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IG20/MADD Plays a Critical Role in GlucoseInduced Insulin Secretion

Liangcheng Li1,3, Yong Wang2, Ryan Carr1, Christine Samir Haddad1, Ze Li2, Lixia Qian1

Jose Oberholzer2, Ajay V. Maker1,2, Qian Wang2, and Bellur S. Prabhakar1,*

1. Department of Microbiology and Immunology, 2. Department of Surgery, College of Medicine. University of Illinois at Chicago. Chicago. IL60612, USA. 3. School of Pharmaceutical Sciences, Xiamen University at Xiang'an, Xiamen, Fujian, 361102, China.

* Corresponding author:

Dr. Bellur S. Prabhakar Professor and Head, Department of Microbiology and Immunology Associate Dean for Research and Training Program College of Medicine, University of Illinois Rm. E705, 835 South Wolcott Ave. (M/C 790) Chicago, IL 60612 Telephone: 3129964945 Fax: 3129966415 Email: [email protected]

Short Title: Deletion of Ig20/Madd causes type 2 diabetes

Word count: 3922; Figures: 6; Supplementary figures: 7; Supplementary tables: 8.

1

Diabetes Publish Ahead of Print, published online December 30, 2013 Diabetes Page 2 of 36

Abstract

Pancreatic βcell dysfunction is a common feature of Type2 diabetes. Earlier, we had cloned IG20 cDNA from a human insulinoma and had shown that IG20/MADD can encode six different splice isoforms that are differentially expressed and have unique functions, but its role in βcell function was unexplored. To investigate

the role of IG20/MADD in βcell function we generated conditional knockout (KMA1ko) mice. Deletion of

IG20/MADD in βcells resulted in hyperglycemia and glucose intolerance associated with reduced and delayed glucoseinduced insulin production. KMA1ko βcells were able to process insulin normally, but had increased insulin accumulation and showed a severe defect in glucoseinduced insulin release. These findings indicated that IG20/MADD plays a critical role in glucoseinduced insulin release from βcells and its functional disruption can cause Type2 diabetes. The clinical relevance of these findings is highlighted by recent reports of very strong association of rs7944584 single nucleotide polymorphism (SNP) of IG20/MADD with fasting hyperglycemia/diabetes. Thus, IG20/MADD could be a therapeutic target for Type2 diabetes, particularly in those with rs7944584 SNP.

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Introduction

Type2 diabetes mellitus affects ~8% of all Americans and 366 million people worldwide with significant

resulting morbidity and mortality. The peripheral insulin resistance and insulin secretion deficit characterize

Type2 diabetes. In an attempt to identify that are involved in pancreatic βcell function, we cloned a

number of genes, including the IG20 (clone number 20 and hence IG20), that were differentially expressed in

a human insulinoma through subtractive hybridization with a human glucagonoma (13). Subsequently, it was

found that IG20 and MADD/DENN cDNAs, which were independently cloned, were nearly identical to each

other and were splice isoforms of the same (4; 5). Alternative splicing allows a single gene to encode

several proteins and thus enhance proteome diversity. The IG20/MADD is one such gene that can undergo

cellspecific alternate splicing and give rise to six different protein isoforms with unique functions (317).

While the IG20pa and IG20SV2 isoforms are not always expressed, the MADD and DENNSV

isoforms are constitutively expressed in all cells and tissues, and are over expressed in many human tumors

and cancer cell lines (3; 14; 15; 17; 18). Though the function of IG20SV2 is not yet known, the MADD isoform

is an Akt substrate and plays an important role in cancer cell survival and confers resistance to TRAIL (TNF

related apoptosisinducing ligand) and TNFαinduced apoptosis (6; 14; 17; 19). The IG20pa isoform is pro

apoptotic and functions like a “tumor suppressor”; in contrast, the DENNSV isoform acts as an “oncogene”

and promotes tumor cell proliferation (9; 10).

The other two isoforms, KIAA0358 and IG20SV4, are expressed only in certain neuronal and

neuroendocrine tissues (7) and play important roles in regulating neurotransmission and neuronal cell survival

(7; 2022). The KIAA0358 isoform of IG20/MADD is a human ortholog of the rat Rab3A GDPGTP exchange

protein (GEP)(12) that regulates neurotransmitter release at the neuromuscular junction (23; 24) during the post

docking process of postsynaptic exocytosis (23).

While the above studies revealed the function of IG20/MADD in neurotransmission and cell survival,

Wide Association Studies (GWAS) showed a strong association of rs7944584 SNP in

IG20/MADD with fasting hyperglycemia in European cohorts (25), and Type2 diabetes in Han Chinese (26).

This suggested that IG20/MADD may play a role in glucose homeostasis by an unknown mechanism. This

finding combined with the fact that IG20 cDNA was cloned from a human insulinoma, and the βcells are of

neuroendocrine origin led us to hypothesize that IG20/MADD may play an important role in βcell function.

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Therefore, we explored the role of IG20/MADD in pancreatic βcells by generating mice in which IG20/MADD

can be selectively deleted in βcells and studied the consequent effects on glucose homeostasis including β

cell function.

Materials and Methods

Generation of Ig20/Maddknockout (KMA1ko) mice. All animal experiments were as per the protocol

approved by the Institutional Animal Care and Use Committee of the University of Illinois at Chicago. Mouse

IG20/MADD homologous sequence was isolated from a 129Sv/Pas BAC library using PCR primers shown in

Table S1. The targeting vector contained exons 3 and 4 flanked by a distal LoxP site at the 3’ end and a LoxP

FRT (flippase recognition target sites)neomycinFRT positive selection cassette at the 5’ end. The neomycin

(Neo) cassette flanking FRT sites allowed for its deletion by crossing with Flp recombinaseexpressing mouse.

The linearized targeting vector (Fig1A) was electroporated into mouse E14 ES cells and G418resistant clones were selected. The primers listed in Tables S2 and S4 were used under the conditions shown in

Tables S3 and S5 respectively for PCR amplification to detect the homologous recombinants at the 5' end (2.6 kb fragment; FigS1) and 3' end (5.4 kb fragment; FigS2). Bgl II and AvrII digested genomic DNA was subjected to Southern blotting with probes generated using PCR primers shown in Tables S6 and S7 for detecting the 5' (8.4 kb fragment) and 3' (7.0 kb fragment) fragments respectively (FigS3 and FigS4).

