with drugs targeting the injurious processes ongoing within the pancreas at onset of type 1 diabetes.
Acknowledgements: The project was founded by South Eastern Norway Regional
Health Authority (Grant to KDJ), The Novo Nordisk Foundation (Grant to KDJ and
OK), through the PEVNET Study Group founded by the European Union's Seventh
Framework Programme [FP7/2007 2013] under grant agreement n°261441 PEVNET
(http://www.uta.fi/med/pevnet/publications.html), Barndiabetesfonden (Swedish
Child Diabetes Foundation), the Swedish Medical Research Council (65X 12219 15
6, K2015 54X 12219 19 4), Diabetes Wellness Sweden, the Swedish Diabetes
Foundation and the Family Ernfors Fund. OK’s position is in part supported by the
National Institutes of Health (U01AI065192 06). Human pancreatic islets were obtained from the Nordic Network for Clinical Islet Transplantation supported by the
Swedish national strategic research initiative EXODIAB (Excellence of Diabetes
Research in Sweden) and the Juvenile Diabetes Research Foundation. The authors would like to acknowledge support of Uppsala Genome Center and UPPMAX for providing assistance in massive parallel sequencing and computational infrastructure.
Work performed at Uppsala Genome Center has been funded by RFI/VR “SNISS”
Swedish National Infrastructure for large Scale Sequencing and Science for Life
Laboratory, Uppsala.
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No potential conflict of interest relevant to this article was reported.
LK was responsible for clinical coordination and recruitment of patient, data
collection, analysis and interpretation, and drafted the manuscript. OS performed the
GSIS analyses, and analysed, interpreted and drafted the manuscript. GS performed
the whole transcriptome sequencing analysis and interpretation and drafted the
manuscript. BE and TB performed the surgery and participated in writing of the
article. KFH, JL, MG and OK contributed to study design, data analysis,
interpretation and writing of the manuscript. KDJ was the principal investigator of the
study, had the initial idea of the DiViD study, and participated in study design,
funding, regulatory issues, international collaboration, data collection, analysis and
interpretation, and in writing of the manuscript. LK and KDJ are the guarantors of this
work and, as such, had full access to all the data in the study and take responsibility
for the integrity of the data and the accuracy of the data analysis.
The authors thank specialist nurse Trine Roald, who has provided invaluable efforts in
coordination of the study, Sofie Ingvast for excellent technical assistance, nurses and
doctors at the local hospitals, providing contact with the patients, and finally the
patients who participated in this study.
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Figure legends
FIG. 1. Glucose-stimulated insulin secretion by isolated islets. Twenty handpicked islets were perifused with low glucose (1.67 mmol/L) for 42 min, high glucose (20 mmol/L) for 48 min, and then low glucose again as indicated. Fractions were collected at 6 min intervals and the secreted insulin was measured by ELISA. A:
Mean insulin secretion from islets isolated from six type 1 diabetes patients (case 1 6) and cultured for 1 (open circles), 3 (black squares) or 6 days (open squares), and from islets isolated from 15 organ donors on day one without pancreatic disease (open triangles). B: Individual insulin secretion from islets from two organ donors with type
1 diabetes (case 7 8) and from six live patients with type 1 diabetes (case 1 6), cultured for 1 (open circles), 3 (black squares), or 6 days (open squares).
Observational data.
FIG. 2. Neighbor-joining trees based on expression similarity. Shown are groupings for all genes (a), the complement system pathway (b), and the insulin secretion pathway (c). With the exception of sample case 6, live and brain dead donors cluster together in (a) and (b), while the insulin secretion pathway (c) splits diabetic from non diabetic samples.
FIG. 3. Insulin secretion pathway for all diabetic samples except case 6 (A) and for case 6 (B). We normalized expression by three brain dead controls and show lower expression in blue and higher expression in red. With the exception of case 6, the majority of genes in the pathway are more lowly expressed in the diabetic samples. Similar figures for the rest of the cases separately are shown in eFigure 1.