To generate chimeric mice, targeted ES cells were injected into C57BL/6 blastocysts and re implanted into pseudopregnant females. KMA1 Flox+/- mice were obtained by mating chimeric males with

C57BL/6 females (FigS5). These mice, generated by GenOway, Lyon, France, were intercrossed to generate

KMA1 (Flox+/+) mice. KMA1 Flox+/- Cre mice were obtained by crossing KMA1 Flox+/+ mice with heterozygous

(Ins2cre/Esr1)1Dam/J (Jackson Laboratory). The KMA1 Flox+/- Cre mice were intercrossed to produce KMA1

Flox+/+Cre mice (KMA1ko). Mice were genotyped using PCR primers listed in Table S8. Expected size of DNA fragments from WT and KMA1ko were 245bp and 363bp respectively (Fig.1B), the Cre fragment was 410 bp and the internal control (IC) MGSCv37 (NT_039606.7) fragment was 200 bp (Fig.1B). Mice were maintained on a C57BL/6 background.

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Genotyping. Tail DNA from 34 week old mice was extracted using the DNeasy Blood & Tissue Kit (Qiagen,

Germantown, MD). The PCR primer sets (Table S8) were used for genotyping. The Flox'd allele and the Cre

allele were detected by PCR.

Induction of KMA1 deletion. Mice were housed in a pathogenfree facility on a 12:12h lightdark cycle and

fed ad libitum with a standard mouse chow diet. To delete KMA1, 40 mg/kg/day of tamoxifen in 100 µL of

peanut oil was injected into the peritoneum (IP) of 45 week old mice for 5 days (27). Blood glucose was

measured between 9:00 to 9:30 AM using a glucometer (Bayer HealthCare LLC, NY). Plasma collected during

the same time was used to test normal fed insulin levels.

RNA Extraction and RTPCR. Total cellular RNA from the islets and hypothalamus of WT, KMA1het and

KMA1ko mice was extracted using TRIzol reagent (Life technologies, Grand Island, NY) and was subjected to

RTPCR using SuperScript® III OneStep RTPCR System with Platinum®Taq reagent (Life technologies,

Grand Island, NY). To detect deletion of IG20/MADD gene, primers flanking exon 4 (Forward: 5'

TCTTCAGGTGCTAACCTGCATCCT3') and 5 (Reverse: 5'AACGCCATCACAGACATGGAGAGT3') were

used. Orexin primers (Forward: 5'ACGGCCTCAGACTTCTTGGGTATT3' and Reverse: 5'TGCTAAAGCG

GTGGTAGTTACGGT3') and Insulin2 (Ins2) primers (forward: 5'ACCATCAGCAAGCAGGAAGGTACT3' and

reverse: 5’GAACCACAAAGGTGCTGCTTGACA3') were used to amplify Orexin and Ins2 as markers for

hypothalamus and pancreatic islets respectively.

Glucose and insulin tolerance tests. Mice (n = 10/group) fasted for 16 hours were injected IP with 2 g/kg

glucose (Sigma, St. Louis, MO) in saline. Blood from the tail vein was obtained and blood glucose levels were

measured using a Bayer Contour Blood Glucose Meter (Bayer HealthCare LLC, NY). Bovine insulin (0.75 U/kg

(Sigma, St. Louis, MO) was injected IP. In both tolerance tests, blood glucose levels were measured at 0, 15,

30, 45,60, and 120 minutes.

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Proinsulin, insulin and cpeptide measurement. Plasma insulin, Proinsulin and Cpeptide levels in mice

fasted for 16 hours were determined using an ultrasensitive mouse insulin (Crystal Chem Inc. Downers Grove,

IL, USA), mouse proinsulin and Cpeptide ELISA kits (ALPCO Diagnostics, NH) respectively.

HOMAIR and HOMAB. Homeostasis model assessment method was used to calculate insulin resistance

(HOMAIR) and βcell function (HOMAB) as described before (28; 29). Fasting glucose and insulin level were used to determine the values of HOMAIR and HOMAB according the following equations: HOMA1IR = (FPI x FPG)/22.5. HOMAB = (20 x FPI)/(FPG3.5) (30). FPI is fasting plasma insulin concentration (mU/L) and

FPG is fasting plasma glucose level (mmol/L).

Islet isolation and culture. Pancreatic islets were isolated as previously described (31) and purified using discontinuous Ficoll (Mediatech, VA) gradients (32). The islets were cultured in RPMI1640 supplemented with

10% fetal bovine serum (Hyclone Inc, MA), 11.2 mM glucose and antibiotics at 37°C in an incubator containing

5% CO2 in air.

Assay for insulin content. Insulin extracted using an acidethanol method (33; 34) from 10 islets/group or total pancreas was measured using an ultrasensitive ELISA kit (Crystal Chem Inc. Downers Grove, IL, USA).

Pancreas staining. Tissue sections were prepared from pancreas fixed in 10% buffered formalin and stained

with H&E or subjected to antigen retrieval procedure (Vector Laboratories, CA). Guinea pig antiinsulin

antibody (1:1000) (Invitrogen, Cat#:180067) and TRITCconjugated antiguinea pig antibody (1:1000, Sigma,

Cat#:T7153) were used to detect insulin.

Apoptosis assay. Paraffin was removed from the tissue sections and pancreatic βcell apoptosis was

assessed using Apoptag® plus fluorescein in situ apoptosis detection kit (Chemicon International Inc., Billerica,

MA). Pancreatic βcells were stained for insulin as above and DAPI was used to label the nucleus.

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Islet perifusion. Simultaneous islet perifusion and fluorescence imaging were performed as described before

(31). Briefly, ten islets per sample were incubated with 5 M Fura2/AM ( Molecular probes, CA) and/or 2.5 M

Rhodamin 123 (Rh123, Sigma, MO) for 30 minutes at 37°C in KrebsRinger buffer (KRB) containing 2 mM

glucose. Islets were introduced into a temperaturecontrolled microfluidic device and mounted on an inverted

epifluorescence microscope (Leica DMI 4000B). Islets were then perifused by a continuous flow of KRB at

37°C (pH 7.4) for 10 minutes. KRB containing 14 mM of glucose or other stimulators was administered to the

islets using a peristaltic pump or a syringe.