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REFERENCES
1. Atkinson,MA, Eisenbarth,GS, Michels,AW: Type 1 diabetes. Lancet
2014;383:69-82
2. In't Veld,P: Insulitis in human type 1 diabetes: a comparison between
patients and animal models. Semin Immunopathol 2014;36:569-579
3. Coppieters,KT, Roep,BO, von Herrath,MG: Beta cells under attack: toward a
better understanding of type 1 diabetes immunopathology. Semin
Immunopathol 2011;33:1-7
4. Marchetti,P, Dotta,F, Ling,Z, Lupi,R, Del,GS, Santangelo,C, Realacci,M,
Marselli,L, Di,MU, Navalesi,R: Function of pancreatic islets isolated from a
type 1 diabetic patient. Diabetes Care 2000;23:701-703
5. Lupi,R, Marselli,L, Dionisi,S, Del,GS, Boggi,U, Del,CM, Lencioni,C, Bugliani,M,
Mosca,F, Di,MU, Del,PS, Dotta,F, Marchetti,P: Improved insulin secretory
function and reduced chemotactic properties after tissue culture of islets
from type 1 diabetic patients. Diabetes Metab Res Rev 2004;20:246-251
6. Planas,R, Carrillo,J, Sanchez,A, Ruiz de Villa,MC, Nunez,F, Verdaguer,J,
James,RFL, Pujol-Borrell,R, Vives-Pi,M: Gene expression profiles for the
human pancreas and purified islets in Type 1 diabetes: new findings at
clinical onset and in long-standing diabetes. Clin Exp Immunol
2010;159:23-44
13
Diabetes Page 14 of 24
7. Nica,AC, Ongen,H, Irminger,JC, Bosco,D, Berney,T, Antonarakis,SE,
Halban,PA, Dermitzakis,ET: Cell-type, allelic, and genetic signatures in the
human pancreatic beta cell transcriptome. Genome Res 2013;23:1554-1562
8. Eizirik,DL, Sammeth,M, Bouckenooghe,T, Bottu,G, Sisino,G, Igoillo-Esteve,M,
Ortis,F, Santin,I, Colli,ML, Barthson,J, Bouwens,L, Hughes,L, Gregory,L,
Lunter,G, Marselli,L, Marchetti,P, McCarthy,MI, Cnop,M: The human
pancreatic islet transcriptome: expression of candidate genes for type 1
diabetes and the impact of pro-inflammatory cytokines. PLoS Genet
2012;8:e1002552
9. Cerasi,E, Luft,R: The plasma insulin response to glucose infusion in healthy
subjects and in diabetes mellitus. Acta Endocrinol (Copenh) 1967;55:278-
304
10. Akirav,E, Kushner,JA, Herold,KC: Beta-cell mass and type 1 diabetes: going,
going, gone? Diabetes 2008;57:2883-2888
11. Drucker,D, Zinman,B: Pathophysiology of beta cell failure after prolonged
remission of insulin-dependent diabetes mellitus (IDDM). Diabetes Care
1984;7:83-87
12. Dupre,J, Jenner,MR, Mahon,JL, Purdon,C, Rodger,NW, Stiller,CR: Endocrine-
metabolic function in remission-phase IDDM during administration of
cyclosporine. Diabetes 1991;40:598-604
14
Page 15 of 24 Diabetes
13. Fonseca,SG, Urano,F, Weir,GC, Gromada,J, Burcin,M: Wolfram syndrome 1
and adenylyl cyclase 8 interact at the plasma membrane to regulate insulin
production and secretion. Nat Cell Biol 2012;14:1105-1112
14. Krogvold,L, Edwin,B, Buanes,T, Ludvigsson,J, Korsgren,O, Hyoty,H, Frisk,G,
Hanssen,KF, Dahl-Jorgensen,K: Pancreatic biopsy by minimal tail resection
in live adult patients at the onset of type 1 diabetes: experiences from the
DiViD study. Diabetologia 2014;57:841-843
15. Korsgren,S, Molin,Y, Salmela,K, Lundgren,T, Melhus,A, Korsgren,O: On the
etiology of type 1 diabetes: a new animal model signifying a decisive role
for bacteria eliciting an adverse innate immunity response. Am J Pathol
2012;181:1735-1748
16. Olbrot,M, Rud,J, Moss,LG, Sharma,A: Identification of beta-cell-specific
insulin gene transcription factor RIPE3b1 as mammalian MafA. Proc Natl
Acad Sci U S A 2002;99:6737-6742
17. Eizirik,DL, Korbutt,GS, Hellerstrom,C: Prolonged exposure of human
pancreatic islets to high glucose concentrations in vitro impairs the beta-
cell function. J Clin Invest 1992;90:1263-1268
18. Brunzell,JD, Robertson,RP, Lerner,RL, Hazzard,WR, Ensinck,JW, Bierman,EL,
Porte,D, Jr.: Relationships between fasting plasma glucose levels and insulin
secretion during intravenous glucose tolerance tests. J Clin Endocrinol
Metab 1976;42:222-229
15
Diabetes Page 16 of 24
19. Jackson RL, Boyd JD, Smith TE: Stabilization of the diabetic child. Am J Dis
Child 1940;59:332-341
20. Ludvigsson,J, Heding,LG, Larsson,Y, Leander,E: C-peptide in juvenile
diabetics beyond the postinitial remission period. Relation to clinical
manifestations at onset of diabetes, remission and diabetic control. Acta
Paediatr Scand 1977;66:177-184
21. Eizirik,DL, Jansson,L, Flodstrom,M, Hellerstrom,C, Andersson,A:
Mechanisms of defective glucose-induced insulin release in human
pancreatic islets transplanted to diabetic nude mice. J Clin Endocrinol
Metab 1997;82:2660-2663
22. Tersey,SA, Nishiki,Y, Templin,AT, Cabrera,SM, Stull,ND, Colvin,SC, Evans-
Molina,C, Rickus,JL, Maier,B, Mirmira,RG: Islet beta-cell endoplasmic
reticulum stress precedes the onset of type 1 diabetes in the nonobese
diabetic mouse model. Diabetes 2012;61:818-827
23. Contreras JL, Jenkins S Smyth C Young C Eckhoff DE. Role of Endoplasmatic
Reticulum (ER)-Stress on Brain-Death (BD)-Induced Human Beta-Cell
Death. In 65th Scientific Sessions of the American Diabetes Association. San
Diego, California, US. 2005.
Ref Type: Conference Proceeding
16
Page 17 of 24 Diabetes
24. Keenan,HA, Sun,JK, Levine,J, Doria,A, Aiello,LP, Eisenbarth,G, Bonner-Weir,S,
King,GL: Residual insulin production and pancreatic ss-cell turnover after
50 years of diabetes: Joslin Medalist Study. Diabetes 2010;59:2846-2853
25. Lee,RH, Carter,J, Szot,GL, Posselt,A, Stock,P: Human albumin preserves islet
mass and function better than whole serum during pretransplantation islet
culture. Transplant Proc 2008;40:384-386
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Page 19 ofa) 24 All genes Diabetes Case 1 live patients Case 2
Case 3
Case 4
Case 5
Case 8 brain-dead donors Case 6*
Control 2
Case 7
Control 1
0.09 Control 3
b) Complement system pathway
Case 7
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0.09 Control 3 Diabetes Page 20 of 24 GIP GLP-1 PACAP A Fatty acid GIPR GLP1R KCNJ11 ABCC8 PACAPR Acetylecholine ATPase FFAR1 Gs TRPM4 depolarization CHRM3 CCK AC VDCC CCKAR Gq/G11 repolarization RAPGEF4 PKA 2+ RIMS2 Ca cAMP ATP PCLO K-CaK- PLC ER MT RYR2 Citric cycle ITPR33 CREB PDX1 DAG RAB3A Pyruvate Ca2+ CAMK nucleus IP3 DNA Glucose 6- Glucose phosphate CAMK INS PKC GCK
Glut1/2/3 Glucose
VAMP2 STX1A SNAP25 INS
GIP GLP-1 PACAP B Fatty acid GIPR KCNC11 ABCC8 PACAPR GLP1R Acetylecholine ATPase FFAR1 Gs TRPM4 depolarization CHRM3 CCK AC VDCC CCKAR Gq/G11 repolarization RAPGEF4 PKA 2+ RIMS2 Ca cAMP ATP PCLO K-CaK- PLC ER MT RYR Citric cycle ITPR33 CREB PDX1 DAG RAB3A Pyruvate Ca2+ CAMK nucleus IP3 DNA Glucose 6- Glucose phosphate CAMK INS PKC GCK Glut1/2/3 Glucose
VAMP2 STX1A SNAP25 INS Page 21 of 24 Diabetes
ONLINE SUPPLEMENTAL MATERIALS
Function of isolated pancreatic islets from patients at onset of type 1 diabetes; Insulin secretion can be restored after some days in a non-diabetogenic environment in vitro. Results from the DiViD study.