Islets were observed with 10 X objectives. Dualwavelength Fura2 was excited ratiometrically at 340

and 380 nm, and changes in [Ca2+] were expressed as F340/F380 (% changes). Rh123 was excited using a

495 nm filter (Chroma Technology) mounted in a Lambda DG4 wavelength switcher. Emission of Fura2/AM

and Rh123 fluorescence was filtered using a Fura2/FITC polychroic beam splitter and double band emission

filter (Chroma Technology; Part number: 73,100bs). The images were captured using RetigaSRV, Fast 1394

(QImaging). Simple PCI software (Hamamatsu Corp) was used for analysis. Aliquots were collected from the

device by a fraction collector to measure insulin secretion.

Immunoelectron microscopy. Immuneelectron microscopy was performed as described previously (35),

Briefly, Islets were cultured overnight in the absence of glucose, and then stimulated with 14 mM of glucose for

5 minutes, fixed in 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer, at room

temperature. The fixed islets were dehydrated in graded ethyl alcohol, placed in a mixture of acrylic resin (LR

White: 70% alcohol, 1:2) for 1 hour, and then in pure acrylic resin for 48 hours at room temperature. Ultrathin

70 nm sections were mounted on 200mesh nickel grids and stained using protein Agold technique (3638).

Sections were stained with a guinea pig antimouse insulin antibody (1:5000) diluted in PBS + 1% bovine

albumin + 1% glycine at pH 7.4 followed by washing and immersion for 1 hour in a solution of 15 nm diameter

colloidal gold particles (CGP) covered with protein A (Janssen; Olen, Belgium) at a dilution of 1:50, pH 8.2, in a

moist chamber at 37°C. The grids were subjected to immunoelectron microscopy (Zeiss CEM 902).

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Statistical analysis

Data are expressed as means ± SE. Significance was tested by unpaired or paired twoway Student’s ttests using Excel, or ANOVA test using SPSS 17. A p < 0.05 was considered significant.

Results

IG20/MADD can be deleted in pancreatic βcells upon tamoxifen injection in KMA1ko mice: Exons 3 and

4 of the IG20/MADD gene were flanked by Flox sites (Fig1A). The homozygous flox’d mice were crossed with

Tg (Ins2cre/Esr1)1Dam/J mice to generate KMA1het (KMA1flox+/- : Cre+) mice, the KMA1het was intercrossed to generate WT (KMA1 flox-/- : Cre), KMA1het and KMA1ko (KMA1flox+/+ : Cre+) mice (Fig1B). WT, KMA1het and

KMA1ko mice were treated with tamoxifen for 5 days (27). Since an earlier report had shown that rat insulin promoter activity may also be seen in the hypothalamus (39), total RNA isolated from pancreatic islets and the hypothalamus were subjected to RTPCR. The flox’d allele was deleted only in pancreatic islets, and not in the

hypothalamus of KMA1ko mice or in either tissue in KMA1het or WT mice (Fig1C).

KMA1ko mice show hyperglycemia with reduced fasting insulin levels: Mice were treated with tamoxifen

and the nonfasting blood glucose level was monitored. Starting on day six, relative to WT and KMA1het mice,

KMA1ko mice showed elevated blood glucose levels, which reached peak levels (321.08 ± 25.96 mg/dL in

KMA1ko mice vs. 140.42 ± 6.94 mg/dL in WT mice) by the tenth day (Fig2A). The body weights were similar

amongst different groups of mice (Fig2B). Interestingly, nonfasting blood insulin levels in the KMA1ko mice

remained unaltered and were comparable to the levels found in the other groups of control mice (Fig2C). The

fasting plasma glucose levels were also elevated in the KMA1ko mice compared to the WT mice (184.55 ±

22.55 mg/dL in KMA1ko vs. 87.1 ± 9.5 mg/dL in WT mice; p < 0.01) (Fig2D). However, in contrast to non

fasting insulin levels, the fasting blood insulin levels in the KMA1ko mice were significantly lower than those in

the WT mice ( 0.12 ± 0.02 ng/mL in KMA1ko vs. 0.26 ± 0.05 ng/mL in WT; p<0.05) (Fig2E).

KMA1ko mice show delayed blood glucose clearance and glucose stimulated insulin release: To

determine the underlying cause of hyperglycemia and fasting hypoinsulinemia in KMA1ko mice, we subjected

these mice to intraperitoneal glucose tolerance test (IPGTT). Fifteen minutes after glucose injection (2g/kg) into 8 Page 9 of 36 Diabetes

the peritoneal cavity of mice that were fasted for sixteen hours the blood glucose levels were dramatically

increased in KMA1ko mice and remained at approximately 500 mg/dL even after 2 hours (Fig3A). The Area

Under the Curve (AUC) of glucose in KMA1ko mice was significantly larger when compared to the WT mice

(Fig3B: 24,550.56 ± 1302.99 min.ng/ml for KMA1ko vs. 15,927.78 ± 2453.12 min.ng/mL for WT; p < 0.01).

Moreover, the rise in blood insulin levels in KMA1ko mice was significantly delayed compared to WT mice.

Interestingly, the highest levels of insulin in KMA1ko did not reach the peak levels found in WT mice even after

120 minutes (Fig3C). Although the serum insulin levels were comparable in WT and KO mice (Fig3C) at two

hours after glucose challenge, it was inappropriately lower in KMA1ko mice given that the serum glucose

concentration at that time point was much higher in those mice (Fig3A). This is reflected in the AUC for insulin

being significantly less for KMA1ko mice compared to the WT mice (7.16±2.71 vs. 23.44±4.17 min.ng/mL; p <

0.05, Fig3D) and suggested that IG20/MADD deletion in βcells can cause hyperglycemia secondary to a

delayed insulin release and inappropriately lower level of circulating insulin in response to a glucose challenge.

KMA1ko mice do not exhibit peripheral insulin resistance but show defective βcell function: Next, we

subjected these mice to both insulin tolerance test (ITT) and homeostasis model assessment (HOMA) test to

detect βcell function and insulin resistance. Both KMA1ko and WT mice showed similar degrees of insulin

sensitivity (Fig3E) and a comparable HOMAIR score (7.27 ± 3.43 vs. 5.68 ± 2.54; p value N.S; Fig3F).

However, the HOMAB score was significantly lower in the KMA1ko mice when compared to the WT mice

(21.05 ± 5.59 vs. 73.62 ± 13.75; p < 0.01; Fig3G). These results indicated that peripheral insulin sensitivity

was preserved but that the KMA1ko mice may have defective βcell function.