Table of contents Islet isolation ...... 2 Islet function ...... 2 Whole transcriptome sequencing and analyses ...... 2 eTable 1.Demographic data, cases and controls...... 3 Legend eFig 1...... 3 References ...... 3
Diabetes Page 22 of 24
Islet isolation The most distal part (0.5-1 cm) of laparoscopic pancreatic tail resections performed at Oslo University Hospital was immediately shipped by air courier in cold organ preservation solution (Viaspan®, UW) to Uppsala University for islet isolation. The islets were isolated by a method based on the procedure used for clinical islet isolation that has been described previously (1). Basically, the pancreatic duct was located under a surgical microscope, cannulated with a fine catheter, and collagenase (Liberase®, Roche) was injected continuously. After about 30 min, the pancreatic tissue was cut into 3–4 pieces, transferred to two 15 mL glass tubes, and digested at 37°C with continuous shaking for 30 min. The digested pancreata was washed twice with cold culture media (CMRL-1066, ICN Biomedicals, Costa Mesa, CA supplemented with 10 mM HEPES, 2 mM L-glutamine, 50 µg/mL gentamycin, 20 µg/mL ciproxfloxacin 10 mM Nicotinamide, and 10% heat-inactivated fetal calf serum) and immediately transferred to culture dishes. 300-700 islets from each patient were handpicked from the digested tissue under a microscope by skilled islet technicians with multiple years of experience. Islets from the brain-dead organ donors were isolated and cultured as described previously (1;2).
Islet function Glucose-stimulated insulin-secretion (GSIS) was assessed in a dynamic perfusion system, Suprafusion 1000 (BRANDEL, Gaithersburg, MD). Twenty handpicked islets cultured for 1, 3, or 6 days were perifused with low glucose (1.67 mM) for 42 min, high glucose (20 mM) for 48 min, and then low glucose again. Fractions were collected at 6 min intervals and the secreted insulin was measured by ELISA.
Whole transcriptome sequencing and analyses RNA was extracted with AllPrep DNA/RNA/Protein Mini Kit (Qiagen) from 50-100 islets per subject, immediately after handpicking from the digested pancreatic tail resections, or after storage of isolated islets from multi-organ donors (case 7 and 8, and controls 1-3) on day 1 after isolation at -80°C in RNAlater (Qiagen). The extracted RNA was of good quality (RIN values between 7.1 and 9.5) and sufficient quantity (>1 µg) for performing whole transcriptome sequencing. For details regarding the methods, see supplementary material. Total RNA samples were depleted from rRNA using the RiboMinus Eukaryote Kit for RNA and the RiboMinus Concentration Module. Total RNA was used to prepare a fragment library using the SOLiD Total RNA-seq Kit (Applied Biosystems). The libraries were sequenced using the AB SOLiD™ 5500xl-W system. The read length was 50 bp for all samples and directionality of RNA molecules was preserved in the sequencing. Reads were aligned to version GRCh37/hg19 of the human genome using version 1.1 of the Applied Biosystems whole transcriptome analysis tool (Uppsala genome Center). The neighbor-joining method, based on the normalized Euclidean distance of reads per kilobase of exon per million fragments mapped (RPKM) between samples was used to analyze differences in expression between all samples, using uniform gene expression to root the tree. Gene lists for the insulin secretion pathway and the complement system pathway were extracted from the KEGG database and relevant literature. RPKM values for all genes in the pathways were calculated (3) and each case was compared to the mean of the three controls in order to identify up- and down-regulated genes. Genes with RPKM values below 0.1 were considered non-expressed.