KMA1ko mice show no apparent change in βcell morphology or insulin processing: Since IG20/MADD

knockdown has been shown to cause spontaneous apoptosis of various cancer cells (6; 7; 14; 15; 17); we

wondered if this accounted for the suboptimal function of βcells in the KMA1ko mice. Our results showed that

pancreatic islets from either the KMA1ko mice or the WT mice had no evidence of significant changes either in

morphology (Fig4A) or apoptosis (Fig4B).

To test if posttranslational processing of insulin in the KMA1ko mice was affected, plasma samples

from KMA1ko or WT mice, fasted overnight, were tested for proinsulin, insulin and Cpeptide levels. There 9 Diabetes Page 10 of 36 was no significant difference in the proinsulin to insulin (Fig4C) and proinsulin to cpeptide (Fig4D) ratios between KMA1ko and WT mice, indicating that IG20/MADD knockout in pancreatic βcells did not affect post

translational processing of insulin.

βcells from KMA1ko mice show insulin accumulation: To test if IG20/MADD knockout affected βcell

insulin content, we stained pancreas sections with an antiinsulin antibody and analyzed them using

immunofluorescence microscopy. There was increased accumulation of insulin in KMA1ko βcells relative to β

cells from WT mice (Fig5A). The ratio of insulin intensity to 4',6diamidino2phenylindole (DAPI) intensity in

the βcells was increased in KMA1ko mice compared to WT mice βcells (Fig5B). Moreover, the total insulin

content was significantly higher in KMA1ko islets relative to WT islets (Fig5C). Electron microscopy of Islet

cells revealed much denser insulin granules in the βcells from KMA1ko mice when compared to those from

WT mice (Fig5D). Similarly, insulin content was increased in the pancreas of KMA1ko mice relative to the

levels found in the pancreas of either KMA1het or WT (FigS6). These results indicated that insulin

accumulation in βcells of KMA1ko mice was not due to increased transcription of the insulin gene (Fig1C) but

likely due to a defect in glucoseinduced insulin release.

KMA1ko islet shows defect in glucoseinduced insulin secretion but normal glucose metabolism: The

islets from WT or KMA1ko mice were subjected to a microperifusion assay and stimulated with 14 mM of

glucose for 45 minutes (Fig6A). 30mM of KCL for 60 minutes (Fig6C), 10µM of Carbochol (Fig6E), or 150µM

Exendin4 (Fig6G) for 45 minutes. The perifusates were collected every five minutes and analyzed for insulin

levels. While the islets from WT mice showed a robust and expected pattern of insulin secretion, the islets

from KMA1ko or KMA1 het mice failed to show a similar timely increase in insulin secretion in response to

glucose challenge (Fig6A). Upon treatment with 30mM KCL, islets from WT mice showed a pattern of insulin

release analogous to that found upon stimulation with 14mM glucose; while KMA1het and KMA1ko islets failed

to show a similar timely increase in insulin release (Fig6C). In contrast, no significant increase in insulin

secretion was noted upon stimulation with 10µM Carbachol alone in the absence of glucose (Fig6E). However,

islets from all three groups of mice showed a similar pattern of insulin release upon stimulation with 150 µM

Exendin4 (Fig6G).

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We carried out repeated stimulation of the islets from WT and KMA1ko mice by treating them

alternatively with 14 mM of glucose for 20 minutes and 2 mM of glucose for 10 minutes. The perifusate was

collected every five minutes and analyzed for insulin levels. While the islets from WT mice showed a robust

and expected pattern of insulin secretion (FigS7A pink line), the islets from KMA1ko failed to increase insulin

secretion in response to glucose challenge (FigS7A, blue line). The AUC of the first peak (FigS7B) and the

second peak (FigS7C) were significantly lower in islets from KMA1ko mice compared to those from WT mice.

These results indicated that targeted deletion of IG20/MADD in pancreatic βcells resulted in defective glucose

stimulated insulin secretion by those cells.

Mitochondrial energetics and intracellular calcium levels play critical roles in insulin secretion in

response to glucose (40), and can serve as reliable indicators of glucose metabolism by βcells. We tested to

determine if the hyperglycemia noted in KMA1ko mice was due to a defect in βcell glucose metabolism. Islet

microperifusion assays showed no significant difference between the groups in their response to stimulation

with 14mM glucose, 30mM KCl, 10Μ, Carbachol, or 150Μ Exendin4 (Fig6B, D, F, H).These data showed

that calcium signaling in islets from KMA1ko mice is normal. Collectively, our results indicated that Ig20/Madd

knockout causes defective glucose stimulated insulin secretion but does not affect glucose metabolism in

pancreatic βcells.

Discussion

The present study clearly showed that targeted deletion of IG20/MADD in mouse βcells can result in

Type2 diabetes phenotype. The nonfasting insulin levels in KMA1ko mice were comparable to those found in

WT mice, but the fasting insulin levels were significantly lower. This suggested that KMA1ko mice suffered

from a severe defect in insulin release in response to normal homeostatic triggers. This was substantiated by

the very slow rise and lower peak in insulin levels in the KMA1ko mice following prolonged glucose challenge,

and further affirmed by a lack of optimal and timely insulin secretion by βcells from KMA1ko mice upon

repeated isletperifusion with glucose (Fig6 and FigS7). Since the total βcell insulin content was higher and

the peripheral proinsulin/insulin and proinsulin/cpeptide ratios in KMA1ko mice were similar to those found in

WT mice, it was unlikely that the defect was in insulin production or processing. Collectively, these results

demonstrated that the hyperglycemia seen in KMA1ko mice was a consequence of slower and suboptimal

glucoseinduced insulin release from βcells.

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Interestingly, KMA1ko islets failed to show normal insulin release upon stimulation with 14mM glucose or 30µM KCL. However, they did respond in a manner similar to those of KMA1het and WT islets in response to stimulation with either 10µM of Carbochol (Fig6E), or 150µM of Exendin4 (Fig6G). These differential responses to stimulation by different secretagogues could be because glucose and KCL induce insulin release by primarily facilitating influx of extracellular Ca++, while Exendin4 and Carbochol cause Ca++ release from intracellular storage by upregulating EPAC2 and PKC respectively (41). Although these results do not definitively show the underlying mechanism of action of MADD, they strongly suggest that the mode 1, and not the mode 2, exocytosis was impaired in the KMA1ko islets(4244).

Although how loss of IG20/MADD function can affect insulin release is not yet known, from earlier studies we do know that IG20/MADD can act as a GDPGTP Exchange Protein (GEP) for both Rab3A (45) and Rab27A (46), which play critical roles in glucosestimulated insulin release from βcells (47; 48). It is therefore possible that the pancreatic βcells of KMA1ko mice likely had reduced levels of “active” Rab3A and

Rab27A, which resulted in inefficient glucoseinduced insulin release. Nevertheless, our findings, for the first

time, show the critical importance of IG20/MADD in regulating glucoseinduced insulin release.

A recent report by the MAGIC (MetaAnalyses of Glucose and Insulinrelated traits Consortium) analyzed the results from twenty one different GWAS with information on fasting glucose and insulin, indices of

βcell function (HOMAB) and insulin resistance (HOMAIR) and identified 16 loci, including MADD rs7944584

SNP, associated with fasting glucose and HOMAB (25). Subsequent analyses found a highly significant

association between IG20/MADD rs7944584 and insulin processing that resulted in higher proinsulin levels

with no effect on insulin secretion (49; 50). Interestingly, the KMA1ko mice showed no apparent reduction in β cell mass or defect in insulin processing, and suggested that the insulin processing defect might be associated with genetic variation/s other than the IG20/MADD rs7944584 SNP or that the rs7944584 SNP might be impacting the function of a yet unidentified gene. However, MADD rs7944584 did not increase the risk for developing Type2 diabetes in European subjects (4951). A notable exception is a study involving a large population of Shanghai Chinese Hans which showed a clear association of IG20/MADD rs7944584 SNP with

Type2 diabetes (26). Although several explanations for the observed differences were suggested, they failed to provide a clear insight into how the IG20/MADD rs7944584 SNP might affect glucose homeostasis in humans (52).

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Localization of IG20/MADD rs7944584 SNP to an intron suggests that it may affect either the

transcription or alternative splicing of IG20/MADD, or might act on the expression of other genes. Additionally,

individuals who harbor this SNP also harbor other mutations and thus it is nearly impossible to attribute the

observed association with a given phenotype to this particular genetic variation. This putative interplay in part

may explain the association of IG20/MADD rs7944584 SNP with related but different phenotypes in different

subpopulations discussed above. Therefore, functional studies have to be carried out to establish causal

relationship between a particular gene and the observed phenotype (53). Our current findings clearly show that

the loss of Ig20/Madd function in βcells alone is sufficient to cause hyperglycemia and diabetes. Our findings

may have clinical relevance in that GLP1 analog may be used to treat IG20/MADD rs7944584 SNP

associated hyperglycemia. Further studies are required to fully elucidate the role of IG20/MADD in clinical

diabetes and its potential to serve as a therapeutic target for Type2 diabetes.

Author Contributions: L.L. designed the experiment, researched data and drafted the manuscript. Y.W., R.C.,

C.H., Z.L., L.Q., Q.W. researched data. Dr. Maker helped with the discussion, J.O., A.M., contributed to

discussion and reviewed/edited manuscript. Dr. Prabhakar conceived the project, designed the experiments,

reviewed the results and wrote the manuscript.

Acknowledgments: This work was supported in part by the grant R01 DK091526 to J.O from the National

Institutes of Health, Bethesda, Maryland. We thank Dr. Jianzhong Qin for his help with some animal studies.

Dr. Bellur Prabhakar is the guarantor of this work and, as such, had full access to all the data in the study and

takes responsibility for the integrity of the data and the accuracy of the data analysis. None of the authors have

any conflict of interest to declare.

References

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5. Schievella AR, Chen JH, Graham JR, Lin LL: MADD, a novel death domain protein that interacts with the type 1 tumor necrosis factor receptor and activates mitogenactivated protein kinase. J Biol Chem 272:12069 12075, 1997 6. Mulherkar N, Ramaswamy M, Mordi DC, Prabhakar BS: MADD/DENN splice variant of the IG20 gene is necessary and sufficient for cancer cell survival. Oncogene 25:62526261, 2006 7. Li LC, Sheng JR, Mulherkar N, Prabhakar BS, Meriggioli MN: Regulation of apoptosis and caspase8 expression in neuroblastoma cells by isoforms of the IG20 gene. Cancer Res 68:73527361, 2008 8. Kurada BR, Li LC, Mulherkar N, Subramanian M, Prasad KV, Prabhakar BS: MADD, a splice variant of IG20, is indispensable for MAPK activation and protection against apoptosis upon tumor necrosis factoralpha treatment. J Biol Chem 284:1353313541, 2009 9. Efimova E, Martinez O, Lokshin A, Arima T, Prabhakar BS: IG20, a MADD splice variant, increases cell susceptibility to gammairradiation and induces soluble mediators that suppress tumor cell growth. Cancer Res 63:87688776, 2003 10. Efimova EV, AlZoubi AM, Martinez O, Kaithamana S, Lu S, Arima T, Prabhakar BS: IG20, in contrast to DENNSV, (MADD splice variants) suppresses tumor cell survival, and enhances their susceptibility to apoptosis and cancer drugs. Oncogene 23:10761087, 2004 11. MurakamiMori K, Mori S, Bonavida B, Nakamura S: Implication of TNF receptorImediated extracellular signalregulated kinases 1 and 2 (ERK1/2) activation in growth of AIDSassociated Kaposi's sarcoma cells: a possible role of a novel death domain protein MADD in TNFalphainduced ERK1/2 activation in Kaposi's sarcoma cells. J Immunol 162:36723679, 1999 12. Brown TL, Howe PH: MADD is highly homologous to a Rab3 guaninenucleotide exchange protein (Rab3 GEP). Curr Biol 8:R191, 1998 13. Levivier E, Goud B, Souchet M, Calmels TP, Mornon JP, Callebaut I: uDENN, DENN, and dDENN: indissociable domains in Rab and MAP kinases signaling pathways. Biochem Biophys Res Commun 287:688 695, 2001 14. Li LC, Jayaram S, Ganesh L, Qian L, Rotmensch J, Maker AV, Prabhakar BS: Knockdown of MADD and c FLIP overcomes resistance to TRAILinduced apoptosis in ovarian cancer cells. Am J Obstet Gynecol 205:362 e312325, 2011 15. Subramanian M, Pilli T, Bhattacharya P, Pacini F, Nikiforov YE, Kanteti PV, Prabhakar BS: Knockdown of IG20 gene expression renders thyroid cancer cells susceptible to apoptosis. J Clin Endocrinol Metab 94:1467 1471, 2009 16. Prabhakar BS, Mulherkar N, Prasad KV: Role of IG20 splice variants in TRAIL resistance. Clin Cancer Res 14:347351, 2008 17. Li L, Jayarama S, Pilli T, Qian L, Pacini F, Prabhakar BSP: Down modulation of IG20/MADD expression or phosphorylation renders TRAIL resistant thyroid cancer cells susceptible to TRAIL treatment. Thyroid, 2012 18. Lim KM, Yeo WS, Chow VT: Antisense abrogation of DENN expression induces apoptosis of leukemia cells in vitro, causes tumor regression in vivo and alters the transcription of genes involved in apoptosis and the cell cycle. Int J Cancer 109:2437, 2004 19. Li P, Jayarama S, Ganesh L, Mordi D, Carr R, Kanteti P, Hay N, Prabhakar BS: Aktphosphorylated mitogenactivated kinaseactivating death domain protein (MADD) inhibits TRAILinduced apoptosis by blocking Fasassociated death domain (FADD) association with death receptor 4. J Biol Chem 285:22713 22722, 2010 20. Miyoshi J, Takai Y: Dual role of DENN/MADD (Rab3GEP) in neurotransmission and neuroprotection. Trends Mol Med 10:476480, 2004 21. Zhang Y, Zhou L, Miller CA: A splicing variant of a death domain protein that is regulated by a mitogen activated kinase is a substrate for cJun Nterminal kinase in the human central nervous system. Proc Natl Acad Sci U S A 95:25862591, 1998 22. Del Villar K, Miller CA: Downregulation of DENN/MADD, a TNF receptor binding protein, correlates with neuronal cell death in Alzheimer's disease brain and hippocampal neurons. Proc Natl Acad Sci U S A 101:42104215, 2004 23. Tanaka M, Miyoshi J, Ishizaki H, Togawa A, Ohnishi K, Endo K, Matsubara K, Mizoguchi A, Nagano T, Sato M, Sasaki T, Takai Y: Role of Rab3 GDP/GTP exchange protein in synaptic vesicle trafficking at the mouse neuromuscular junction. Mol Biol Cell 12:14211430, 2001 24. Schluter OM, Schmitz F, Jahn R, Rosenmund C, Sudhof TC: A complete genetic analysis of neuronal Rab3 function. J Neurosci 24:66296637, 2004