Page 23 of 24 Diabetes
eTable 1.Demographic data, cases and controls.
Time from HbA1c at Age Sex Insulin Anti Anti- T1D biopsy anti-ZnT8 Anti IA2 Category No (years (M/ (U/kg/d GAD insulin diagnosis (% (<0,12ai)* (<0,10ai)* ) F) ay) (<0,08ai)* (<0,08ai)* (weeks) mmol/mol) Case 1 25 F 4 6.7 (50) 0,5 1,76 0,7 0,28 0,16 DiViD- Case 2 24 M 3 10.3 (89) 0,35 0,79 <0,01 0,44 >3,00 cases, living newly Case 3 34 F 9 7.1 (54) 0,17 1,77 < 0,05 1,45 > 3,00 diagnosed Case 4 31 M 5 7.4 (57) 0,4 0,77 0,1 < 0,01 2,54 T1D Case 5 24 F 5 7.4 (57) 0,36 0,46 0,1 0,06 >3 patients Case 6 35 M 5 7.1 (54) 0,52 1,85 < 0,05 < 0,01 < 0,04
Organ- Case 7 40 M 0 nd - Neg. Neg. Neg. Neg. donors died at onset of T1D Case 8 29 M 0 10.4 (90) - Neg. Neg. Neg. Neg.
Organ- Control 1 22 M - 5.5 (37) - Neg. Neg. Neg. Neg. donors Control 2 20 M - 5.8 (40) - Neg. Neg. Neg. Neg. without T1D Control 3 25 M - 5.9 (41) - Neg. Neg. Neg. Neg.
*Arbitrary units according to Diabetes Antibody Standardization Program (DASP) (4)
Legend eFig 1
Insulin secretion pathway for all diabetic cases (1-8) shown separately. We normalized expression by three brain dead controls and show lower expression in blue and higher expression in red. With the exception of case 6, the majority of genes in the pathway are more lowly expressed in the diabetic samples.
References
1. Goto,M, Eich,TM, Felldin,M, Foss,A, Kallen,R, Salmela,K, Tibell,A, Tufveson,G, Fujimori,K, Engkvist,M, Korsgren,O: Refinement of the automated method for human islet isolation and presentation of a closed system for in vitro islet culture. Transplantation 2004;78:1367-1375 2. Korsgren,S, Molin,Y, Salmela,K, Lundgren,T, Melhus,A, Korsgren,O: On the etiology of type 1 diabetes: a new animal model signifying a decisive role for bacteria eliciting an adverse innate immunity response. Am J Pathol 2012;181:1735-1748 3. Mortazavi,A, Williams,BA, McCue,K, Schaeffer,L, Wold,B: Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 2008;5:621-628 4. Bingley,PJ, Bonifacio,E, Mueller,PW: Diabetes Antibody Standardization Program: first assay proficiency evaluation. Diabetes 2003;52:1128-1136
Diabetes Page 24 of 24
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Fatty acid gipr glp-1r kir6.2 sur1 Fatty acid gipr glp-1r kir6.2 sur1 pacapr pacapr Acetylecholine Case 1 Acetylecholine Case 2 atpase atpase gpr40 gs nscc depolarization gpr40 gs nscc depolarization m3r m3r CCK ac CCK ac vdcc vdcc cckar cckar gq/g11 repolarization gq/g11 epac pka repolarization epac pka 2+ rim2 Ca2+ rim2 Ca cAMP ATP cAMP ATP piccolo k-caK- piccolo k-caK- plc plc ER MT ER MT ryr ip3r ryr ip3r3 creb pdx-1 Citric cycle 3 creb pdx-1 Citric cycle rab3 rab3 DAG Pyruvate DAG Pyruvate Ca2+ camk Ca2+ camk nucleus nucleus IP3 DNA Glucose 6- Glucose IP3 DNA Glucose 6- Glucose phosphate phosphate camk ins camk ins pkc gck pkc gck