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Nat Genet 42:105116, 2010 26. Hu C, Zhang R, Wang C, Wang J, Ma X, Hou X, Lu J, Yu W, Jiang F, Bao Y, Xiang K, Jia W: Variants from GIPR, TCF7L2, DGKB, MADD, CRY2, GLIS3, PROX1, SLC30A8 and IGF1 are associated with glucose metabolism in the Chinese. PLoS One 5:e15542, 2010 27. Andersson KB, Winer LH, Mork HK, Molkentin JD, Jaisser F: Tamoxifen administration routes and dosage for inducible Cremediated gene disruption in mouse hearts. Transgenic Res 19:715725, 2010 28. Song Y, Manson JE, Tinker L, Howard BV, Kuller LH, Nathan L, Rifai N, Liu S: Insulin sensitivity and insulin secretion determined by homeostasis model assessment and risk of diabetes in a multiethnic cohort of women: the Women's Health Initiative Observational Study. Diabetes Care 30:17471752, 2007 29. Wallace TM, Levy JC, Matthews DR: Use and abuse of HOMA modeling. Diabetes Care 27:14871495, 2004 30. 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34. Matsui K, Oda T, Nishizawa E, Sano R, Yamamoto H, Fukuda S, Sasase T, Miyajima K, Ueda N, Ishii Y, Ohta T, Matsushita M: Pancreatic function of spontaneously diabetic torii rats in prediabetic stage. Exp Anim 58:363374, 2009 35. Saccomanno K, Taccagni G, Bosi E, Preti P, Dozio N, Cantaboni A: Immunoelectron microscopy: a new method for detection of insulin antibodies. J Histochem Cytochem 41:12331239, 1993 36. Bendayan M, Garzon S: Protein Ggold complex: comparative evaluation with protein Agold for high resolution immunocytochemistry. J Histochem Cytochem 36:597607, 1988 37. Roth J: Subcellular organization of glycosylation in mammalian cells. Biochim Biophys Acta 906:405436, 1987 38. Roth J: Postembedding cytochemistry with goldlabelled reagents: a review. J Microsc 143:125137, 1986 39. Wicksteed B, Brissova M, Yan W, Opland DM, Plank JL, Reinert RB, Dickson LM, Tamarina NA, Philipson LH, Shostak A, BernalMizrachi E, Elghazi L, Roe MW, Labosky PA, Myers MG, Jr., Gannon M, Powers AC, Dempsey PJ: Conditional gene targeting in mouse pancreatic ssCells: analysis of ectopic Cre transgene expression in the brain. Diabetes 59:30903098, 2010 40. MacDonald PE, Joseph JW, Rorsman P: Glucosesensing mechanisms in pancreatic betacells. Philos Trans R Soc Lond B Biol Sci 360:22112225, 2005 41. Kahn SE, Hull RL, Utzschneider KM: Mechanisms linking obesity to insulin resistance and type 2 diabetes. Nature 444:840846, 2006 42. Chepurny OG, Kelley GG, Dzhura I, Leech CA, Roe MW, Dzhura E, Li X, Schwede F, Genieser HG, Holz GG: PKAdependent potentiation of glucosestimulated insulin secretion by Epac activator 8pCPT2'OMe cAMPAM in human islets of Langerhans. Am J Physiol Endocrinol Metab 298:E622633, 2010 43. Kasai H, Suzuki T, Liu TT, Kishimoto T, Takahashi N: Fast and cAMPsensitive mode of Ca(2+)dependent exocytosis in pancreatic betacells. Diabetes 51 Suppl 1:S1924, 2002 44. Tian Y, Laychock SG: Protein kinase C and calcium regulation of adenylyl cyclase in isolated rat pancreatic islets. Diabetes 50:25052513, 2001 45. Coppola T, PerretMenoud V, Gattesco S, Magnin S, Pombo I, Blank U, Regazzi R: The death domain of Rab3 guanine nucleotide exchange protein in GDP/GTP exchange activity in living cells. Biochem J 362:273 279, 2002 46. Yoshimura S, Gerondopoulos A, Linford A, Rigden DJ, Barr FA: Familywide characterization of the DENN domain Rab GDPGTP exchange factors. J Cell Biol 191:367381, 2010 47. Kasai K, OharaImaizumi M, Takahashi N, Mizutani S, Zhao S, Kikuta T, Kasai H, Nagamatsu S, Gomi H, Izumi T: Rab27a mediates the tight docking of insulin granules onto the plasma membrane during glucose stimulation. J Clin Invest 115:388396, 2005 48. Yaekura K, Julyan R, Wicksteed BL, Hays LB, Alarcon C, Sommers S, Poitout V, Baskin DG, Wang Y, Philipson LH, Rhodes CJ: Insulin secretory deficiency and glucose intolerance in Rab3A null mice. J Biol Chem 278:97159721, 2003 49. Strawbridge RJ, Dupuis J, Prokopenko I, Barker A, Ahlqvist E, Rybin D, Petrie JR, Travers ME, Bouatia Naji N, Dimas AS, Nica A, Wheeler E, Chen H, Voight BF, Taneera J, Kanoni S, Peden JF, Turrini F, Gustafsson S, Zabena C, Almgren P, Barker DJ, Barnes D, Dennison EM, Eriksson JG, Eriksson P, Eury E, Folkersen L, Fox CS, Frayling TM, Goel A, Gu HF, Horikoshi M, Isomaa B, Jackson AU, Jameson KA, Kajantie E, KerrConte J, Kuulasmaa T, Kuusisto J, Loos RJ, Luan J, Makrilakis K, Manning AK, MartinezLarrad MT, Narisu N, Nastase Mannila M, Ohrvik J, Osmond C, Pascoe L, Payne F, Sayer AA, Sennblad B, Silveira A, Stancakova A, Stirrups K, Swift AJ, Syvanen AC, Tuomi T, van 't Hooft FM, Walker M, Weedon MN, Xie W, Zethelius B, Ongen H, Malarstig A, Hopewell JC, Saleheen D, Chambers J, Parish S, Danesh J, Kooner J, Ostenson CG, Lind L, Cooper CC, SerranoRios M, Ferrannini E, Forsen TJ, Clarke R, Franzosi MG, Seedorf U, Watkins H, Froguel P, Johnson P, Deloukas P, Collins FS, Laakso M, Dermitzakis ET, Boehnke M, McCarthy MI, Wareham NJ, Groop L, Pattou F, Gloyn AL, Dedoussis GV, Lyssenko V, Meigs JB, Barroso I, Watanabe RM, Ingelsson E, Langenberg C, Hamsten A, Florez JC: Genomewide association identifies nine common variants associated with fasting proinsulin levels and provides new insights into the pathophysiology of type 2 diabetes. Diabetes 60:26242634, 2011 50. Wagner R, Dudziak K, HerzbergSchafer SA, Machicao F, Stefan N, Staiger H, Haring HU, Fritsche A: GlucoseRaising Genetic Variants in MADD and ADCY5 Impair Conversion of Proinsulin to Insulin. PLoS One 6:e23639, 2011

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Figure legends

Figure1. Generation of mice with conditional knock out of IG20/MADD in βcells. (A): Shows schematic

representation of the strategy used for generating flox’d KMA1 mice. (B). Shows genotyping results from off

springs of KMA1het (KMA1flox+/- : Cre) mice intercrossing. Primers used for PCR are shown in Table S8.

Representative figure from 98 mice are shown. (C). Shows results from RTPCR using RNA isolated from

hypothalamus and islets from WT, heterozygous (KMA1het) and KMA1ko mice. Orexin and insulin2 served as

specific markers of hypothalamus and pancreatic isles respectively. Representative data from 3 mice/group are

shown.

Figure2 Conditional knockout IG20/MADD gene in β cells leads to hyperglycemia in KMA1ko mice.

Mice were injected with 40 mg/kg/day of tamoxifen in 100 µL of peanut oil for 5 consecutive days and then

tested for body weight, and levels of blood glucose and insulin. (A). Shows nonfasting blood glucose levels;

(B). Shows body weight. (C). Shows nonfasting blood insulin levels. (D). Shows fasting (16 hours) blood

glucose levels. (E). Shows fasting blood insulin levels. Xaxis shows days postfirst injection of tamoxifen. Data are expressed as means ± SE; Oneway ANOVA analysis was performed, *p < 0.05, **p < 0.01, ***p<0.01 with

KMA1ko mice vs WT; n = 1012 mice per genotype.

Figure3 Glucose and Insulin tolerance test. Mice were injected with 40 mg/kg/day of tamoxifen in 100 µL of peanut oil for 5 consecutive days, and 1422 days later they were subjected glucose and insulin tolerance tests.

(A). Shows blood glucose levels at different times after glucose injection via i.p.; (B). Shows area under the

curve for blood glucose; (C). Shows insulin levels at different times after glucose injection via i.p; (D). Shows

area under the curve for insulin levels. (A)(D) mice were fasted overnight. (E). Shows blood glucose levels at

different times after insulin injection into KMA1ko and WT mice; Overnight fasting blood glucose and plasma

insulin was used to assess HOMAIR (F) or HMOAB (G). Male and female mice of 6 to 8wk age were used.

Data are expressed as means ± SE. *p<0.05, and **p < 0.01 with KMA1ko mice vs WT; N.S, not significant; n

= 7–10 mice per group.

Figure 4 Islet morphology and apoptosis, and insulin processing. Mice were injected with 40 mg/kg/day of tamoxifen in 100 µL of peanut oil for 5 consecutive days, and thirty days later their pancreas were examined.

(A). H and E stained sections of islets from KMA1ko and WT mice show normal morphology; (B).TUNEL

staining shows no difference in the frequency of apoptotic cells in the islets of KMA1ko and WT mice. n = 3 18 Page 19 of 36 Diabetes

mice per group. Fourteen to twenty two days after the last tamoxifen injection blood was collected to determine

the fasting levels of plasma proinsulin, insulin and cpeptide by ELISA. (C). Shows the ratio of proinsulin to

insulin; (D). Shows the ratio of proinsulin to Cpeptide. N.S, not significant, n = 10 mice per group.