glut1/2 Glucose glut1/2 Glucose
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Fatty acid gipr glp-1r kir6.2 sur1 Fatty acid gipr glp-1r kir6.2 sur1 pacapr Case 3 pacapr Case 4 Acetylecholine atpase Acetylecholine atpase gpr40 gs nscc depolarization gpr40 gs nscc depolarization m3r m3r CCK ac CCK ac vdcc vdcc cckar cckar gq/g11 repolarization gq/g11 repolarization epac pka 2+ epac pka 2+ rim2 Ca rim2 Ca cAMP ATP cAMP ATP piccolo k-caK- piccolo k-caK- plc plc ER MT ER MT ryr ryr 3 Citric cycle ip3r3 Citric cycle ip3r creb pdx-1 creb pdx-1 DAG rab3 Pyruvate DAG rab3 Pyruvate Ca2+ camk Ca2+ camk nucleus nucleus IP3 DNA Glucose 6- Glucose IP3 DNA Glucose 6- Glucose phosphate phosphate camk ins camk ins pkc gck pkc gck
glut1/2 Glucose glut1/2 Glucose
vamp2 vamp2 syntaxin syntaxin snap25 snap25 ins ins
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Fatty acid gipr glp-1r kir6.2 sur1 Fatty acid gipr glp-1r kir6.2 sur1 pacapr pacapr Acetylecholine Case 5 Acetylecholine Case 6 atpase atpase gpr40 gs nscc depolarization gpr40 gs nscc depolarization m3r m3r CCK ac CCK ac vdcc vdcc cckar cckar gq/g11 epac pka repolarization gq/g11 epac pka repolarization Ca2+ Ca2+ rim2 rim2 ATP cAMP ATP cAMP piccolo k-caK- piccolo k-caK- plc plc MT ER MT ER ryr ip3r ryr Citric cycle ip3r3 creb pdx-1 Citric cycle 3 creb pdx-1 rab3 rab3 DAG Pyruvate DAG Pyruvate 2+ camk Ca2+ camk Ca nucleus nucleus IP IP3 DNA Glucose 6- Glucose 3 DNA Glucose 6- Glucose phosphate phosphate camk ins camk ins pkc gck pkc gck
glut1/2 Glucose glut1/2 Glucose
vamp2 vamp2 syntaxin syntaxin snap25 snap25 ins ins
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Fatty acid gipr glp-1r kir6.2 sur1 Fatty acid gipr glp-1r kir6.2 sur1 pacapr pacapr Acetylecholine Acetylecholine Case 8 atpase Case 7 atpase gpr40 gs nscc depolarization gpr40 gs nscc depolarization m3r m3r CCK ac CCK ac vdcc vdcc cckar cckar gq/g11 epac pka repolarization gq/g11 epac pka repolarization Ca2+ Ca2+ rim2 rim2 ATP cAMP ATP cAMP piccolo k-caK- piccolo k-caK- plc plc MT ER MT ER ryr ip3r ryr Citric cycle ip3r3 creb pdx-1 Citric cycle 3 creb pdx-1 rab3 DAG rab3 Pyruvate DAG Pyruvate Ca2+ camk Ca2+ camk nucleus nucleus IP3 DNA Glucose 6- Glucose IP3 DNA Glucose 6- Glucose phosphate phosphate camk ins camk ins pkc gck pkc gck
glut1/2 Glucose glut1/2 Glucose
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