Figure 5 Insulin content in pancreatic βcells. Mice were injected with 40 mg/kg/day of tamoxifen in 100 µL

of peanut oil for 5 consecutive days, and another 2831 days later the animals were sacrificed. (A). Pancreatic

sections were stained with antiinsulin antibody, and further stained using a TRITCconjugated antiguinea pig

antibody; DAPI was used to stain the nucleus. (B). Shows the average intensity of insulin staining relative to

nuclear DAPI staining. (C). Shows insulin content in the freshly isolated islets from KMA1ko and WT mice. (D).

Electron micrograph shows insulinProteinAGold staining of insulin granules within the selected area of β

cells. *p < 0.05, N.S, not significant, n = 34 mice per group.

Figure 6 KMA1ko in pancreatic βcells results in defective glucose induced insulin secretion but

normal Calcium influx. Mice were injected with 40 mg/kg/day of tamoxifen in 100 µL of peanut oil for 5

consecutive days. Pancreatic islets isolated on day seven from WT, KMA1 het and KMA1ko mice were

cultured overnight and then preincubated with 2 mM glucose for 5 min and stimulated with 14 mM glucose (A,

B), 30mM KCl for 60 minutes (C, D), 10Μ Carbachol (E,F) or 150Μ Exendin4 (G,H) for 45 minutes. The

Insulin secretion (A, C,E and G) and Calcium influx (B, D, F and H) were measured. Percentage changes in

mean value from the initial levels are expressed. Representative figure from three mice are shown.

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Figure1. Generation of mice with conditional knock out of IG20/MADD in βcells. 148x172mm (300 x 300 DPI)

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Figure2 Conditional knockout IG20/MADD gene in β cells leads to hyperglycemia in KMA1ko mice. 119x80mm (300 x 300 DPI)

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Figure-3 Glucose and Insulin tolerance test. 155x252mm (300 x 300 DPI)

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Figure 4 Islet morphology and apoptosis, and insulin processing. 132x134mm (300 x 300 DPI)

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Figure 5 Insulin content in pancreatic βcells. 123x228mm (300 x 300 DPI)

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Figure 6 KMA1ko in pancreatic βcells results in defective glucose induced insulin secretion but normal Calcium influx 155x221mm (300 x 300 DPI)

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Supplementary Information

IG20/MADD Plays a Critical Role in GlucoseInduced Insulin Secretion

Liang-cheng Li, Yong Wang, Ryan Carr, Christine Samir Haddad, Ze Li, Lixia Qian, Jose Oberholzer, Ajay V. Maker, Qian Wang, and Bellur S. Prabhakar

Supplementary Figs. S1 to S5 Supplementary Tables. S1 to S8

Supplementary Figures and Figure Legends

Fig-S1: PCR identification of the homologous recombination event at the 5' end. (a) The figure indicates the PCR screening strategy for the 5’ homologous recombination event. Green arrows illustrate the locations of primers. (b) Testing the sensitivity of the screening PCR using the primers shown on Table S2 with the PCR conditions shown on Table S3. The optimised PCR screen was tested on 0.1, 1 and 10 copies of the KMA1-SA-C+ vector, alone or in the presence of 10 ng genomic C57BL/6J DNA as template. PCR amplification buffer was used as a negative control. M: 1 kb DNA Ladder (NEB). Page 27 of 36 Diabetes

Fig-S2: PCR identification of the homologous recombination event at the 3’ end, including the distal loxP integration. (a) The figure indicates the PCR screening strategy for the 3’ homologous recombination event. Green arrows illustrate the primer localisations. (b) Testing the sensitivity of the screening PCR using the primers shown on Table S4 with the PCR conditions shown on Table S5. The optimised PCR screen was conducted using 10 ng genomic 129Sv DNA from ES cell as template. PCR amplification buffer was used as a negative control. M: 1 kb DNA-Ladder (NEB) Diabetes Page 28 of 36

Fig-S3: Southern blot analysis for 5’ homologous recombination in ES cells. (a) Schematic representation of the wild-type MADD allele and the recombined allele with the relevant restriction sites for the Southern blot analysis is shown. The strategy for the 5’ Southern blot analysis is indicated in blue. (b) The genomic DNA of the tested ES cell clones were compared with wild-type DNA (129ES). The digested DNAs were blotted on nylon membrane and hybridized with the external 5’-probe SA-E-A to screen for 5’ homologous recombination events. The primer set for generation 5’-probe SA-E-A was listed in Table S6. Page 29 of 36 Diabetes

Fig.S4: Southern blot analysis for 3’ homologous recombination in ES cells. (a) Schematic representation of the wild-type Madd allele and the recombined allele with the relevant restriction sites for the Southern blot analysis. The strategy for the Southern blot detection of the 3’ recombination event is indicated in red. (b) The genomic DNA of the tested ES cell clones was compared with wild-type DNA (129ES). The digested DNAs were blotted on nylon membrane and hybridized with the internal 3’-probe LA-I-B to screen for 3’ homologous recombination events. The primer set for generation 3’-probe LA-I-B was listed in Table S7.

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Fig.S5: Detection of the Flp-mediated excision event by PCR. The genotypes of the agouti pups derived from the F1 breeding with Flp deleter mice were tested by PCR using the primer combination KMA1-Flp-F/ KMA1-Flp-R (listed in Table S8) (A) to analyze the excision status of the MADD allele (B). PCR using DNA wild-type ES cells (WT) was used as positive control. PCR without template served as a negative control. M: 100 bp DNA-Ladder (NEB).

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Fig.S6 Insulin content in the total pancreas. Pancreas from WT, KMA1het and KMA1ko mice were

collected after 5 days of Tamoxifen treatment and homogenized in a cold acid/ethanol mixture containing 75%

ethanol, 23.5% distilled water, 1.5% 2N hydrochloric acid to extract insulin. The level of insulin in the extract

was measured using an Ultrasensitive Insulin ELISA kit (Crystal Chem, Downers Groove, IL). Data are

expressed as means ± SE, n = 2 mice per group.

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Fig.S7 pancreatic β-cells from KMA1ko mice show defective glucose induced insulin secretion. Mice

were treated with Tamoxifen consecutively for 5 days, and sacrificed thirty days later. (A). Shows results from

the perifusion assay. Islets isolated from WT or KMA1ko mice were used in perifusion assays. Every twenty minutes the islets were stimulated with 14 mM of glucose and perifusates were collected to determine the

levels of insulin. Representative results from 4 mice per group are shown (B). and (C) show area under the

curve for peak 1 and peak 2 respectively, (indicated in panel A) for each of the groups. *p < 0.05, **p<0.01, n =

4 mice per group.

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Supplementary Tables

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